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Doctoral Thesis

Fungal lifestyle: analysis of the cell wall and secretedantibacterial proteins

Author(s): Essig, Andreas

Publication Date: 2014

Permanent Link: https://doi.org/10.3929/ethz-a-010279227

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ETH Library

DISS. ETH NO. 21789

Fungal lifestyle: analysis of the cell

wall and secreted antibacterial proteins

A thesis submitted to attain the degree of

DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich)

presented by

ANDREAS ESSIG

MSc ETH Biology

born on June 10, 1982 citizen of Mettauertal, Aargau

accepted on the recommendation of

Prof. Dr. Markus Aebi Dr. Markus Künzler

Prof. Dr. Ruedi Aebersold Prof. Dr. Gerald Hart

Prof. Dr. Hans-Georg Sahl

2014

Cover image NMR structure of Copsin

Acknowledgments First of all, I would like to thank my two supervisors Prof. Dr. Markus Aebi and Dr. Markus

Künzler for giving me the opportunity to perform my PhD thesis in their group. I greatly

appreciated their support and the freedom they gave me to design my projects. Fruitful

discussions and a stimulating working atmosphere they created had certainly a major impact on

the outcome of this thesis.

Many thanks go to my PhD committee members Prof. Gerald Hart, Prof. Hans-Georg Sahl, and

Prof. Ruedi Aebersold for their helpful inputs and feedbacks during my PhD.

Furthermore, I would like to acknowledge my scientific collaborators for their extensive

contributions to my projects, especially Paolo Nanni, Peter Gehrig, Bernd Roschitzki, and René

Brunisholz from the FGCZ, Prof. Gerhard Wider and Daniela Hofmann from the institute of

molecular biology and biophysics, and Tanja Schneider and Daniela Münch from the university

of Bonn.

I owe many thanks to my two Master students Savitha Gayathri and John Hintze for their

valuable support. It was a great pleasure to work with them.

I would like to thank members of the molecular life sciences PhD program, in particular, Susanna

Bachmann.

Many thanks go to past and present members of the Aebi Group and the institute of microbiology

for a very nice time inside and outside the lab. Especially, I want to thank Susanne, Andreas,

David, Martina, Stefanie, Niels, Ivan, Ramon, Jörg, Sonja, Palmira and Pauli.

Finally, I am deeply thankful to my parents, my brother and Alessia for their love and constant

support.

Table of Contents

Summary …………………………………………………………………………………. i

Zusammenfassung …………………………………………………………………… iii

Chapter 1 ………………………………………………………………………………… 1

Introduction - Antimicrobial peptides

Chapter 2 ……………………………………………………………………………….. 17

Copsin, a novel peptide-based fungal antibiotic interfering with the

peptidoglycan synthesis

Chapter 3 ……………………………………………………………………………….. 51

Studies towards the mode of action of copsin and its homologs in C. cinerea

Chapter 4 ……………………………………………………………………………….. 75

Fungal lysozyme identified in the secretome of C. cinerea

Chapter 5 ……………………………………………………………………………..... 99

O-mannosylated cell wall proteins of S. cerevisiae

Chapter 6 ……………………………………………………………………………... 131

Discussion and future perspectives

Curriculum vitae ...…………………………………………………………………... 139

Summary

Fungi and bacteria coexist in a variety of biological niches, including soil, plants, and animals. In

these environments, the cell wall and secreted substances provide fungi with an effective

machinery to interact and to compete with bacteria. The discovery of penicillin by Alexander

Fleming, a fungal antibacterial metabolite, demonstrated the importance of understanding such

defense mechanisms.

Antimicrobial peptides (AMPs) are a highly diverse group of defense molecules identified in

prokaryotes and eukaryotes. As a part of the innate immune system, AMPs act microbicidal and

possess immunomodulatory properties. The first chapter provides an overview of the structure

and activity of AMPs including pharmaceutical applications.

The identification and characterization of a novel fungal AMP is described in chapter 2.

Coprinopsis cinerea is a basidiomycete that naturally occurs in herbivorous dung. Based on a

defined analytical setup, we studied the interactions of C. cinerea with different bacterial species

and analyzed secreted fungal proteins. A novel fungal defensin, copsin, was identified by

quantitative mass spectrometry (MS) and recombinantly produced in Pichia pastoris. The

structure of copsin was solved by NMR, which revealed an α/β-fold stabilized by six disulfide

bonds. Both the structural compactness and its terminal modifications render copsin extremely

heat stable and insensitive towards proteases. Characterization of the antibacterial activity

showed that copsin acts bactericidal by binding to the cell wall precursor lipid II predominately in

Gram positive species. Its high stability and potent activity against bacteria resistant to

conventional antibiotics make copsin to a valuable candidate for a novel antibacterial drug.

Further characterization of copsin and its homologs is reported in chapter 3. A sequence

comparison of copsin exhibited several homologous AMPs encoded in the C. cinerea genome.

Copsin and the homologous defensin CC82 were successfully produced in a bioreactor using P.

pastoris as the heterologous host. The distinct antibacterial profiles determined for both

polypeptides indicate that C. cinerea expresses a diversified group of AMPs against a multitude

of microbial competitors found in herbivorous dung.

Saprophytic fungi such as C. cinerea secrete an arsenal of enzymes to digest dead organic

matter. The secretome of C. cinerea was analyzed by MS, as described in chapter 4. Besides

numerous proteases and glycosidases, we discovered a novel lysozyme, for which we optimized

the heterologous expression in P. pastoris. This fungal lysozyme has a low sequence identity to

known peptidoglycan cleaving enzymes and possesses a very specific antibacterial profile.

i

The cell wall of fungi is a unique and complex network of polysaccharides and glycoproteins.

Besides the function as a determinant of the cell shape, it acts as a protective line for invading

and competing microbes. In order to gain more insights in the cell wall structure, we developed a

MS based workflow for the analysis of O-mannose (O-Man) glycans, a structurally and

functionally important modification of cell wall proteins. In chapter 5, a comprehensive yeast cell

wall O-Man glycoproteome is described with an in-depth analysis of the enormous heterogeneity

of this type of O-glycosylation. Furthermore, we implemented SILAC (stable isotope labeling by

amino acids in cell culture), which is a valuable tool for quantitative MS measurements of O-Man

glycans in yeast and other organisms.

ii

Zusammenfassung

Pilze und Bakterien interagieren in einer Vielzahl von biologischen Nischen; einschliesslich dem

Erdreich, Pflanzen und Tieren. Dabei dienen die Zellwand und sekretierte Substanzen den

Pilzen unter anderem zur Abwehr von Bakterien. Die Entdeckung des pilzlichen Metaboliten

Penicillin durch Alexander Fleming und dessen medizinische Anwendung hat gezeigt, wie

wichtig das Verständnis solcher Abwehrmechanismen ist.

Antimikrobielle Peptide (AMPs) sind eine vielfältige Gruppe von Molekülen, die von Prokaryoten

und Eukaryoten exprimiert werden. Als Teil der angeborenen Immunabwehr besitzen AMPs

antimikrobielle und immunomodulatorische Eigenschaften. Das erste Kapitel gibt einen

Überblick über die Struktur und Aktivität von bekannten AMPs und deren pharmazeutische

Anwendungen.

Die Identifizierung und Charakterisierung eines neuen pilzlichen AMP wird in Kapitel 2

beschrieben. Coprinopsis cinerea ist ein Ständerpilz, der natürlicherweise auf Dung von

pflanzenfressenden Tieren vorkommt. Die Interaktion von C. cinerea mit verschiedenen

Bakterienarten wurde in einem Modellsystem studiert, welches es ermöglichte sekretierte

Pilzproteine zu analysieren. Dabei wurde das Defensin Copsin mittels quantitativer

Massenspektrometrie identifiziert und in Pichia pastoris rekombinant produziert. Die durch NMR

gelöste 3D Struktur ergab eine α/β-Faltung, die durch sechs Disulfidbrücken stabilisiert ist. Die

strukturelle Kompaktheit und die terminalen Modifikationen machen Copsin extrem

hitzebeständig und unempfindlich gegenüber Proteasen. Die Charakterisierung der

antibakteriellen Aktivität zeigte, dass Copsin an den Peptidoglykan-Vorläufer Lipid II bindet und

dabei die Zellwandsynthese von vorwiegend Gram positiven Bakterien hemmt. Die enorme

Stabilität und eine starke Aktivität gegen Bakterien, die gegenüber herkömmlichen Antibiotika

resistent geworden sind, machen Copsin zu einem vielversprechenden Kandidaten für ein neues

Antibiotikum.

Die weitere Charakterisierung von Copsin und dessen homologen AMPs wird in Kapitel 3

diskutiert. Ein Sequenzvergleich von Copsin zeigte, dass im Genom von C. cinerea mehrere

homologe AMPs kodiert sind. Copsin und das homologe Defensin CC82 wurden erfolgreich in

einem Bioreaktor in P. pastoris produziert. Die unterschiedlichen antibakteriellen Profile von

Copsin und CC82 deuten darauf hin, dass C. cinerea eine diversifizierte Gruppe von AMPs

exprimiert, die gegen spezifische Mikroorganismen aktiv sind.

Saprophytisch lebende Pilze wie C. cinerea sekretieren ein Arsenal von Enzymen die dem

Abbau von totem organischem Material dienen. Das Sekretom von C. cinerea wurde mittels

iii

Massenspektrometrie analysiert und ist in Kapitel 4 beschrieben. Unter zahlreichen Proteasen

und Glykosidasen identifizierten wir ein neuartiges Lysozym, das wir in P. pastoris heterolog

exprimierten. Dieses pilzliche Lysozym zeigt eine niedrige Sequenzidentität mit bereits

bekannten Peptidoglykan-spaltenden Enzymen und besitzt ein sehr spezifisches antibakterielles

Profil.

Die Zellwand von Pilzen ist ein einzigartiges und komplexes Netzwerk von Polysacchariden und

Glykoproteinen. Sie dient nicht nur zur Stabilisierung der Zelle, sondern auch zum Schutz vor

invasiven und konkurrierenden Mikroben. Basierend auf einem massenspektrometrischen

Ansatz haben wir eine Methode zur Analyse von O-Mannose (O-Man) Glykanen entwickelt.

Diese O-Glykane stellen eine strukturell und funktionell wichtige Modifikation von

Zellwandproteinen dar und ermöglichten uns weitere Einblicke in die Zellwandstruktur. In Kapitel

5 beschreiben wir ein umfassendes Hefe-Zellwand O-Man Glykoproteom und analysieren

eingehend die enorme Heterogenität dieser Art von O-Glykosylierung. Zur quantitativen

Messung von O-Man Glykanen in Hefe integrierten wir SILAC (stable isotope labeling by amino

acids in cell culture) in die Analyse.

iv

Chapter 1

Introduction

Antimicrobial peptides

1

Introduction

More than 2300 antimicrobial peptides (AMPs) are described and are cataloged in the AMP

database (http://aps.unmc.edu/AP/main.php). AMPs are expressed by all organism

examined and they play a key role in defense strategies against bacteria, fungi, and viruses 1,2. Especially organisms that lack an adaptive immune system rely heavily on the action of

AMPs as first line of defense. In addition to directly killing microbes, AMPs are frequently

involved in modulation of the immune system, for example, by attracting and activating

immune cells, by supporting wound repair and angiogenesis, or by neutralizing bacterial

endotoxins 3,4.

The diversity of AMPs with regard to primary sequences and structural elements makes a

proper classification difficult. Nevertheless, there are some general features that count for

most AMPs. AMPs are gene encoded and are often synthesized as prepro-proteins that are

proteolytically processed to release an active AMP. They are short with a length of

approximately 6 to 80 amino acids and are rich in arginine and lysine residues and thus,

have an overall positive charge. However, there are anionic peptides active against

microbes. But only little is known about their targets and mechanism of action 5. They are not

further covered in this review. Hancock and Sahl classified AMPs according to their primary

amino acid sequence and structural features into four classes: (i) β-sheet peptides stabilized

by two to four disulfide bonds; (ii) α-helical peptides; (iii) extended structures enriched in

certain amino acids; (iv) loop peptides with one or more disulfide bond 2.

The three biggest classes of AMPs, bacteriocins, cathelicidins and defensins, are described

in more detail in the following sections with a special emphasize on defensins.

Chapter 1

2

Bacteriocins

Bacteriocins are a heterogeneous subgroup of AMPs exclusively produced by bacteria. They

mainly act as growth inhibitors of other microorganisms competing for nutrients 6.

Bacteriocins expressed by Gram positive bacteria are commonly categorizes in modified

(class I) and unmodified peptides (class II). Both classes are further divided according to

their primary sequences and structural features 7.

Amino acids side chains of class I peptides undergo extensive post translational

modifications. An example is the group of lantibiotics, characterized by the dehydration of

serine and threonine residues and subsequent formation of lanthionine thioether bonds 8.

Nisin and mersacidin are two very prominent members of lantibiotics. Both are synthesized

as an inactive precursor containing a leader peptide that directs them to the transport and

modification machinery 9. Nisin is produced by strains of Lactococcus lactis and exhibits

potent activity against different Gram positive bacteria 10. For the mode of action, different

models are proposed. In the lipid II independent mechanism, nisin aggregates in the outer

leaflet of the membrane, which leads to the formation of short-lived pore-like structures. In

the second model, nisin integrates into the membrane and forms a complex with lipid II,

which leads to an oligomerization and subsequent pore formation 11. The lipid II dependent

pores exhibit a much higher stability, increasing the activity of nisin approximately by three

orders of magnitude 9. Due to its low toxicity and high antibacterial activity, nisin is approved

as food additive since 1988. Mersacidin, a lantibiotic with globular structure, kills bacteria

also via interaction with lipid II 12. However, in comparison to nisin, it does not lead to pore

formation.

Class II bacteriocins lack modified residues. They include the class IIa one-peptide pediocin-

like bacteriocins and the class IIb two-peptide bacteriocins 13. Pediocin PA-1 is a plasmid

encoded member of the class IIa peptides initially characterized from Pediococcus acidilactici 14. As it shows activity in the nanomolar range against human pathogens like Listeria

monocytogenes, it is currently tested as food preservative. Lactococcin G was the first two-

peptide class IIb bacteriocin identified in 1992 15. It consists of the 39 residue α-peptide and

the 35 residue β-peptide encoded next to each other in the same operon. Both peptides

together form a membrane penetrating helix-helix structure that renders the membranes

permeable for small molecules, leading to cell death. An optimal activity requires the

presence of both peptides in equal amounts. Class IIb bacteriocins have to be further

characterized and optimized for making them valuable candidates for applications in clinics

and in food industry 13.

Chapter 1

3

In some classifications, proteins with an enzymatic activity are categorized as class III

bacteriocins (e.g. enterolysin A) and circular bacteriocins as class IV 6. These two classes

are not covered in this review.

AMPs that are ribosomally synthesized by Gram negative bacteria can be divided in larger

proteins such as colicins and smaller peptides such as microcins 6.

Colicins are organized in a C-terminal catalytic domain, an N-terminal translocation domain

and a central receptor binding domain 16. They are able to cross the outer membrane of

Gram negative bacteria by interacting with specific receptors and finally execute their

antibacterial activity on a periplasmic or intracellular compound. However, it is not fully

understood how colicins cross the membrane and finally kill the cells. Due to their high

molecular weight, they are not considered suitable for drug development and are often not

assigned to the group of AMPs similar to class III bacteriocins.

Microcins are peptides with a mass below 10 kDa secreted by enterobacteria. They are

synthesized as precursor peptides similar to bacteriocins of Gram positive bacteria and they

act mainly in regulation of the intestinal microflora 17. Their killing mechanism is based on

pore formation or interaction with periplasmic or intracellular compounds such as the RNA

polymerase. Microcins are extremely heat and pH stable and insensitive towards proteases.

Cathelicidins

Cathelicidins are ubiquitously expressed in vertebrates, such as the fowlicidins in chicken 18,

protegrins in pigs 19, or cathelicidins identified in snake venoms 20. One of the first

cathelicidins described was a dodecapeptide isolated from bovine neutrophils, where it is

stored in secretory granules and released upon inflammatory stimuli 21. So far, no cathelicidin

has been identified in an invertebrate.

Cathelicidins are synthesized as precursor proteins that are characterized by a highly

conserved pro-sequence linked to a heterogenic C-terminal domain corresponding to the

mature antimicrobial peptide. The pro-sequence (~100 amino acids) shows a high similarity

to cathelin, an inhibitor of cathepsin L that gave the name to this group of AMPs 22,23. The C-

terminal antimicrobial domain (~10-80 amino acids) comprises, in comparison to the cathelin

domain, a wide repertoire of structures including linear and cyclic molecules (Fig. 1).

Chapter 1

4

Fig. 1. Gene and protein structures of cathelicidins. Cathelicidin genes are composed of four

exons and three introns. The exons 1-3 specify the prepro-region including the cathelin pro-sequence.

The antimicrobial domain is encoded in exon 4. Mature AMPs include α-helical (a, LL-37), cysteine-

rich (b, protegrin-1), and tryptophan-rich (c, indolicidin) peptides and proline-rich peptides (prophenin).

Structures obtained from www.rcsb.org (adapted from Zanetti, M., 2005) 22.

In higher concentrations, cathelicidins are detected at sites of inflammation, where they act

as a part of the innate immune system to kill bacteria, fungi or viruses 24. In addition to the

direct antimicrobial activity, cathelicidins possess different immunomodulatory properties, for

example, the chemoattractive action on neutrophils and monocytes or binding and

inactivation of LPS and thus, preventing an endotoxic shock 25,26.

In humans, one cathelicidin gene has been identified, which encodes a precursor protein

called human cationic antimicrobial peptide-18 (hCAP18) with in total 170 amino acid

residues 27. After removal of the signal peptide, hCAP18 is stored as inactive precursor in

secondary granules. The pro-peptide is cleaved off by the serine proteinase 3 after secretion

and the active AMP LL-37 is released. The hCAP18/LL37 protein is widely expressed in

humans, for example, in mucous epithelia, sweat, skin, or salivary glands. It is also secreted

into wound fluid and airway surface fluid 22.

LL-37 adopts a cationic amphipathic helix-helix structure, which is composed of an N- and C-

terminal α-helix and a C-terminal tail. The positively charged residues allow the peptide to

interact with negatively charged head groups of phospholipids of the bacterial membrane.

The subsequent pore forming activity is based on a toroidal pore carpet-like mechanism

Chapter 1

5

proposed 28,29. LL-37 is active against a wide range of bacteria such as Escherichia coli,

Staphylococcus aureus, and Mycobacterium tuberculosis. LL-37 is involved in a multitude of

other functions of the immune system, such as an anti-endotoxic activity, immunostimulation,

or wound repair 24.

Several attempts were undertaken to develop cathelicidins and their derivatives in

antimicrobial or immunomodulatory drugs, but development was hindered by a high toxicity

and low solubility. One of the few promising candidates is omiganan, an indolicidin based

peptide variant. It is currently undergoing Phase III clinical trials mainly for topical applications

against acne and rosacea 6,30.

Defensins

More than 50 years ago, leukin, an extract of rabbit leukocytes, was shown to be able to kill

Gram positive bacteria 31. Shortly after this discovery, another extract from rabbit leukocytes

exhibited activity against Gram positive and Gram negative bacteria. It was termed

phagocytin 32. In the following decades, both extracts were further characterized and the

active component was called cationic antimicrobial proteins (CAPs). In 1985, the term

defensin was introduced for natural peptide antibiotics 33. Nowadays, more than 250

defensins are described in the literature covering producing organisms from fungi, plants,

insects, to mammals (Fig. 2).

Defensins are synthesized as prepro-proteins, which are processed to release an active

AMP. In comparison to cathelicidins, the mature peptides are much more conserved

throughout evolution, which made a more accessible classification possible. Considering all

biological kingdoms defensins can be roughly divided in two big groups, fungal / plant /

invertebrate defensins and vertebrate defensins. This classification is mainly based on

structural motifs, which is explained in more detail in the following sections.

Vertebrate defensins

Vertebrate defensins are divided in linear α- and β-defensins and cyclic θ-defensins.

Predominantly expressed by epithelial cells, they often act as a first line of defense against

invading pathogens such as bacteria, fungi, or viruses. Similar to cathelicidins, vertebrate

defensins have a variety of other biological effects such as immunomodulation, neutralization

of endotoxins, or induction of angiogenesis and wound repair 4,34.

α- and β-defensins share the common structural motif of a triple stranded antiparallel β-sheet

structure stabilized by three disulfide bonds. A differentiation between these two groups is

based on their amino acid composition and the pattern of pairing between the cysteine

residues. α-defensins (cysteine pairing: 1-6, 2-4, 3-5) are relatively rare in lysine residues and

Chapter 1

6

are generally 5-10 amino acids shorter than β-defensins (cysteine pairing: 1-5, 2-4, 3-6),

which contain a high number of lysine and arginine residues.

In humans, six α-defensins have been identified, called HNP (human neutrophil peptides) 1-4

and HD (human defensin) 5-6 4. HNP 1-4 are fully processed to the active form before they

are stored in primary granules of leukocytes. HD 5-6 are expressed by Paneth cells of the

small intestine. Upon stimulation, α-defensins are released from granules into the

extracellular space together with other defense molecules such as lysozymes, tumor

necrosis factor-α (TNF-α), and immunoglobulin A 4.

Four β-defensins (HBD 1-4) have been described in more detail and more than 30 have

been predicted by bioinformatics tools in humans, but little is known about their biological

functions. HBDs are predominantly expressed in epithelial cells and differ substantially in

their activity profile against Gram negative and Gram positive bacteria 34. In comparison to α-

defensins that have been identified in mammals only, AMPs with close homology to β-

defensins could be tracked back to reptiles and birds such as the crotamine-myotoxin family

of snake venom or the avian β-defensin family (AvBD) 35,36,37.

The only cyclic peptides known from mammals are θ-defensins, first isolated from rhesus

macaque leukocytes and bone marrow 38. They are produced through a head-to-tail ligation

of two truncated α-defensins, which results in a cyclic peptide stabilized by three disulfide

bonds. The antimicrobial spectrum is similar to the one of α-defensins with an enhanced

activity against viruses. Humans express mRNA encoding for θ-defensins, but premature

stop codons prevent the production of a functional peptide 39.

Due to the fact that most vertebrate defensins possess an amphiphilic structure with a high

net positive charge, mode of actions are proposed, where defensins accumulate and embed

into the lipid bilayer of bacterial membranes. Finally, this leads to membrane disruption and

cell death 28. However, recent studies support a more targeted mode of action for different

AMPs. HBD-2 and crotamines are two examples that are able to bind to ion channels and

perturb the membrane potential similar to cobatoxins, a scorpion toxin family 36,40.

Furthermore, for several α-and θ-defensins it could be shown that they interact with specific

glycan structures, indicating that binding of carbohydrates mediated by a lectin domain could

play a central role in recognition and killing of microbes 4.

Chapter 1

7

Fig. 2. Major classes of defensins. Plectasin, Pseudoplectania nigrella; Rs-AFP1, Raphanus

sativus; MGD-1, Mytilus galloprovincialis; HNP-1, HBD-2, RTD-1, Homo sapiens. Disulfide bonds

shown in red form the core structural motif CSαβ. All 3D structures were obtained from the protein

data bank (http://www.rcsb.org) 41,42,43,44,45,46.

Chapter 1

8

Invertebrate, plant, and fungal defensins

The majority of invertebrate, fungal, and plant defensins share a core structure that consists

of a cysteine stabilized α-helix β-strand motif (CSαβ) 47. The α-helix is connected over two

disulfide bonds to the second β-strand (cysteine pairing: 2-5, 3-6) and a cysteine residue of

the N-terminal part forms a disulfide bond with a cysteine of the first beta-strand (1-4).

Plant defensins are a diverse group of AMPs with regard to their amino acid composition,

with the exception of eight conserved cysteine residues that form four disulfide bonds 48,49. In

the Arabidopsis thaliana genome more than 300 defensin-like peptides could be identified, of

which 78% comprise the CSαβ fold 50. The majority of plant defensins have been isolated

from seeds such as the radish Raphanus sativus defensins (Rs-AFPs) 51. Rs-AFP1 and Rs-

AFP2 are composed of a CSαβ motif extended by an additional N-terminal β-strand and a

fourth disulfide bond. Upon disruption of the seed coat, Rs-AFPs are released and represent

up to 30% of secreted seed proteins. Unlike most vertebrate and invertebrate defensins that

have an antibacterial impact, the majority of plant defensins exert their antimicrobial action on

fungi, including plant and human pathogenic species. It is proposed that Rs-AFP2 interacts

with glucosylceramides in fungal membranes, inducing Ca2+ influx and thus inhibition of

hyphal growth 52,53. In recent studies, several other biological activities of plant defensins

have been described such as the inhibition of α-amylases, inhibition of protein translation or

an influence on growth and development of plant roots. Due to their high stability and potent

activity against fungi, they are considered as valuable candidates for new antimycotic

drugs 54.

Defensins play a pivotal part in the innate immune system of all invertebrates investigated,

such as scorpions, spiders, mussels, and flies. Drosomycin is one of the best characterized

insect defensins isolated from Drosophila melanogaster 55. Similar to the plant defensin Rs-

AFP, it consists of an extended CSαβ motif and is the major weapon of D. melanogaster

against fungi. However, a structure-function relationship could not be shown experimentally,

so far, mainly due to lack of an efficient expression system to produce an adequate amount

of peptide 56. MGD-1 was originally isolated from the edible mediterranean mussel Mytilus

galloprovincialis and is composed of a CSαβ fold stabilized by four disulfide bonds 43. The

activity of MGD-1 is directed against bacteria, mainly Gram positive species.

With the publication of plectasin in 2005, fungi were added to the list of defensin producing

organisms, as the last biological kingdom 41. Plectasin is expressed by the saprophytic

ascomycete Pseudoplectania nigrella and is strongly active against Gram positive bacteria

such as Streptococcus spp. and Staphylococcus spp. Resembling a prototypic CSαβ fold

stabilized by three disulfide bonds, plectasin shows a high similarity to invertebrate and plant

defensins. This relation led to the assumption that there is a common ancestor of plant,

fungal, and invertebrate defensins, further supported by the recent finding of defensin-like

Chapter 1

9

peptides in bacteria 57. In the following years, several other fungal defensins have been

identified, predominantly in the phyla of ascomycota and zygomycota 58,59,60. These findings

indicate that AMPs are key components of the innate immune system of fungi active against

competing microbes and are very likely expressed by the majority of fungal species. Binding

studies of plectasin with different precursors of the peptidoglycan synthesis revealed that

plectasin binds to lipid II, a target of different antibiotics like nisin or vancomycin 9,61,62.

However, in comparison to the bacteriocin nisin, it did not show any pore forming activity.

NMR based modeling exhibited that plectasin binds specifically to the pyrophosphate moiety

and to the D-γ-glutamate of the pentapeptide side chain of lipid II and interacts with the

membrane surface (Fig. 3). Due to a high efficacy in vivo against pneumococcal infections in

mice, efforts have been done to develop plectasin further to a commercial drug. Therefore,

different expression systems were successfully optimized to get a maximum yield of peptide,

such as in E. coli, Pichia pastoris, and Aspergillus oryzae 41,63,64. Plectasin entered clinical

phase 1 trials and was considered as one of the most promising drug candidates tested.

Nonetheless, due to commercial and scientific reasons Novozymes and Sanofi-Aventis, both

companies involved in the development, decided to quite the studies on plectasin 65.

Fig. 3. Lipid II/plectasin/DPC complex based on NMR spectroscopy. (A) Yellow indicates residues

that interact with a dodecylphosphocholine (DPC) membrane. (B) Residues of plectasin (magenta)

that show substantial chemical shifts upon titration with lipid II. (C) Hydrogen bonds are formed

between the pyrophosphate and F2, G3, C4, and C37. The N-terminus of plectasin and the side chain

of His18 form a salt bridge with D-γ-glutamate of lipid II (from Schneider, T. et al., 2010) 61.

Chapter 1

10

Summary and concluding remarks

AMPs are produced by prokaryotes and eukaryotes, where they are an important part of the

innate immune system. The diversity at the sequence and structure level is immense from

highly derivatized peptides like the lantibiotics nisin and mersacidin to defensins with

secondary structural elements like MGD-1. Therefore, it is often rather difficult to categorize

and define groups of AMPs. In this regard, Yeaman and Yount discovered a structural motif,

which is preserved in all classes of cysteine stabilized AMPs 66. The so-called γ-core is

defined by a hallmark Gly-X-Cys motif (X can be any amino acid), which is integrated in a

Cys-stabilized anti-parallel β-sheet structure (Fig. 4).

Fig. 4. γ-core structural motif. Shown is a Cys-stabilized anti-parallel β-sheet structure with the Gly-

X-Cys motif (X can be any amino acid). Positive charges are commonly found at the poles of the motif.

Peptides shown: mβD-8 (1), a murine β-defensin; BNBD-12 (2), bovine neutrophil β-defensin-12;

DEF3 (3), a human neutrophil defensin; protegrin (4), an antimicrobial peptide from porcine

neutrophils; plectasin (5); MGD-1 (6); and hPF-4 (7), human platelet factor-4 kinocidin (from Yeaman

and Yount, 2010) 67.

Chapter 1

11

The γ-core alone is sufficient for an antimicrobial activity (e.g. protegrin), but can also be

extended by additional α-helices and β-strands (e.g. plectasin). Recently, it has been shown

that the γ-core motif appears also in other classes of disulfide stabilized defense molecules

such as cytokines and chemokines with a direct antimicrobial activity. Interestingly, the γ-core

topologies show a clear pattern related to the complexity of the host immune system.

Invertebrates and fungi mainly produce molecules with a γ-α composition, often

characterized as the CSαβ fold. AMPs of higher organisms with an adaptive immune system

possess an altered structural composition of a γ-β fold, reflecting a co-evolution of AMPs with

the development of the immune system 67.

In contrast to the manifold appearance, most AMPs execute their antibacterial action in a

similar way of disrupting the bacterial cell membrane and forming pore-like structures. Only

for a few AMPs a more specific target has been identified, for example, plectasin and nisin

that both use lipid II as a specific anchoring point in the bacterial membrane. Nevertheless,

bacteria developed several strategies to get resistant against AMPs. For example,

esterification of teichoic acid phosphate groups with D-alanine is one way to reduce the net

negative charge of the cell wall and thus, repelling the overall positively charged AMPs 9,28.

Antimicrobial applications discussed in this review are manifold, directed against bacteria,

fungi, and viruses. We did not address the immunomodulatory properties of AMPs, which

could provide an additional pharmaceutical benefit in comparison to conventional antibiotics.

Altogether, AMPs are promising candidates for medical applications in animals and humans.

Chapter 1

12

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Chapter 1

16

Chapter 2

Copsin, a novel peptide-based

fungal antibiotic interfering with the

peptidoglycan synthesis

Andreas Essiga, Daniela Hofmannb, Daniela Münchc, Savitha Gayathria, Hans-Georg

Sahlc, Gerhard Widerb, Tanja Schneiderc, Markus Aebia

a: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland

b: Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland

c: Institute of Medical Microbiology, Immunology, and Parasitology, Pharmaceutical Microbiology Section,

University of Bonn, Bonn 53105, Germany

Patent application (21.10.2013)

Essig A et al. (2013) Antimicrobial polypeptides, EP13005013 Patent pending.

Contributions

Co-cultivation of C. cinerea and bacteria

Discovery and purification of copsin

Recombinant expression in P. pastoris

MS based N-terminal sequencing

Determination of MICs and kill curve

TLC assays for binding to lipid I and lipid II

17

Abstract

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche.

This repertoire represents a rich and inexhaustible source for antibiotics and fungicides.

Antimicrobial peptides (AMPs) are an emerging class of fungal defense molecules that are

promising candidates for pharmaceutical applications. Based on a co-cultivation system, we

studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different

bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly

expressed in Pichia pastoris and the 3D structure was solved by NMR. The cysteine stabilized

α/β-fold (CSαβ) with a unique disulfide connectivity and an N-terminal pyroglutamate rendered

copsin extremely stable against high temperatures and protease digestion. Copsin was

bactericidal against a diversity of Gram positive bacteria, including human pathogens such as

Enterococcus faecalis and Listeria monocytogenes. Characterization of the antibacterial activity

revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore

interfered with the cell wall biosynthesis. In particular, the third position of the lipid II pentapeptide

was identified as a binding site for copsin. The unique structural properties of copsin make it a

possible scaffold for new antibiotics.

Chapter 2

18

Introduction

Fungi and bacteria coexist in a variety of environments, where fungi directly compete with

heterotrophic bacteria. However, bacterial-fungal interactions (BFIs) can also be commensal or

symbiotic associations. One of the best studied types of BFIs is the antibiosis, where secreted

substances play a key role in combating other microorganisms to defend a nutritional niche (1).

Secondary metabolites are the best characterized group of defense molecules, with their most

prominent member penicillin from the Penicillium chrysogenum mold (2). Numerous other

metabolites discovered in studies of BFIs have pharmaceutical applications, emphasizing the

importance of gaining knowledge on molecular mechanisms of BFIs.

Antimicrobial peptides (AMP) are a class of defense molecules that participate in competing

bacteria and other microbes as a part of an innate immune system. AMPs are a large and highly

diverse group of low molecular mass proteins (<10 kDa), expressed by both prokaryotes and

eukaryotes. They are broadly classified according to their structure and amino acid sequence.

Often, they are characterized by an amphipathic composition resulting in hydrophobic and

cationic clusters (3). Plectasin was the first fungal defensin identified in the secretome of the

ascomycete Pseudoplectania nigrella (4). It shows high similarity to plant and insect defensins

with a core structural motif of a cysteine stabilized α/β-fold (CSαβ). Plectasin acts bactericidal by

binding to the lipid II precursor and by inhibiting the peptidoglycan synthesis of predominantly

Gram positive bacteria (5). The cell wall biosynthetic pathways are a very effective target for

antibacterial substances, as shown for a number of clinically applied antibiotics. The far biggest

group of these drugs are the β-lactams, which inhibit the transpeptidation step of the

peptidoglycan layer (6). In an era of increasing bacterial resistance against many commercially

available antibiotics, AMPs are considered promising candidates for a new generation of

antibiotics.

Here, we studied the interaction of the basidiomycete C. cinerea with different bacterial species.

To model the lifestyle of this coprophilous fungus, a system was developed, where the fungus

was grown on medium-submerged glass beads. This method allowed for a co-cultivation with

different bacterial species and made it possible to analyze the secretome of both fungus and

bacteria.

The analysis of secreted proteins revealed that C. cinerea secreted AMPs that acted as a

defense line against bacteria. One of these peptides, hereafter called copsin, was characterized

further at a molecular and structural level and its mode of action was determined both in vivo and

in vitro.

Chapter 2

19

Results

Interaction of C. cinerea and bacteria

Dung of herbivores, the natural substrate of C. cinerea, is a rather complex environment. The

fungus grows on a solid substrate in high humidity and is confronted with a complex diversity of

bacteria and other microbes (7). To mimic this substrate and to study the interactions of C.

cinerea with bacteria, an artificial system was applied, in which the fungus was grown on glass

beads submerged in liquid medium (8). Because the fungal mycelium was kept in place by the

beads layer, the medium supporting fungal growth could be replaced and also allowed for the

co-cultivation with either Bacillus subtilis (strain 168), Pseudomonas aeruginosa (strain PA01), or

Escherichia coli (strain BL21). In addition, the growth of both organisms in competition could be

monitored easily (Fig. 1).

The Gram negative bacterium P. aeruginosa and E. coli had an inhibitory effect on the growth of

C. cinerea (Fig. 1A). B. subtilis, the Gram positive species tested, did not affect the expansion of

the fungal mycelium. But C. cinerea had an inhibitory impact on B. subtilis (Fig. 1B). P.

aeruginosa was not affected at the beginning of the co-cultivation, but the culture reached the

stationary phase much earlier than the fungal-free control. The growth of E. coli did not display

any dependency on C. cinerea. Our results showed that C. cinerea was indeed interacting in a

species-specific way with bacteria. In particular, we noticed an antagonistic behavior in

competition with B. subtilis and P. aeruginosa.

Chapter 2

20

Fig. 1. Interactions between C. cinerea and bacteria. (A) Vegetative mycelium of C. cinerea was grown

on submerged glass beads in minimal medium. After 60 h, 4 ml medium was replaced by a suspension of

Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. 0 h and 48 h after addition of the bacteria,

the plates were photographed. Fungal growth is indicated by the white mycelium. (B) Bacterial growth was

monitored by measuring OD600 over 48 h. As controls, bacteria and fungus were grown independently in

the beads system. All data points were acquired in three biological replicates and are displayed with the

standard deviation. C. cinerea exhibited a bactericidal effect in competition with B. subtilis and was

strongly inhibited by P. aeruginosa.

Chapter 2

21

Identification and recombinant expression of secreted AMPs

The co-cultivation experiments suggested a secreted fungal substance with a negative effect on

B. subtilis growth (Fig. 1B). To characterize this fungal activity, it was purified from conditioned

medium: Unchallenged C. cinerea was grown in the glass beads system in minimal medium.

After five days, the medium was collected, concentrated and the proteins precipitated with

ammonium sulfate. After a proteinase K treatment at 60 °C, the remaining proteins were

separated on a cation exchange column and evaluated according to their activity against B.

subtilis in a standard disk diffusion assay (Fig. S1 A and B). Fractions that displayed an

inhibition zone against B. subtilis were treated with reducing agent followed by a tryptic digest

and subjected to a mass spectrometry (MS) measurement. Five C. cinerea-derived proteins

were identified in all of the active fractions. These proteins were quantified by spectral counting

and the relative amount of each protein was correlated to the activity in the disk diffusion assay

(Fig. S1C). The relative concentration of the protein CC1G_13813 (copsin) was congruent to the

activity profile.

The transcript of the copsin locus was analyzed by reverse transcriptase-initiated PCR and the

results revealed an open reading frame encoding a protein with in total 184 amino acids. In silico

analysis suggested a signal peptide (position 1-23), a pro-peptide (position 24-127) and a

carboxy-terminal domain of 57 amino acids corresponding to the mature AMP (Fig. S2).

To obtain pure copsin, the cDNA encoding the native signal sequence was cloned in a pPICZA

expression vector and transformed in Pichia pastoris. The secreted recombinant product was

purified by cation-exchange chromatography and yielded a mature polypeptide with a

monoisotopic mass of 6059.5 Da, determined by electrospray ionization (ESI)-MS (theoretical

monoisotopic mass: 6059.5 Da).

Sequencing of the N-terminus by ESI-MS/MS of recombinant copsin and of the native

polypeptide from C. cinerea revealed the same peptide, both with an N-terminal glutamine

converted to a pyroglutamate (Fig. S3). These findings showed that the recombinant prepro-

protein was processed in P. pastoris in the same way as in the host fungus C. cinerea. Mature

copsin contained 12 cysteine (Cys) residues, all involved in a disulfide bond as displayed by a

mass shift of 12 Da after a reduction with dithiothreitol. This was further confirmed by typical 13C

NMR chemical shifts, which differ from reduced Cys residues. [1H, 15N]-HMQC NMR spectra

recorded at pH 6.8 and 7.4 exhibited a stable positive charge on the His26 side chain and a

delta protonated, uncharged His21. Thus, a net positive charge of +7 was assigned to the

polypeptide.

A sequence comparison on the AMP database and by the Blastp algorithm exhibited maximum

identities of 20-27% of copsin with defensins from invertebrates, fungi, and plants, such as

plectasin from P. nigrella or eurocin from Eurotium amstelodami, both assigned to the fungal

phylum of ascomycota (Fig. 2A) (4, 9, 10).

Chapter 2

22

Fig. 2. 3D structure of copsin and similarity to other AMPs. (A) A sequence alignment of copsin with

AMPs exhibited the highest sequence identities with defensins from plants, fungi, and invertebrates (4, 10-

12). Disulfide bonds shown in solid lines form the core structural motif CSαβ. Disulfide bonds in dashed

lines were additionally detected in copsin. Regions of the α-helix and the two β-strands as well as the

positions of the Cys residues are indicated according to the sequence of copsin. The alignment was

performed with the ClustalW algorithm and visualized with the Jalview software (13, 14). (B) Cartoon

representation of the structure of copsin determined by NMR spectroscopy. Left: ribbon diagram of the

structure with the lowest energy after energy refinement with AMBER (15), highlighting secondary

structure elements; α-helical region (red) and the β-sheet (green). N and C terminus are denoted by N and

C, respectively, and selected residues are indicated with the residue type and sequence number; middle:

bundle of 20 conformers after energy refinement; right: Cys residues are denoted by the sequence

position and are colored in yellow for the conserved disulfide bonds and in orange for the three additional

disulfide bonds of copsin.

Chapter 2

23

Structural features of copsin

The three dimensional structure was determined using nuclear magnetic resonance (NMR)

spectroscopy. An almost complete assignment of resonances was obtained (99.1% backbone

resonances, 85.7% sidechain resonances). The structure calculation was performed as

described in the Supporting Information (SI) Materials and Methods. The input data for the

structure calculation and the characterization of the NMR structure are summarized in Table S1.

The structure of copsin contains one α-helix (residues 15-23) followed by two β-strands and is

stabilized by disulfide bonds (CSαβ) (Fig. 2B). The two β-strands (residues 36-40; 46-50) form a

small antiparallel sheet structure. These secondary structural elements exhibited a high

sequence identity to known CSαβ defensins, whereas the loop regions and the termini were very

unique in their length and composition. Standard identification of disulfide bonds following

proteolytic digest could not be applied, due to lack of single disulfide bond cleavage products.

Therefore, the disulfide bond pattern was investigated based on NMR data using target function

analysis (16), ambiguous disulfide restraint (17), and cysteine-cysteine distance measurements

(18), described in SI Materials and Methods. A comparison of an initial bundle of structures

calculated without disulfide constraints (Fig. S4A) with the three dimensional structures of other

defensins strongly supports the assumption of three conserved disulfide bonds. These bonds

connect the α-helix to the second β-strand (C18-C48; C22-C50) and the N-terminal region to the

first β-strand (C10-C40). Also, these disulfide bonding connectivities converged to a slightly

better target function, than structures obtained for other possible disulfide patterns (Table S2).

Three additional disulfides stabilize copsin, linking the termini to the loop between the α-helix

and the β-sheet. Given the conserved disulfide bonds, it follows that the two cysteines C35 and

C54 are connected. Calculating bonding probabilities for the remaining cysteines, based on

cysteine Cβ-Cβ distances (Table S2), the disulfides C3-C32 and C25-C57 seem most probable.

This assignment of all six disulfide bonds was further validated using ambiguous disulfide

restraints, where the same connectivity was obtained for all calculated structures (Fig. S4B and

C).

The compactness of the structure with its six disulfide bonds and the modifications at the termini

are mainly responsible for the very high stability of copsin. The activity was completely retained

after heat treatment or incubation at different pHs shown in a disk diffusion assay (Fig. S5A and

B). Copsin exhibited a strong resistance towards different proteases, such as proteinase K,

trypsin, or pepsin (Fig. S5C). However, antibacterial activity was lost when treated with a

reducing agent, supporting the pivotal role of disulfide bonds for the structural integrity of copsin.

Chapter 2

24

Antibacterial profile of copsin

The antibacterial activity of recombinant copsin was tested in disk diffusion assays against a

variety of Gram positive and Gram negative bacteria. For selected species the minimal inhibitory

concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the

standard microdilution broth method in Mueller Hinton Broth (MHB) at pH 7.3 (Table S3). Copsin

exhibited MIC values in the low microgram per milliliter range for Gram pos. bacteria, such as B.

subtilis, Listeria spp., and Enterococcus spp., including a vanA type vancomycin resistant E.

faecium strain. Most potent activity was detected against Listeria monocytogenes with MIC

values of 0.25-0.5 µg/ml. A change in pH to 6 in MHB did not affect the activity of copsin,

determined against B. subtilis and S. carnosus. Gram negative species such as E. coli were not

affected in viability when exposed to copsin.

MICs and MBCs did not differ more than twofold in their value indicating a bactericidal effect of

copsin on Gram pos. bacteria. To verify this finding, a kill curve assay using B. subtilis was

performed in MHB at pH 6 and 7.4 (Fig. 3). Independent of the pH, a clear reduction of the

viable count after 30 min of incubation was observed, supporting a bactericidal activity of copsin.

Fig. 3. Killing kinetics of copsin. B. subtilis grown in MHB at pH 6 and 7.3 and incubated with and

without (Ctrl) 4 µg/ml copsin (4xMIC). The OD600 is shown above the spectrum, measured at 0, 2 and 5 h.

Chapter 2

25

Molecular target of copsin in bacteria

The antibacterial activity and specificity of copsin was comparable to cell wall biosynthesis

interfering antibiotics such as plectasin or vancomycin (5). We, therefore, determined the cellular

localization of copsin on B. subtilis and S. carnosus cells during exponential growth phase, using

TAMRA labeled copsin and BODIPY-FL labeled vancomycin as a control. Fig. S6 shows a dual

staining of BODIPY-vancomycin and TAMRA labeled copsin. Vancomycin is known to localize

specifically to sites of active cell wall synthesis (19). Copsin exhibited binding at the cell surface,

preferably in curved regions of the rod-shaped bacterium B. subtilis and similarly distributed on

the spherical-shaped S. carnosus cells. A co-localization with vancomycin was found at the cell

septa.

To gain further insights on the molecular targets, we studied the binding affinity of copsin to

essential precursors of bacterial cell wall synthesis in vitro. Lipid I was synthesized as N-

acetylmuramic acid-pentapeptide (MurNAc-pentapeptide) linked to the

bactoprenolpyrophosphate lipid carrier (C55-PP). The pentapeptide was composed of L-alanyl-γ-

D-glutamyl-L-lysyl-D-alanyl-D-alanine, a composition found in Staphylococcus spp. For lipid II, an

N-acetylglucosamine (GlcNAc) was attached to the MurNAc moiety of lipid I (Fig. 4A). Binding

studies included bactoprenolphosphate (C55-P) and lipid III, a teichoic acid synthesis precursor

consisting of GlcNAc linked to C55-PP (20). Copsin was incubated with the synthesized

compounds in a defined molar ratio for 20 min and the free substrate was analyzed by thin layer

chromatography (TLC) (Fig. 4B). It was found that copsin bound in a 1:1 molar ratio to lipid I and

II, but had no affinity for C55-P and lipid III.

This findings suggested specific interaction of copsin with lipid I and lipid II and indicated that

MurNAc-pentapeptide might be pivotal for a stable complex. To examine whether the peptide

chain was necessary for binding to copsin, truncated versions of the pentapeptide were

synthesized and the binding affinity to copsin was analyzed (Fig. 4C). Copsin showed a strongly

reduced affinity for the lipid I-dipeptide (L-Ala-D-Glu) in comparison to the lipid I-tripeptide (L-Ala-

D-Glu-L-Lys). This result demonstrated that the third position of the pentapeptide is essential for

stable binding of copsin to the peptidoglycan precursor.

Chapter 2

26

Fig. 4. Binding of copsin to cell wall precursors. (A) Schematic of the peptidoglycan precursor lipid II

and the teichoic acid precursor lipid III (M: MurNAc; G: GlcNAc; P: Phosphate). (B) Purified cell wall

precursors were incubated with increasing molar ratios of copsin. After extraction with n-butanol / pyridine

acetate (pH 4.2), samples were analyzed by TLC, which displayed a binding of copsin to lipid I and lipid II.

(C) Truncated versions of lipid I (lipid I-dipeptide, lipid I-tripeptide) as well as lipid I-pentapeptide were

synthesized, using purified enzymes from S. aureus. Copsin was added in increasing molar ratios to the

lipid I versions. After incubation for 20 min at room temperature, samples were extracted as described and

analyzed by TLC. The result showed that copsin is binding to the third position of the pentapeptide.

Based on the hypothesis that copsin bound to lipid II, we wanted to know, whether copsin was

able to permeabilize the bacterial membrane, similar to the mode of action of nisin, a pore

forming lantibiotic (21, 22). Two assays were performed: The first relied on Carboxyflourescein

(CF) efflux from lipid II containing liposomes (Fig. 5A), the second on the potassium efflux of B.

subtilis cells (Fig. S7). In both assays, no pore formation was detected. Importantly, pre-

incubation of the lipid II containing liposomes with copsin inhibited the action of nisin, which was

used as a positive control (Fig. 5B). These results showed that copsin interacted specifically with

lipid II at the extracellular site of the bacterial membrane, prevented the binding of nisin, but did

not lead to pore formation.

Chapter 2

27

Fig. 5. Carboxyfluorescein efflux from lipid II containing liposomes. Activity of copsin and nisin

against unilamellar liposomes made of DOPC supplemented with 0.1 mol% lipid II. Peptide-induced

marker release from liposomes with entrapped CF was measured. The 100% leakage level was

determined by addition of Triton X-100 after 300 sec. (A) 1 µM copsin (solid line) or nisin (dashed line)

were added after 100 sec. (B) First, 1 µM copsin was added (100 sec) following the addition of 1 µM nisin

(200 sec) to the same sample. Copsin did not permeabilize the membrane, but it blocked the action of

nisin.

Chapter 2

28

Discussion

Co-cultivation studies of C. cinerea with bacteria led to the identification of the peptide-based

antibiotic copsin, to our knowledge the first defensin identified in the fungal phylum of

basidiomycota.

Growing fungi on the surface of inert glass beads submerged in liquid medium provides a

reproducible and easy to handle model system for studying the interaction with bacteria. In

comparison to a confrontation assay on an agar plate or in a pure liquid culture, this setup has

the big advantage of combining a solid surface and a humid environment, two pivotal factors for

growth of bacteria and fungi in nature. Furthermore, it allows for a repeatable extraction of

secreted fungal and bacterial substances to perform an appropriate analysis at the metabolomic

and proteomic level. For our assays, B. subtilis, E. coli, and P. aeruginosa were selected as

competitors, which are well characterized and are known to interact with fungi (1, 23, 24). Under

the conditions tested, we observed an antagonistic behavior of the fungus with B. subtilis and P.

aeruginosa and a more commensal association with E. coli. These differentiated interactions

suggest an interdependent community of C. cinerea and bacteria on herbivorous dung to

preserve their nutritional niche. Different studies demonstrated that AMPs act as regulators of

microbial diversity, for example, α-defensins in mice or arminin peptides in Hydra species (25,

26). Based on the inhibition found against B. subtilis we developed a novel workflow for the

purification and identification of highly stable and active antibacterial peptides and proteins. This

analytical method involved a quantitative MS measurement, where we finally selected copsin for

further investigations.

Pichia pastoris, chosen for the production of copsin, is a highly efficient system for the

heterologous expression of secreted proteins at high yields in shake flasks and bioreactors (27).

It ensured also a correct processing of copsin, as P. pastoris expresses a Kex2 protease that

cleaves the pro-region at the lysine-arginine site, frequently identified in defensins (28). The 3D

structure of copsin was solved by NMR and revealed a core CSαβ fold with three disulfide

bonds. The CSαβ structural motif is a major characteristic of defensins of plants, invertebrates,

and fungi and is differentiating them from vertebrate defensins with a common motif of a triple-

stranded β-sheet structure (29). Copsin contains three additional disulfide connectivities not

described in a defensin, so far. The structural compactness with an N-terminal pyroglutamate

and a C-terminal cysteine involved in a disulfide bond render copsin extremely stable in a wide

pH and temperature range and insensitive towards proteases. Apart from the conserved α-helix

and β-strand regions, sequence comparisons exhibited no significant alignments with known

defensins.

Microscopy studies and binding assays with cell wall precursors revealed lipid II as the molecular

target of copsin. Lipid II is highly conserved molecule throughout the bacterial kingdom. Binding

Chapter 2

29

to this essential building block is an effective way to interfere with a proper cell wall synthesis

and consequently to kill a bacterium (30). Furthermore, it is unlikely that toxic effects on, for

example, mammalian cells would occur due to the unique structural composition of this

peptidoglycan precursor and specific binding pattern of copsin. Antibiotics found to interact with

lipid II exert their actions over defined binding sites, such as vancomycin, which interacts with the

D-Ala residues of the pentapeptide (19). In depth studies of plectasin exhibited that the N-

terminal GFGC part is essential for a correct binding to pyrophosphate and glutamate of lipid II

(5). This motif is found in many other defensins such as MGD-1 or eurocin, but is absent in

copsin (10, 11). Binding studies of copsin with truncated versions of lipid I revealed that the third

amino acid in the pentapeptide side chain of lipid II is crucial for binding to copsin, independently

of whether lysine or diaminopimelic acid are located at this position. This finding is consistent

with the strong activity of copsin against a vancomycin resistant Enterococcus faecium strain,

where D-lactate is located at the C-terminal site of lipid II instead of D-Ala (31). Similar to other

fungal defensins as plectasin or eurocin, copsin exhibited a distinct antibacterial profile

predominantly active against Gram positive bacteria. Besides Enterococcus spp., the most

potent activity was determined against L. monocytogenes, a food-borne pathogen causing

severe forms of listeriosis in animals and humans (32).

The exceptional stability of copsin together and potent activity against bacteria are important

features for further applications in clinics or food industry.

Chapter 2

30

Materials and Methods

Chemicals and fungal strains

The C. cinerea strain AmutBmut (A43mut B43mut pab1.2) was used for all experiments involving

the fungus. All chemicals, if not otherwise mentioned, were bought at the highest available purity

from Sigma-Aldrich.

C. cinerea in competition with bacteria on glass beads

Preparation of the glass bead plates was adapted from van Schöll L et al. 2006 (8). In brief, 40 g

of sterile borosilicate glass beads (5 mm) were poured into a Petri dish and 15 ml of C. cinerea

minimal medium (CCMM) was added (composition per liter medium: 5 g glucose, 2 g

asparagine, 50 mg adenine sulfate, 1 g KH2PO4, 2.3 g Na2HPO4 (anhydrous), 0.3 g Na2SO4, 0.5

g C4H12N2O6, 40 µg thiamine-HCl, 0.25 g MgSO4 x 7H2O, and 5 mg p-aminobenzoic acid).

C. cinerea was grown on 1.5% (w/v) agar plates containing YMG (0.4% (w/v) yeast extract

(Oxoid AG, England), 1% (w/v) malt extract (Oxoid AG, England), 0.4% (w/v) glucose) for 4 days

in the dark at 37 °C. Afterwards, two mycelial plugs were cut from the margin of the mycelium

and transferred two the center of a glass bead plate in a distance of 1 cm. After 60 h of letting

the fungus grow on the beads (37 °C, dark), 4 ml of the medium underneath the fungus was

replaced by 4 ml of a bacterial suspension (Bacillus subtilis 168, Pseudomonas aeruginosa 01,

or Escherichia coli BL21) grown previously in CCMM to an optical density (OD600) of 0.2. Both

organisms were grown in competition for 48 h (37 °C, dark). For the fungal growth control, the

medium was replaced by the same amount of CCMM (4 ml). As control for the bacterial growth,

the three bacterial strains were grown in the beads system independently for 48 h. The growth of

the bacteria was monitored by measuring the OD600 of the medium.

Purification of AMPs from fungal secretome

C. cinerea was grown in a glass bead plate (19 cm diameter) with 200 g glass beads and 80 ml

CCMM for 5 days in the dark. Subsequently, the medium was extracted and concentrated to 4 ml

by lyophilization. The proteins were then precipitated with 2.2 M ammonium sulfate at 4 °C for 30

min. After centrifugation at 20000 x g for 15 min, the pellet was dissolved in phosphate buffered

saline (PBS) pH 7.4 and the proteins digested with 100 ng/ul proteinase K (Roche Applied

Science, Germany) at 60 °C for 2 h. The solution was loaded in a 10 kDa MWCO dialysis

cassette (Thermo Scientific, USA) and dialyzed against PBS pH 7.4 at 4 °C for 24 h. The

remaining proteins were applied to a Resource S cation exchange column (GE Healthcare,

England) and eluted with a 100-600 mM NaCl gradient in 50 mM Na-phosphate buffer pH 7.4.

0.5 ml fractions were collected and the effluent monitored by absorbance at 280 nm. The flow

through and fractions showing activity against B. subtilis in a standard disk diffusion assay were

Chapter 2

31

subjected to a reduction with 10 mM DTT at 37 °C for 45 min and alkylation with 40 mM

iodoacetamide at 25 °C for 30 min in the dark. A proteolytic digest was performed with 0.25

µg/ml trypsin (Promega AG, USA) for 16 h at 37 °C and the peptides were desalted on C18

ZipTip columns (Millipore, Germany). The MS analyses were performed on a hybrid Velos LTQ

Orbitrap mass spectrometer (Thermo Scientific, USA) coupled to an Eksigent-nano-HPLC

system (Eksigent Technologies, USA). Separation of peptides was done on a self-made column

(75 µm x 80 mm) packed with C18 AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were

eluted with a linear gradient from 2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250

nl/min. MS and MS/MS spectra were acquired in the data dependent mode with up to 20 collision

induced dissociation (CID) spectra recorded in the ion trap using the most intense ions. All

MS/MS spectra were searched against the p354_filteredMod_d C. cinerea database using the

Mascot search algorithm v2.3 (Matrix Science Inc. , USA) with oxidation (methionine) as variable

modification and carbamidomethyl (cysteine) as fixed modification. Further statistical validation

was performed with Scaffold 4.0 (Proteome Software, USA) with a minimum protein probability of

90% and a minimum peptide probability of 50%. This program was also used for determining the

total non-normalized spectral counts for a protein identified in the fractions active against B.

subtilis and in the flow through.

cDNA synthesis and cloning of the copsin precursor

To extract RNA, 20 mg of lyophilized mycelium was lysed with 25 mg of 0.5 mm glass beads in

three FastPrep steps of 45 s at 4.5, 5.5 and 6.5, cooling the sample for 5 min on ice between

each step. RNA was extracted with 1 ml Qiazol (Qiagen, Germany) and 0.2 ml chloroform. After

a centrifugation at 12000 x g at 4 °C for 15 min, RNA was recovered in the aqueous phase,

washed on-column using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) and eluted in

RNase-free water. cDNA was synthesized from 2 µg of extracted RNA using the Transcriptor

Universal cDNA Master kit (Roche Applied Science, Germany) following the manufacturer

instructions.

The coding sequence of the copsin precursor protein was amplified from cDNA by PCR with the

Phusion high-fidelity DNA polymerase (Thermo Scientific, USA) according to standard protocols

(Sambrook J, Russell D, 2001, Molecular cloning, 3rd edition).

FP: 5’-CCGGAATTCATGAAACTTTCTACTTCTTTGCTCG-3’;

RP: 5’-ACGCGTCGACTTAACAACGAGGGCAGGGG-3’;

The PCR product was cloned into the pPICZA expression plasmid (Life Technologies, USA)

containing a zeocin resistance gene using the EcoRI and SalI (Fermentas GmbH, Switzerland)

restriction sites. The resulting plasmid was linearized by the SacI restriction enzyme and

transformed into the P. pastoris strain NRRLY11430 by electroporation with 1.2 kV of charging

voltage, 25 µF of capacitance and 129 Ω resistance (33). Positive clones were selected on YPD

Chapter 2

32

plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar) containing

100 µg/ml zeocin (LabForce, Switzerland).

Expression and purification of recombinant copsin

P. pastoris transformants were inoculated in BMGY medium (1% (w/v) yeast extract, 2% (w/v)

peptone, 1.3% (w/v) YNB w/o amino acids (Becton Dickinson, USA), 100 mM K-phosphate

buffer pH 6, 1% (v/v) glycerol) and cultured at 30 °C for 48 h. The cells were harvested by

centrifugation at 3000 x g for 10 min, resuspended in P. pastoris minimal medium (1.3% (w/v)

YNB w/o amino acids, 100 mM K-phosphate buffer pH 6, 0.4 µg/ml biotin, 0.5% (w/v) NH4Cl, 1%

(v/v) MeOH) and cultured at 30 °C for 72 h. Methanol was added to 1% (v/v) in a time interval of

12 h and NH4Cl was added to 0.5% (w/v) in a time interval of 24 h.

The culture broth was centrifuged at 3000 x g for 10 min and the supernatant concentrated in a

3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc., USA) by a treatment with

polyethylene glycol 6000 at 4 °C. The concentrated supernatant was dialyzed against 20 mM Na-

phosphate, 50 mM NaCl buffer pH 7 (buffer A) at 4 °C for 24 h. The protein solution was sterile-

filtered and loaded on a self-made SP sephadex cation exchange column equilibrated with buffer

A. The column was washed with 180 mM NaCl and bound proteins were eluted with 400 mM

NaCl in 20 mM Na-phosphate buffer pH 7. The eluent was subjected to a size exclusion

chromatography for further polishing. The separation was performed on a Superdex75 column

(HiLoad 16/60; GE Healthcare, USA) equilibrated with 20 mM Na-phosphate, 50 mM NaCl buffer

pH 6. The effluent was monitored by absorbance at 210 nm. The fractions containing copsin

were combined and the molecular mass was determined by ESI-MS.

Expression of 15N/13C labeled copsin in Pichia pastoris

The expression of labeled copsin was adapted from a previously described protocol (34). In

brief, P. pastoris transformants were inoculated in minimal medium supplemented with 0.5%

(w/v) 15NH4Cl (98 atom% 15N) and 13C3-glycerol (99 atom% 13C), respectively, and cultivated in

shake flasks at 30 °C to an OD600 of approximately 35. After centrifugation at 3000 x g for 10

min, the cell pellet was dissolved in P. pastoris minimal medium containing 0.5% (w/v) 15NH4Cl

(98 atom% 15N) and 13C-methanol (99 atom% 13C). The culturing was performed at 30 °C for 3 d. 13C-methanol was added to 1% (v/v) in a time interval of 12 h and 15NH4Cl was added to 0.5%

(w/v) in a time interval of 24 h. Purification of 15N/13C copsin was performed as described for the

unlabeled product.

NMR

Preparation of NMR Samples. Uniformly 15N/13C isotope-labeled copsin in 20 mM Na-phosphate,

50 mM NaCl buffer was prepared as described above.

Chapter 2

33

NMR Spectroscopy. Sequence specific resonance assignments were obtained using a set of

two-dimensional (2D) [15N,1H], and [13C,1H] heteronuclear single quantum coherence (HSQC),

constant-time [13C,1H] HSQC, [1H,1H] total correlated spectroscopy (TOCSY) (mixing time

τm=80ms), exclusive correlation spectroscopy (ECOSY), [1H,1H] ] nuclear Overhauser

enhancement spectroscopy (NOESY) (τm=40ms), three-dimensional (3D) HN(CO)CA and 15N-

resolved [1H,1H]-TOCSY (τm=40ms) spectra recorded on Bruker Avance III 500, 600, 700 and

900 MHz at a temperature of 293K. Data was processed with TopSpin 3.0 (Bruker, Germany)

and analyzed with CARA 1.8.4 (cara.nmr.ch). The protonation state of histidine residues was

determined by recording a [15N,1H]-HMQC spectrum with a long (22 ms) INEPT transfer period

(35). Proline residues were assigned to cis and trans conformation based on chemical shifts of 13Cβ and 13Cγ nuclei (36).

Structure calculation. Structure calculation was performed using 2D H20 and D2O [1H,1H]-

NOESY and 3D 13C-, 15N-resolved [1H,1H]-NOESY spectra (τm=40 and 60ms), recorded on

Bruker Avance 700 and 900 MHz spectrometers. Eight hydrogen bonds, identified based on

slow exchange of amide protons in D2O and based on correlations in long-range HNCO spectra

(37), were included as restraints. The NOEs were manually picked; the resulting peak list, the

amino acid sequence and the NOESY spectra were used as input for a structure calculation with

the software Cyana 3.0 (38), using the simulated annealing protocol. In each of the seven cycles,

800 structures were calculated. The 40 structures with the lowest target function were then

selected and used to calculate the final structure. At the outset of the structure calculation, no

assumptions on disulfide bond topology were used and the cysteines defined as negative

charged without a H atom. After mapping of the disulfide bonds, a last round of structure

calculation was performed and the best calculated structures were subjected to constrained

energy minimization using the software AMBER (15). The non-standard N-terminal amino acid

pyroglutamate was specially included into the library of AMBER.

Identification of disulfide bonds. Three methods for mapping of the disulfide bonds were used:

(A) ambiguous intersulfur restraint (17), (B) probability of a certain disulfide bond ensemble (18),

and (C) the averaged target function (16). (A) The cysteines were forced to form disulfide bonds

by the Cyana algorithm using ambiguous distance restraints, for every cysteine, between one

specific cysteine and all other cysteines (38). Clustering of at least two sulfur atoms together

satisfies the restraint. Standard distances between the sulfur and carbon atoms Sγ-Sγ (2.1/2.0 Å),

Sγ-Cβ (3.1/3.0Å) and Cβ-Sγ (3.1/3.0Å) were used for upper and lower limit, respectively. The

topology of the disulfide bond of the best 40 structures selected on the basis of their energy was

analyzed and visualized by PyMOL (www.pymol.org) (Fig. S4B-C). (B) An initial set of 800

conformers was calculated with no explicit constraint for disulfide bonds and the sulfur Hγ atoms

removed (Fig. S4A), subsequently the interatomic distances between all cysteine Cβ atoms were

extracted and averaged over the 40 final lowest energy structures. Based on the initial structure

Chapter 2

34

ensemble, all conceivable disulfide patterns were formed and a weighting factor representing the

probability of a specific disulfide bond topology was then calculated for all these possibilities

(Tab. S2). (C) The averaged target function of the 40 best Cyana structures was compared for

different fixed disulfide bond connectivities and violation were analyzed.

Effect of temperature, pH, and proteases on copsin activity

The pH stability of copsin was determined after incubation for 1 h at room temperature in a pH

gradient of buffered solutions, including 250 mM KCl/HCl buffer (pH 2), 100 mM Na-acetate

buffer (pH 4 and 5), 250 mM Na-phosphate buffer (pH 6) and 250 mM HEPES buffer (pH 8). As

a control, copsin form stock solution in 20 mM Na-phosphate,50 mM NaCl buffer pH 6 was

loaded on one. Thermal stability was tested in PBS buffer pH 7.4 at 4, 25, 50, 70, and 90 °C for

1 h incubation.

The effect of proteases as pepsin, trypsin, and proteinase K on the activity of copsin was

investigated by incubation with respective proteases at 37 °C for 3 h in a reaction mixture

containing a ratio of 1:10 (w/w) of protease to copsin. For pepsin the reaction was performed in

250 mM KCl/HCl buffer pH 2 and for trypsin, proteinase K, and for the control without any

protease in 100 mM Tris-HCl buffer pH 8. Copsin was also incubated in 5 mM DTT at pH 8. In

order to ensure that the proteases and buffers themselves do not contribute to any inhibition,

each of the proteases alone in the corresponding buffer was used a control.

After incubation, the samples were centrifuged at 12000 x g and the antibacterial activity of the

supernatant was tested by a disk diffusion assay on B. subtilis.

Antimicrobial activity

The MIC/MBCs were determined by the microdilution broth method (39). In brief, bacteria were

grown to an OD of 0.1-0.2 in Mueller-Hinton broth (MHB; Becton Dickinson, USA). The bacterial

suspension was diluted to 105-106 cfu/ml and added to a two-fold dilution series (0.25-64 µg/ml)

of copsin in a 96-well microtiter plate (Enzyscreen B.V., Netherlands). The plates were incubated

at 37 °C for 20-24 h. For all bacteria, the assays were performed in MHB pH 7.3. For Listeria

spp. and C. diphtheria, MHB was supplemented with 3% laked horse blood (Oxoid AG,

England). For B. subtilis and S. carnosus, the assay was additionally performed in MHB buffered

with 50 mM Na-phosphate pH 6. The cfu/ml was determined by plating serial dilutions on LB-

agar plates. The MIC is defined as the lowest concentration of copsin where no visible growth

was observed. The MBC is defined as the concentration of polypeptide that killed 99.9% of

bacteria after 20 – 24 h. All determinations were performed at least in duplicates.

Chapter 2

35

Killing kinetics

B. subtilis was grown in MHB to an OD600 of 0.2 and diluted to an OD600 of 0.1 in MHB

supplemented with 50 mM Na-phosphate buffer adjusted to pH 6 and 7.3. Copsin was added to

4 µg/ml (4xMIC) and the cultures incubated at 37 °C for 5 h. The cfu/ml were determined in

intervals of 30 min by plating serial dilutions on LB-agar plates. The OD600 was measured at 0, 2

and 5 h.

Light microscopy

For TAMRA labeling, copsin was dissolved in 1 M NaHCO3 buffer pH 8.0 to 1.6 mg/ml. TAMRA

(Life Technologies, USA) was added to a final concentration of 1 mg/ml and incubated at room

temperature for 1 h. The excess dye was removed by passing the solution through a pre-packed

Sephadex G-25M PD-10 desalting column (GE Healthcare, USA) in 20 mM Na-phosphate, 50

mM NaCl buffer pH 6.0.

Bacteria were grown in LB medium to OD600 of 0.3 and incubated with TAMRA-copsin at 0.5xMIC

(0.5 µg/ml) for Bacillus subtilis and 2xMIC (16 µg/ml) for S. carnosus for 10 min. The cells were

washed twice with PBS and then immobilized on a poly-L-lysine coated cover slip. After washing

away the unbound cells with sterile water, BODIPY-vancomycin (Life Technologies, USA) was

used at 1 µg/ml concentration for staining on the cover slip. The cells were observed under a

100x oil-immersion objective (Zeiss, Germany) on a spinning-disk confocal microscope (Visitron,

Germany) and imaged with Evolve 512 EMCCD camera (Photometrics, USA) using standard

filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine (540/575 nm). Images were

processed using ImageJ v1.46.

Binding of copsin to cell wall precursors

2 nmol of each purified C55P, lipid I, lipid II (40) or lipid III (41) were incubated in the presence of

increasing molar copsin concentrations from 0.5:1 - 4:1 (copsin:lipid precursor) in a total volume

of 30 μl. After incubation for 20 min at 25 °C, the mixture was extracted with 2:1 (v/v) n-butanol/

pyridine acetate (pH 4.2), analyzed by thin layer chromatography (TLC) as described earlier (42,

43). Analysis was carried out by PMA staining.

Synthesis of UDP-MurNAc-peptides by S. aureus MurA-F enzymes

UDP-MurNAc-pp was synthesized as described (5) with modifications. UDP-GlcNAc (100 nmol)

was incubated with 15 µg of each of the recombinant histidine-tagged muropeptide synthetases

MurA to MurF in 50 mM Bis-Tris propane, pH 8, 25 mM (NH4)2SO4, 5 mM MgCl2, 5 mM KCl, 0.5

mM DTT, 2 mM ATP, 2 mM phosphoenolpyruvate, 2 mM NADPH, 1 mM of each amino acid (L-

Ala, D-Glu, L-Lys and D-Ala), 10% DMSO in a total volume of 100 µl for 120 min at 30 °C. 33 µl

of the reaction mixture, corresponding to 25 nmol of UDP-MurNAc-pp, were used in a MraY

Chapter 2

36

synthesis assay without further purification (42). UDP-MurNAc-peptide variants with shortened

stem peptide, i.e. UDP-MurNAc-dipeptide and -tripeptide, were synthesized in the presence of

the corresponding subset of muropeptide synthetases and were used for the synthesis of lipid I

(dipeptide) and lipid I (tripeptide), respectively. After synthesis, lipid intermediates were

incubated at room temperature with increasing molar ratios of copsin for 20 min. TLC analysis

was performed as described above.

CF-efflux from lipid II containing liposomes

Large unilamellar vesicles were prepared by the extrusion technique, essentially as described by

Wiedemann I et al. 2001 (22). Vesicles were made of DOPC supplemented with 0.5 mol% Lipid

II (referring to the total amount of phospholipid). Carboxyfluorescein (CF)-loaded vesicles were

prepared with 50 mM CF and then diluted in 1.5 ml of buffer (50 mM MES-KOH, 100 mM K2SO4,

pH 6.0) at a final concentration of 25 μM phospholipid on a phosphorous base. After addition of

1 µM peptide (nisin or copsin), the increase of fluorescence intensity was measured at 520 nm

(excitation at 492 nm) on an RF-5301 spectrophotometer (Shimadzu, Japan) at room

temperature. Leakage was documented relative to the total amount of marker release after

solubilization of the vesicles by addition of 10 μl of 20% Triton X-100.

Potassium efflux from whole cells

For potassium efflux experiments a microprocessor pH meter (pH 213; Hanna Instruments,

Germany) with a MI-442 potassium electrode and MI-409F reference electrode was used. In

order to obtain stable results, the electrodes were pre-conditioned by immersing both the

potassium selective and the reference electrodes in choline-buffer (300 mM choline chloride, 30

mM MES, 20 mM Tris, pH 6.5) for at least 1 hour before starting calibration or measurements.

Calibration was carried out before each determination by immersing the electrodes in fresh

standard solutions containing 0.01, 0.1 or 1 mM KCl in choline buffer. Cells of B. subtilis were

grown in MHB and harvested at an OD600 of 1.0 to 1.5 (3300 x g, 4 °C, 3 min), washed with 50 ml

cold choline buffer, and resuspended in the same buffer to an OD600 of 30. The concentrated cell

suspension was kept on ice and used within 30 min. For each measurement the cells were

diluted in choline buffer (25°C) to an OD600 of about 3. Calculations of potassium-efflux in

percent were performed according to the equations established by Orlov D. S. et al. 2002 (44).

Peptide-induced leakage was monitored for 5 min, with values taken every 10 sec, and was

expressed relative to the total amount of potassium release induced by addition of 1 µM nisin.

Copsin was added at 10xMIC.

Chapter 2

37

References

1. Frey-Klett P, et al. (2011) Bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists. Microbiol Mol Biol Rev 75(4):583-609.

2. Fleming A (1929) On the antibacterial action of cultures of a Penicillium, with special reference

to their use in the isolation of B. influenzae. Brit J Exp Pathol 10:226-236. 3. Hancock RE & Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective

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38. Güntert P (2004) Automated NMR structure calculation with CYANA. Methods Mol Biol 278:353-378.

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44. Orlov DS, Nguyen T, & Lehrer RI (2002) Potassium release, a useful tool for studying

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Chapter 2

40

Supplementary Information

Fig. S1. Identification of an AMP in the secretome of C. cinerea. (A) Proteins were extracted from the

unchallenged C. cinerea secretome, digested with proteinase K, and the remaining proteins fractionated

on a cation exchange column. The effluent was monitored at 280 nm (solid line) and the conductivity was

measured (dashed line). (B) Fractions collected (0.5 ml) during the run were spotted in a disk diffusion

assay against B. subtilis. Activity was detected from 7 to 8.5 ml elution volume. (C) Proteins in the active

fractions and flow through (FT) were identified by an electrospray ionization tandem mass spectrometry

(ESI-MS/MS) measurement and quantified by spectral counting. The relative counts corresponding to the

protein CC1G_13813 (copsin) were best correlating to the diameter of the inhibition zones displayed

against B. subtilis.

Chapter 2

41

1 ATG AAA CTT TCT ACT TCT TTG CTC GCT ATC GTC GCT GTG GCG TCT ACC TTC ATT GGG AAC

M K L S T S L L A I V A V A S T F I G N

>-------------------------------- Signal peptide ------------------------------

61 GCC CTC TCA GCC ACC ACC GTC CCC GGA TGC TTC GCT GAG TGC ATT GAC AAG GCT GCC GTA

A L S A T T V P G C F A E C I D K A A V

----------< >-------------------- Pro peptide ---------------------------------

121 GCC GTC AAT TGC GCC GCG GGG GAT ATC GAC TGC CTC CAG GCT TCC TCG CAG TTC GCT ACT

A V N C A A G D I D C L Q A S S Q F A T

-------------------------------------------------------------------------------

181 ATC GTT AGT GAA TGC GTC GCT ACC AGC GAC TGC ACT GCA CTT TCT CCT GGC TCG GCT TCT

I V S E C V A T S D C T A L S P G S A S

-------------------------------------------------------------------------------

241 GAC GCG GAC TCC ATC AAC AAG ACC TTC AAC ATT CTC TCG GGT CTT GGT TTC ATT GAC GAA

D A D S I N K T F N I L S G L G F I D E

-------------------------------------------------------------------------------

301 GCC GAC GCC TTC AGC GCC GCC GAT GTT CCC GAA GAG CGC GAT CTC ACT GGG TTG GGC CGT

A D A F S A A D V P E E R D L T G L G R

-------------------------------------------------------------------------------

361 GTT TTG CCC GTT GAA AAG CGC CAG AAC TGC CCT ACC CGT CGT GGT TTG TGT GTC ACC TCA

V L P V E K R Q N C P T R R G L C V T S

--------------------------< >--------------------------------------------------

421 GGC TTG ACG GCG TGT CGA AAC CAC TGT CGC TCA TGC CAC CGG GGA GAT GTA GGT TGT GTT

G L T A C R N H C R S C H R G D V G C V

--------------------------------- Mature copsin --------------------------------

481 AGG TGC AGT AAT GCA CAG TGC ACG GGC TTT TTG GGC ACC ACA TGC ACC TGC ATT AAC CCC

R C S N A Q C T G F L G T T C T C I N P

-------------------------------------------------------------------------------

541 TGC CCT CGT TGT TAA

C P R C -

--------------<

Fig. S2. Prepro-protein of copsin. Prediction of the signal peptide was performed with SignalP 4.0 (45).

Chapter 2

42

Fig. S3. Pyroglutamate at the N-terminus of copsin. A tryptic digest of purified recombinant copsin and

the extracted defense peptide of C. cinerea (CC1G_13813) was measured by ESI-MS/MS and the

detected fragments analyzed by the Xcalibur software. The recombinant and natural version of copsin

exhibited both the same N-terminal peptide with a glutamine converted to a pyroglutamate (pE). The

peptide with an N-terminal glutamine was not detectable. y* is a y ion with a loss of an ammonia molecule.

Chapter 2

43

Fig. S4. Structure calculations for the delineation of the six disulfide bonds. Cartoon representation

of the structure of copsin. Cysteine residues are marked with their residue number and colored in orange

for the new and in yellow for the conserved disulfides (see main text). N and C indicate N and C termini,

respectively. (A) Structure calculated with cysteine residues defined as negative charged without any use

of disulfide constraints. (B) Bundle of 40 best structures calculated by Cyana using ambiguous disulfide

constraints (38). Each individual cysteine was forced to build a disulfide bond to at least one of the

remaining eleven cysteines by an upper and lower distance restraint. For all calculated structures the

same disulfide connectivity was obtained. (C) Same as (B) but 90 degrees rotated into the plain of the

paper showing the terminal disulfides.

Chapter 2

44

Fig. S5. Temperature, pH, and protease stability of copsin. (A and B) Shown are disk diffusion assays

on B. subtilis with copsin that was exposed to different temperatures (4, 25, 50, 70, 90 °C) and in a pH

range of 2 to 8 for 1 h. (C) To evaluate the protease resistance, copsin/protease mixtures in a ratio of 10:1

(w/w) were incubated at pH 8 for trypsin and proteinase K and at pH 2 for pepsin for 3 h. Copsin was also

subjected to a treatment with 5 mM DTT. Additionally, the reaction mixtures were spotted without copsin.

As control (Ctrl), untreated copsin was loaded on a disc. The activity of copsin was retained in the whole

temperature and pH range tested and it showed no sensitivity in a treatment with proteases. The only way

to delete the activity of copsin was by disrupting the disulfide bonds.

Chapter 2

45

Fig. S6. Co-localization of copsin and vancomycin. B. subtilis and S. carnosus cells in the exponential

growth phase were stained with TAMRA-copsin (red) and subsequently stained with BODIPY-vancomycin

(green). Cells showed a co-localization of copsin with vancomycin at the cell septa (scale bar: 2 µm).

Fig. S7. Potassium efflux from living cells. Potassium efflux from B. subtilis cells was monitored with a

potassium-sensitive electrode. Ion leakage is expressed relative to the total amount of potassium released

after addition of 1 µM pore-forming lantibiotic nisin (100%, circles). Copsin was added at 10xMIC

(squares). Controls were without peptide antibiotics (triangles). Peptides were added after 60 sec. Copsin

was unable to form pores in the cytoplasmic membrane of B. subtilis cells.

Chapter 2

46

Supplementary Tables

Table S1. Structural statistics of the 20 best NMR structures of copsin

A: calculated without disulfide constraints, B: with defined ensemble of disulfide bonds, C: after energy refinement NMR Restraints

Distance Restraints 823 823 intraresidual sequential (|i-j|=1) 224 237 medium range (1<|i-j|<5) 129 long range (|i-j|>=5) 225 hydrogen bondsa 8 Torsion Anglesb 0 Disulfide bonds Energy Statisticsc

Average distance constraint violations 0.1-0.2 Å 8.9 +/- 2.5 0.2-0.3 Å 3.1 +/- 1.5 0.3-0.4 Å 0.7 +/- 0.6 >0.4 Å 0.1 +/- 0.4 Maximal (Å) 0.33 +/- 0.07 Average angle constraint violations <5 degree 0.0 +/- 0.0 >5 degree 16.0 +/- 0.0 Maximal (degree) 96.57 +/- 0.20 (needs to be improved) Mean AMBER Violation Energy Constraint (kcal mol-1) 1470.8 +/-3.3 Distance (kcal mol-1) 11.3 +/-3.0 Torsion (kcal mol-1) 1459.6 +/-0.8 (needs to be improved) Mean AMBER Energy (kcal mol-1) -2380.0 +/- 8.8 Mean Deviation from ideal covalent geometry Bond Length (Å) 0.0043 +/- 0.0001 Bond Angle (degrees) 1.790 +/- 0.014 Ramachandran plot Statisticsc,d,e

Residues in most favored regions (%) 83.6 +/- 2.5 Residues in additionally allowed regions (%) 15.7 +/- 2.5 Residues in generously allowed regions (%) 0.7 +/- 1.2 Residues in disallowed regions (%) 0.0 +/- 0.0 RMSD to mean structure Statisticsc,d

Backbone atoms (Å) 0.42 +/- 0.09 Heavy atoms (Å) 0.86 +/- 0.11 Target function

A(A)

809 462 115 232 8 8 0 0 0 1 70.4 29.6 0.1 0.0 0.50 +/- 0.09 0.98 +/- 0.16 0.21 +/-0.003

B(B)

823 223 236 126 230 8 0 6 0 1 64.2 35.8 0.0 0.0 0.43 +/- 0.09 0.87 +/- 0.11 0.14 +/-0.003

C(C)

823 223 236 126 230 8 0 6 8.8 +/- 2.0 2.2 +/- 1.2 0.2 +/- 0.4 0.0 +/- 0.0 0.28 +/- 0.03 0.0 +/- 0.0 0.0 +/- 0.0 0.0 +/- 0.0 9.1 +/-1.2 9.1 +/-1.2 0.0 +/- 0.0 -2331.2 +/- 5.2 0.0043 +/- 0.0001 1.728 +/- 0.009 79.2 +/- 1.9 20.7 +/- 2.1 0.1 +/- 0.5 0.0 +/- 0.0 0.34 +/- 0.09 0.78 +/- 0.08

Structure calculation by simulated annealing protocol of Cyana (38) (A), (B), 40 structures were selected on the basis of lowest target function and energy minimized using Amber (15)(C). (A) No constraint for disulfide linkage was used (B) Each disulfide bond was explicitly defined by upper and lower distance limit restraints between the sulfur and carbon atoms of the two linked cysteins: C3-C32, C10-C40, C18-C48, C22-C50, C25-C57, C35-C54 (C) Energy refinement of the structure (B) using Amber (15). a H-bond constraints were identified from HNCO (37) experiments and slow exchanging amide protons in D2O c Statistics computed for the best structures d Based on structured residue range, res 3-57 e Ramachandran plot, as defined by the program Procheck (46)

Chapter 2

47

Table S2. Elucidation of disulfide network by target function a and intercysteine distances b analysis

Disulfide bond pattern Target function a Violations a Weight factor b

Variation of conserved disulfide c

10-40; 18-48; 22-50; 35-54; 3-32; 25-57 * 0.14 1 VdWe 0.0648

10-40; 18-48; 22-35; 50-54; 3-32; 25-57 0.18 1 VdWe 0.0283

10-40; 18-48; 22-54; 35-50; 3-32; 25-57 0.60 5 distancef/ 1 VdWe 5.3654e-7

10-48; 18-40; 22-50; 35-54; 3-32; 25-57 0.16 1 VdWe 1.3644e-4

10-48; 18-40; 22-35; 50-54; 3-32; 25-57 0.20 1 VdWe 5.9653e-4

10-48; 18-40; 22-54; 35-50; 3-32; 25-57 0.95 6 distancef/ 2 VdWe 1.1294e-9

10-18; 48-40; 22-50; 35-54; 3-32; 25-57 0.15 1 VdWe 0.0044

10-18; 48-40; 22-35; 50-54; 3-32; 25-57 0.22 1 VdWe 0.0019

10-18; 48-40; 22-54; 35-50; 3-32; 25-57 0.82 8 distancef/ 1 VdWe 3.6487e-8

10-40; 18-22; 48-50; 35-54; 3-32; 25-57 0.54 3 distancef/ 1 VdWe 1.7643e-5

10-40; 18-50; 48-22; 35-54; 3-32; 25-57 1.29 9 distancef/ 2 VdWe 3.1976e-10

Variation of new disulfides d

10-40; 18-48; 22-50; 35-54; 3-32; 25-57 * 0.14 1 vdWe 0.0648

10-40; 18-48; 22-50; 35-54; 3-25; 32-57 0.15 1 vdWe 0.0091

10-40; 18-48; 22-50; 35-54; 3-57; 32-25 0.30 5 distancef/ 1 vdWe 7.5591e-5 Characterization of bundles of 40 conformers of copsin with different disulfide topologies. The cysteine bond pattern of the final structure (Fig 2B) is represented in the top row of the first column and denoted by a star. Different other conceivable disulfide patterns are listed below and deviations in cysteine connectivities from the favored one are indicated in bold. Target function (second row), amount of violations (third row) and weights based on averaged Cβ-Cβ distances (forth row) are shown for the different cysteine pairings. Worse target function and weight factors were obtained for all remaining combinations of cysteine pairing. a Target function and violation analysis. For every topology a structure calculation was performed using Cyana (38). The disulfides were given as fixed constraints determined by the standard three upper and lower distance limits. Target function and violations are shown averaged over the 40 selected best energy structures. b Characterization of disulfide bond pattern by cysteine Cβ-Cβ distances analysis as described by Klaus (18). Averaged distances were extracted from the structure ensemble generated without any disulfide constraints. Subsequently weights were assigned to every disulfide pattern. The weight is directly proportional to the likelihood of certain disulfide bond pattern to be realized in the final structure. c All possible combination of disulfide bond patterns for the conserved six cysteines are shown with the respective calculated target function, number of violations and the weight factor. d Analysis of the disulfide bond connectivity of the new disulfides in copsin. The bond between C35-C54 is taken as fix, based on the initial structure and the assigned conserved cysteines (see main text). All possible combinations of the remaining four cysteines (C3, C25, C32, C57) are listed and analyzed. e Van der Waal violations f Distance violations * Disulfide bonding connectivity of the final structure as described in results (main text). Variations of single disulfide bonds from this assignment are shown in bold.

Chapter 2

48

Table S3. Antibacterial profile of copsin

Bacterium Source Copsin [µg/ml]

MIC MBC

Bacillus subtilis 168 1 1

Streptococcus

S. pneumoniae DSM 20566 active n.d.

S. pyogenes DSM 20565 active n.d.

Staphylococcus

S. carnosus DSM 20501 8 8

S. epidermidis DSM 20044 64 >64

S. aureus 113 DSM 4910 >64 >64

S. aureus ATCC 29213 >40 >40

Listeria

L. monocytogenes WSLC 1042 0.5 1

L. monocytogenes WSLC 1001 0.25 0.5

L. ivanovii WSLC 3009 0.25 0.5

L. innocua WSLC 2012 0.5 1

Enterococcus

E. faecium DSM 20477 4 8

E. faecium VRE DSM 13590 2 2

E. faecalis DSM 20478 8 16

Micrococcus luteus DSM 1790 0.6 n.d

Escherichia coli BL21 >64 >64

Corynebacterium diphtheriae DSM 44123 4 n.d MICs and MBCs were determined with the microdilution broth method in MHB pH 7.3. The MBC is defined as the concentration of copsin where 99.9% of bacteria are killed within 20-24 h. Active, displayed an inhibition zone in a standard disk diffusion assay; n.d. not determined.

Chapter 2

49

50

Chapter 3

Studies towards the mode of

action of copsin and its homologs

in C. cinerea

Contributions

Identification and characterization of homologous proteins of copsin

Further experiments were performed by Savitha Gayathri and John Hintze during their

Master studies at the ETH Zürich under the supervision of Andreas Essig and Pauli Kallio.

51

Introduction

The following chapter is divided in three sections: 1. Homologous proteins of copsin encoded in

the genome of Coprinopsis cinerea. 2. The recombinant expression of copsin in a bioreactor.

3. Further characterization of the mode of action of copsin.

AMPs are an ancient part of the innate immune system known from all biological kingdoms.

Their evolution strongly contributed to immunological diversity and is essential for host survival 1.

Amplification of defensins is achieved by gene duplications, an event well described for

mammalian β-defensins 2,3. Followed by a positive selection, gene duplications can diversify and

specify the defense machinery, particularly important for invertebrates and fungi that lack an

adaptive immune system. However, little is known about defensins in fungi and their gene copy

numbers.

Pichia pastoris demonstrated to be an appropriate host for the heterologous production of

defensins such as plectasin 4. A big advantage of this expression system is the easy way of

upscaling from shake flask cultures to a fermenter culture, using defined and low cost basal salts

media. A bioreactor system provides a highly controlled environment that allows for growing

P. pastoris to cell densities of approximately 500 OD600 U/ml or 100 g/l cell dry weight 5. A high

biomass is especially important for secreted proteins, due to the fact that the cell density is

roughly proportional to amount of product in the culture supernatant 6. Furthermore, transcription

induced over the AOX1 promoter, is more efficient when methanol is continuously provided at

growth limiting rates, instead of methanol excess commonly added in shake flask cultures.

Deciphering the mode of action of an antibiotic is often difficult and includes a variety of

biochemical assays. A common first step is to investigate, which macromolecular biosynthesis

pathway is affected by an antibiotic, for example, through the incorporation of radioactively

labeled precursors (e.g. DNA replication, protein synthesis, cell wall (peptidoglycan)

synthesis) 7,8. Besides complementary approaches like genetic assays or transcriptional profiling,

recent publications showed that fluorescence microscopy is a powerful tool to get a first idea

how an antibiotic works 9,10. In combination with specific labeling strategies, microscopy allows

for a bacterial cytological profiling (BCP) and discrimination between different antibacterial

substances.

Chapter 3

52

Results and Discussions

1. Homologous proteins of copsin

Sequence comparisons of copsin based on the Blastp algorithm revealed the highest identities

to proteins encoded by C. cinerea itself (Fig. 1). Comparing the homologs at the sequence level,

it was remarkable that the overall scaffold of the peptides was highly conserved with the cysteine

residues and the N-terminal pyroglutamate.

Fig. 1. Copsin and homologs. The highly conserved cysteine pattern is indicated in red. The mature

peptides are shown, including the Kex2 protease recognition site (KR) at the N-terminus. The alignment

was performed with the ClustalW algorithm and visualized with the Jalview software 11,12.

The homolog CC1G_08260, hereafter called CC82, was recombinantly expressed in P. pastoris

(Fig. S1). The antibacterial profiles of recombinant copsin and CC82 were similar with an overall

weaker activity of CC82, determined in a disk diffusion assay (Table 1). Copsin revealed its most

potent inhibition on L. monocytogenes and L. ivanovii, whereas CC82 was not at all active

against these two species. These findings indicated that C. cinerea encodes a diversified group

of AMPs, which are active against specific bacteria and potentially also against other microbes.

The expression of other homologs, such as CC1G_15644 and further investigations on the

antibacterial profiles is needed to confirm this hypothesis. In addition, the high similarity of copsin

and CC82 at the sequence level in contrast to a distinct activity against bacteria can give further

insights, how they interact with lipid II and other compounds of the bacterial cell wall.

Chapter 3

53

Table 1. Antibacterial profile of copsin and its homolog CC82

Bacterium Source Copsin [µg/ml] CC82

MIC MBC

Bacillus subtilis 168 1 1 active

Streptococcus

S. pneumoniae DSM 20566 active n.d. active

S. pyogenes DSM 20565 active n.d. active

Staphylococcus

S. carnosus DSM 20501 8 8 active

S. epidermidis DSM 20044 64 >64 n.a.

S. aureus 113 DSM 4910 >64 >64 n.a.

S. aureus N315 >40 >40 n.d.

S. aureus HG003 >40 >40 n.d.

S. aureus ATCC 29213 >40 >40 n.d.

Listeria

L. monocytogenes WSLC 1042 0.5 1 n.a.

L. monocytogenes WSLC 1001 0.25 0.5 n.d.

L. ivanovii WSLC 3009 0.25 0.5 n.a.

L. innocua WSLC 2012 0.5 1 n.d.

Enterococcus

E. faecium DSM 20477 4 8 active

E. faecium VRE DSM 13590 2 2 active

E. faecalis DSM 20478 8 16 active

Micrococcus luteus DSM 1790 0.6 n.d n.d.

Escherichia coli BL21 >64 >64 n.a.

Corynebacterium diphtheriae DSM 44123 4 n.d n.d

MICs and MBCs were determined with the standard microdilution broth method in MHB pH 7.3. The MBC is defined as the concentration of copsin where 99.9% of bacteria are killed within 20-24 h. Active, displayed an inhibition zone in a standard disk diffusion assay; n.a. no activity in disk diffusion assay; n.d. not determined.

2. Recombinant expression in a bioreactor

The expression of copsin was performed in a 3.6 l bioreactor with the open reading frame (ORF)

encoding the native signal sequence. In general, we followed standard procedures with an initial

biomass production step, using glycerol as the carbon source 13,14. After a transition phase, the

expression of copsin was initiated with methanol and continued over 90 h.

Chapter 3

54

Fig. 2. Copsin production in a bioreactor. Samples were taken from the fermenter culture at defined

time points after starting the induction with methanol. (A) After a centrifugation step, the supernatant was

directly loaded on a SDS-PAGE and a silver-staining was performed. (B) The antibacterial activity was

determined in a disk diffusion assay on Bacillus subtilis. The increase of copsin concentration detectable

at ~10 kDa is clearly correlating with the antibacterial activity.

After methanol induction, we reached a cell wet weight of 320 g/l and a final yield of purified

copsin of 20 mg/l, which is a tenfold increase compared to shake-flask cultures. A silver staining

displayed a very low amount of endogenous proteins secreted by Pichia and a continuously

increasing amount of copsin correlating with its antibacterial activity (Fig. 2).

Taken together, performing the Pichia expression in a bioreactor is absolutely crucial to achieve

high amounts of heterologous product. Based on the setup and protocol that we designed,

optimization is needed at different steps, for example, with a codon optimized version of copsin

(Fig. S2).

3. Mode of action of copsin

Localization of copsin and induced morphological changes

In a first experiment, we studied the morphological effect of copsin on B. subtilis. Therefore, we

treated bacteria with sub-MIC concentrations of copsin and imaged them after 2 h. Differential

interference contrast (DIC) microscopy images revealed bacteria, which were severely swollen

and curved in comparison to untreated cells (Fig. 3). Furthermore, chains of non-separated and

shortened cells were visible with a higher frequency of septation. Some bacteria were

undergoing lysis, most likely due to a lethal concentration of copsin molecules encountered by

that particular cell. Since the cell wall is the major determinant of the bacterial cell shape, the

altered morphology indicated an indirect or direct impact of copsin on cell wall formation.

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Fig. 3. Morphological effect of copsin on B. subtilis. Cells at OD600 of 0.2 were exposed to sub-MIC

concentrations (0.5 µg/ml) of copsin in LB medium for 2 h and mounted on 1% agarose pads. (B - E)

Treated cells showed severe malformations compared to the untreated control sample (A). White arrows

point to swollen and shortened cells and the black arrows point to cells that have lyzed (scale bar: 4 µm).

Peptidoglycan, a polymer of alternating amino sugars of N-acetylglucosamine (GlcNAc) and N-

acetylmuramic acid (MurNAc), is the major constituent of the cell wall of Gram positive bacteria 15,16. To visualize better the underlying features of the morphological effect, fluorescently labeled

BODIPY-FL vancomycin was used as a reporter for sites of active peptidoglycan synthesis on

sub-MIC treated B. subtilis cells. Vancomycin specifically binds to the D-Ala-D-Ala part of newly

synthesized lipid II precursor 17. Phase contrast and the corresponding fluorescence microscopy

images are shown in figure 4. In the control staining, vancomycin was specifically located at the

cell septa and only faintly at the sidewalls of B. subtilis cells (Fig. 4A). A dispersed binding

pattern of vancomycin was visible on copsin treated cells, correlating with the phenotype of bend

cells and multiple septation events in close proximity (Fig. 4B-F). These findings suggested that

copsin induced a disturbed synthesis of peptidoglycan that resulted in the display of vancomycin

targets at the cell surface.

A B C

D

E

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Fig. 4. Localization of vancomycin on sub-MIC treated B. subtilis. Cells at OD600 of 0.2 were

incubated with copsin at 0.5xMIC for 2 h and subsequently stained with BODIPY-vancomycin either in

culture (A - B) or after immobilization on a mounting cover slip (D - F). (A) Control cells that were not

treated with copsin showed an intense staining at the cell septa. (B - F) Copsin treated B. subtilis with an

abnormal septation displayed a localization of vancomycin at bend/swollen sites (scale bar: 5 µm).

Next, we studied the localization of TAMRA labeled copsin on B. subtilis cells. TAMRA is a red

fluorescent dye that specifically reacts with terminal alkyne groups. Importantly, the fluorescent

derivative of copsin retained its activity, as shown in a disk diffusion assay on B. subtilis (Fig.

5A). Additionally, we tested, whether the TAMRA dye itself is leading to an unspecific binding on

B. subtilis. Therefore, we treated cells with TAMRA labeled Marasmius oreades agglutinin

(MOA), a Galα1,3Gal/GalNAc-specific lectin that has no affinity for the cell wall of Gram positive

bacteria 18. As shown in figure 5B, there was no binding of TAMRA dye detectable. Incubation of

bacterial cells with TAMRA-copsin exhibited a staining exclusively at the cell wall. Copsin was

similarly distributed on the spherical-shaped S. carnosus cells (Fig. 5D). For the rod-shaped

bacterium B. subtilis, the fluorescence intensity was slightly increased at the septa and poles of

the cells, suggesting that copsin is preferably binding to curved membrane regions (Fig. 5C).

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Fig. 5. Localization of TAMRA-copsin on bacteria. (A) Copsin labeled with or without TAMRA displayed

the same inhibition zone in a disk diffusion assay on B. subtilis. (B) TAMRA attached to the lectin MOA

was used as a negative control and showed that there is no affinity of the dye itself for B. subtilis. At OD600

of 0.3, B. subtilis (C) and S. carnosus (D) cells were incubated with TAMRA-copsin at 2xMIC for 10min.

For both bacterial species, the fluorescence was located at the cell wall, with an accumulation at the poles

and septa of B. subtilis (scale bar: 8 µm).

A co-staining of B. subtilis and S. carnosus cells with BODIPY-vancomycin and TAMRA-copsin

revealed that both dyes indeed co-localize at the cell septa (Fig. 6). Differences were detectable

in the overall distribution of the fluorescence intensity, as copsin stained the cells more uniformly

than vancomycin that was focused at the cell septa and poles.

C

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Fig. 6. Co-localization of copsin and vancomycin. B. subtilis and S. carnosus cells in the exponential

growth phase were stained with TAMRA-copsin (red) and subsequently stained with BODIPY-vancomycin

(green). Cells showed a co-localization of copsin with vancomycin at the cell septa (scale bar: 6 µm).

To investigate, whether there is a correlation between binding sites of copsin and the

morphological phenotype, B. subtilis cells were incubated with sub-MIC concentrations of

TAMRA-copsin for 2 h. Subsequently, the cells were in addition stained with BODIPY-

vancomycin to compare the binding pattern. Interestingly, copsin was fully covering cells that

were undergoing lysis, where vancomycin was almost completely absent (Fig. 7). These cells did

not show the expected aberrant morphology. On the other hand, cells that received only a low

number of copsin molecules developed severe malformations with mislocalization of

vancomycin, as shown in figure 4. On particular cells, red fluorescence was clearly detectable at

bend regions, suggesting that copsin affects directly the cell wall synthesis.

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Fig. 7. Correlation between binding and effect of copsin on B. subtilis cells. Cells were incubated

with 0.5xMIC TAMRA-copsin for 2 h and subsequently stained with BODIPY-vancomycin. The white

arrows point to cells that encountered a lethal dose of copsin and are about to lyse.

Taken together, copsin had a strong impact on the morphology and viability of bacterial cells. For

localization studies, copsin was fluorescently labeled with the TAMRA dye. B. subtilis treated

with supra-MIC concentrations of TAMRA-copsin for 10 min, exhibited a binding at the

membrane and cell wall with a stronger intensity at the cell poles and septa, similar to

vancomycin. Reducing the concentration of copsin to sub-MIC values with a prolonged exposure

of about 2 h led to an aberrant morphology with severely bend, swollen and shortened cells.

Moreover, vancomycin exhibited a disturbed binding pattern, mainly detectable in regions of

malformations, correlating with multiple septa formations in close proximity. Interestingly, copsin

was then preferably binding to cells that lyzed. These observations indicated that cells, which

received a critical number of copsin molecules and did not further divide, were not able to

develop the aberrant phenotype, but lyzed rapidly. Furthermore, it suggested that copsin

interferes with the cell division machinery or with compounds of the cell membrane or wall. A kill

curve performed for B. subtilis exhibited that there is indeed a strong drop of the viable count

after one generation time (30 min) of growth.

The morphological changes are rather different to phenotypes seen for cell wall interfering

agents, such as vancomycin or penicillin, as we never observed blebbing or spheroblasts.

Similarities can be found with antibiotics that affect the cell membrane. One example is nisin, a

type A lantibiotic produced by Lactococcus lactis with a potent activity against Gram positive

species 19. Nisin exerts its antibacterial action by binding to the pyrophosphate moiety of lipid II

precursor and subsequently penetrating into the cytoplasmic membrane of a bacterium 20. This

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process is followed by pore formation and efflux of vital molecules such as ATP. A recent study

showed that B. subtilis treated with nisin displays multiple septations and septal malformations

with shortened minicells 21. They correlated these observations with a deregulation of the ATP

dependent Min system, which controls and suppresses the formation of new septa 22. The

lipopeptide daptomycin is another example, which even closer resembles our findings.

Daptomycin is naturally produced by the soil bacterium Streptomyces roseosporus and is

approved for the treatment of infections caused by Gram positive pathogens 23. Even though

clinically used for almost one decade, the mode of action is still heavily discussed. Known is that

daptomycin acts in a calcium dependent manner on the cell envelope through interference with

the cell wall biosynthesis and/or the cell membrane integrity 24,25. Treatment of B. subtilis with

sub-MIC concentrations of daptomycin exhibited a phenotype as observed for copsin with bend,

shortened, and swollen cells 9. Fluorescent microscopy studies showed that daptomycin co-

localizes with the cell division protein DivIVA at bend regions. DivIVA is an essential protein in

the cell division machinery of B. subtilis that localizes to curved regions of the cell membrane 26,27. It is proposed that DivIVA causes local changes in peptidoglycan synthesis, leading to an

asymmetric extension of the cell wall and consequently to bending 9. Analog to copsin, B. subtilis

cells treated with a supra-MIC concentration of daptomycin did not develop the malformations,

but lyzed rapidly due to membrane damages.

Membrane permeabilization of copsin

The morphological malformations induced by copsin indicated a strong similarity to antibiotics

that permeabilize the cell membrane or even lead to pore formation, such as nisin. To examine,

whether copsin has an impact on the cell membrane integrity, we used Sytox Green, a

fluorescent dye of about 600 Da. Sytox Green can only enter bacteria with a compromised

plasma membrane, often used for Live/Dead staining 28. Upon binding to nucleic acids, the

fluorescence emission of Sytox Green is approximately 500-fold enhanced.

First, we used B. subtilis cells in the early exponential growth phase to compare the influx of

Sytox Green upon treatment with copsin and nisin. Nisin is known to induce uptake of Sytox

Green, resulting from its pore forming activity. As a negative control, we exposed the cells to

vancomycin, which does not directly affect the plasma membrane. The three antibiotics were

applied in supra-MIC concentrations. We monitored an immediate increase in fluorescence

emission after adding copsin to a B. subtilis suspension pre-incubated with Sytox Green, similar

to nisin (Fig. 8). After treatment with vancomycin, there was no change in fluorescence

detectable.

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61

Fig. 8. Sytox Green uptake after antibiotic treatment. After pre-incubation of B. subtilis cells with Sytox

Green, the antibiotics were added at 4xMIC (arrow). The fluorescence emission measured at 520 nm

showed a comparable increase after treatment with nisin and copsin. Vancomycin and the medium control

had no impact on the Sytox Green uptake.

To confirm these results, the Sytox Green uptake was analyzed by microscopy (Fig. 9). The

percentage of B. subtilis cells emitting green fluorescence was calculated manually in the field of

view. After 10 min of treatment with the corresponding antibiotic, images obtained for nisin and

copsin showed a strong signal with 50% and 85%, respectively, of cells affected. Only a minor

fluorescence was detectable for the control (1.6%) and cells treated with vancomycin (1.5%).

The immediate influx of Sytox Green indicated that copsin permeabilized the cell membrane of

B. subtilis.

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62

Fig. 9. Visualization of Sytox Green uptake. B. subtilis cells in the exponential growth phase were

treated with antibiotic for 10 min and immobilized on a 1% agarose pads. Nisin and copsin induced an

influx of Sytox Green shown by cells with green fluorescence signal. The untreated control cells and cells

exposed to vancomycin were not permeabilized.

Next, we investigated whether the permeabilization of the bacterial membrane induced by copsin

depends on the growth stage of a bacterial culture. Therefore, B. subtilis cells in the early and

late exponential growth phase were treated with supra-MIC concentrations of copsin and nisin. A

4-fold decrease in fluorescence emission at higher optical density was monitored for both

antibiotics (Fig. 10).

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63

Fig. 10. Cell stage dependency of copsin and nisin. B. subtilis cell cultures at OD600 of 0.15 and 1.2

were diluted to the same OD600 (0.1) and pre-incubated with Sytox Green. Copsin and nisin were added to

4xMIC (arrow). At a later growth stage, a clear drop of fluorescence emission (520 nm) was visible.

We can summaries that copsin had a strong impact on the integrity of the bacterial cytoplasmic

membrane when applied in lethal concentrations. Together with the microscopy studies, it

indicated a pore forming mechanism similar to nisin, dependent on a cell wall precursor.

However, the killing kinetics of copsin is more similar to cell wall interfering agents than to rapidly

lytic antibiotics 8. Furthermore, our experiments exhibited that the permeabilization ability of

copsin is strongly dependent on fast dividing cells, which made an interaction solely with the cell

membrane more unlikely.

These results are not consistent with the findings for the carboxy-fluorescein efflux from lipid II

containing liposomes and the K+ release from B. subtilis cells. In both assays, there was no

permeabilization of the bacterial membrane detectable within 3 min after exposure to copsin, in

comparison to nisin that was immediately active on the membrane. However, factors such as

buffer or medium conditions, properties of the reporter ion, or technical setups potentially had a

strong impact on the outcome of these assays. Further experiments are required to give a

conclusive reason for the differences in cell membrane permeabilization. Nevertheless, one fact

that was clearly demonstrated by these assays was that copsin acts differently on lipid II than

nisin, as indicated by the kinetic measurements.

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Synergy of copsin with lysozyme and EDTA

The synergistic effect of antibiotics together with other substances is widely known and exploited

to kill resistant pathogens 29. Microscopy studies and Sytox Green assays showed that copsin

interacts with a compound of the cytoplasmic membrane, leading to cell lysis. In Gram positive

bacteria, this membrane is covered and protected by a thick layer of murein and in Gram

negative species even by an additional outer membrane that hinders the penetration of many

antibiotics. Here we tested, whether copsin acts synergistically with lysozyme or

ethylenediaminetetra-acetic acid (EDTA). Lysozyme specifically hydrolyses the β1,4-linkages

between MurNAc and GlcNAc and thus, is degrading the peptidoglycan layer of mainly Gram

positive bacteria 30. EDTA is known to damage the outer membrane of Gram negative bacteria

making it permeable for extracellular molecules 31.

Fig. 11. Double disk diffusion synergy test. (A) Copsin (C; 23 µg) spotted on B. subtilis and two

adjacent disks with each 23 µg of hen egg white lysozyme HEWL (L). The inhibitory halo of copsin was not

affected by lysozyme. (B) Copsin together with EDTA (E; upper row: 0.5 M; lower row: 0.1 M) on E .coli

cells. A slight halo is visible around the disks loaded with copsin, showing that EDTA renders E. coli

susceptible for copsin.

A double disk synergy test showed that the antibacterial activity of copsin was not supported by

the action of lysozyme against B. subtilis (Fig. 11A) 32. EDTA in combination with copsin induced

an inhibitory halo on E. coli that we could not detect when copsin was applied independently

(Fig. 11B). This result showed that the target molecule of copsin is not specific for Gram positive

species.

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65

Materials and Methods

All chemicals, if not otherwise mentioned, were bought at the highest available purity from

Sigma-Aldrich.

Antimicrobial activity

The MIC/MBCs for copsin were determined by the microdilution broth method and as described

in chapter 2 33. For disk diffusion assays, 15 µg of purified CC82 and copsin was spotted on an

individual disk and plates incubated at appropriate growth temperatures for 24 h.

Expression in bioreactor

Bioreactor and control software

A Labfors 5 bioreactor with a 3.6 l working volume (WV) (Infors AG, Switzerland) was used for P.

pastoris fermentations. The bioreactor was equipped with a jacketed glass vessel, pH probe

(Mettler-Toledo, Switzerland), dissolved oxygen probe (Mettler-Toledo, Switzerland), foam probe,

air-flow meter, 2x Rushton impellers, baffles, sparger, pumps for acid, base, feed and anti-foam.

An auxiliary peristaltic pump was used for the initial glycerol feed. PC running IRIS NT version 5

software (Infors AG, Switzerland) was used for implementation of fermentation control

sequences.

Bioreactor operation

Expression was performed following the P. pastoris fermentation process guidelines of

Invitrogen 13 with slight modifications according to Diethard Mattanovich, BOKU, Vienna: Instead

of a direct change from glycerol to methanol feed, a pulse of 5 ml/l methanol is added before the

start of the methanol feed. This is to avoid accumulation of methanol in the medium as the cells

change their carbon metabolism. Initial volume of fermentation was 1.2 l expecting a final volume

of 1.5-2 l depending on length of the methanol fed-batch.

The fermentation process has two distinct phases. The first phase consists of a 24 h batch and 4

h fed-batch using glycerol as a carbon source with the purpose of generating a high cell density

without any recombinant protein production. The second phase is a 100 h methanol fed-batch,

where production of recombinant protein is induced under the control of the AOX1 promoter.

1.2 l of basal salts medium (per liter: 26.7 ml H3PO4, 0.93 g CaSO4, 18.2 g K2SO4, 14.9 g

MgSO4*7H20, 4.13 g KOH, 40 g glycerol) was added to the reactor, the pH probe calibrated and

the vessel with probes and medium autoclaved at 121 °C for 20 min. The medium was

maintained at the desired temperature (30°C) and the pH was set to 5 by sterile addition of 25%

NH4OH followed by addition of 4.35 ml/l of PTM1 trace salts solution (PTM1 trace salts per liter:

6.0 g CuSO4*7H20, 0.08 g NaI, 3.36 g MnSO4*H20, 0.2 g Na2MoO4*2H20, 0.02 g boric acid,

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66

0.82 g CoCl2*6H20, 20 g ZnCl2, 65 g FeSO4, 5 ml 95% H2SO4) and 0.87 mg/l biotin of a fresh

0.2 g/l biotin stock. The vessel was flushed with N2 allowing the calibration of the dissolved

oxygen probe. Stirrer speed and airflow rate was set to 750 rpm and 1.2 l/min.

50 ml BMGY (1% (w/v) yeast extract, 2% (w/v) peptone, 1.3% (w/v) YNB w/o amino acids

(Becton Dickinson, USA), 100 mM potassium phosphate buffer pH 5, 1% (v/v) glycerol)

supplemented with 100 µg/ml zeocin was inoculated with P. pastoris NRRLY11430 transformed

with native copsin. The culture was grown overnight at 30°C to an OD600 of 6-10 and 40 ml of the

culture was used to inoculate the bioreactor.

The fermentation was run in batch mode for approximately 24 h. We attempted to maintain the

dissolved oxygen above 20 % of air saturation by regulating the stirrer speed and air flow rate

using a PID cascade loop. When all glycerol was consumed, indicated by a rise in dissolved

oxygen levels to > 90%, a limiting glycerol feed was started at a rate of 15.5 ml/h*l. After 4 h, a

total of 62 ml of feed was added and the pump was stopped. Depletion of glycerol in the reactor

was indicated by an increase of dissolved oxygen to > 90%. Methanol is toxic to P. pastoris at

too high concentrations. To avoid methanol accumulation while cells changed their metabolism

from glycerol to methanol a pulse of 5 ml/l of methanol was added to the culture. The methanol

was allowed to be consumed before the methanol feed pump was started approximately 4 h

later. Using a control sequence written in the IRIS software (Infors AG, Switzerland), the addition

of the methanol pulse and subsequent start of the methanol feed were automated. The methanol

feed was also regulated by the software to maintain the dissolved oxygen level above 10%, while

the stirrer speed and air flow rate were kept at 1170 rpm and 1.2 to 2 l/min, respectively. If the

oxygen level could not be maintained above 10% the feed was regularly turned off to see, if

methanol was accumulating in the reactor. After 90 h of methanol feed and addition of

approximately 700 ml of methanol the culture was harvested. An overview of the process is

shown in table 2.

Table 2: Pichia pastoris fermentation process scheme

Step Carbon source Duration h Stirrer (rpm) Aeration (l/min)

Batch Glycerol ~24 750 - 1170 1.2

Fed-batch 1 Glycerol ≥4 1170 1.2

Transition Methanol ~4 750 - 1170 1.2

Fed-batch 2 Methanol 90 1170 1.2-2

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67

Purification of copsin and CC82

The post-fermentation broth was centrifuged at 3000 x g for 20 min and the supernatant

concentrated in a 3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc., USA) by

a treatment with polyethylene glycol 6000 at 4 °C. The concentrated supernatant was dialyzed

against 20 mM Na-phosphate, 50mM NaCl buffer pH 7 (buffer A) at 4 °C for 24 h. The protein

solution was sterile-filtered and loaded on a self-made SP sephadex cation exchange column

equilibrated with buffer A. The column was washed with 180 mM NaCl and bound proteins were

eluted with 400 mM NaCl in 20 mM Na-phosphate buffer pH 7. The eluent was subjected to a

size exclusion chromatography for further polishing. The separation was performed on a

Superdex75 column (HiLoad 16/60, GE Healthcare, USA) equilibrated with 20 mM Na-

phosphate, 50 mM NaCl buffer pH 6. The effluent was monitored by absorbance at 210 nm. The

fractions containing the active peptide were combined.

Copsin induced morphological changes of B. subtilis

B. subtilis cells were grown in LB medium to an OD600 of 0.2 and treated with 0.5 µg/ml copsin

for 2 h. Untreated B. subtilis cells were used as control. For microscopy, 10 µl of the cultures

were immobilized on 1% agarose slabs. DIC images were acquired with an Axioscope 2

Apotome microscope with a 100x/1.4 Zeiss objective using an AxioCam MR (Zeiss, Germany).

Poly-L-lysine coating of coverslips

20 x 20 mm cover slips were washed thoroughly with ethanol for 5 min and air dried. The sterile

cover slips were then incubated in 0.01% poly-L-lysine solution for 5 min at RT. The cover slips

were washed with sterile water and air dried for 1 h.

Confocal microscopy

Bacterial cells were observed under a 100x oil-immersion objective (Zeiss, Germany) on a

spinning-disk confocal microscope (Visitron, Germany) and imaged with Evolve 512 EMCCD

camera (Photometrics, USA) using standard filter sets for GFP (excitation/emission:

488/525 nm) and rhodamine (540/575 nm). Images were processed using ImageJ v1.46.

Localization of TAMRA-copsin on bacteria

Bacteria were grown in LB medium to an OD600 of 0.3 and incubated with TAMRA-copsin at

2xMIC (B. subtilis: 2 µg/ml; S. carnosus: 16 µg/ml) for 10 min. The cells were washed twice with

phosphate buffered saline (PBS) at pH 7.4 and immobilized on a poly-L-lysine coated cover slip.

Unbound cells were washed away with sterile water. The cover slip was then mounted on a

microscopic glass slide with 90% (v/v) glycerol. The cells were observed as described. The red

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68

fluorescence signal from the TAMRA dye was visualized by an excitation/emission wavelength of

540/575 nm with a standard filter set for rhodamine.

Co-localization of copsin and vancomycin

Bacteria were grown in LB medium to OD600 of 0.3 and incubated with TAMRA-copsin at 0.5xMIC

(0.5 µg/ml) for Bacillus subtilis and 2xMIC (16 µg/ml) for S. carnosus for 10 min. The cells were

washed twice with PBS and then immobilized on a poly-L-lysine coated cover slip. After washing

away the unbound cells with sterile water, BODIPY-vancomycin (Life Technologies, USA) was

used at 1 µg/ml concentration for staining on the cover slip. The cells were observed as

described, using standard filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine

(540/575 nm).

Localization of copsin and vancomycin on sub-MIC treated B. subtilis cells

B. subtilis cells were grown in LB medium to an OD600 of 0.2 and treated with 0.5 µg/ml of

unlabeled copsin or TAMRA-copsin for 2 h. Untreated B. subtilis cells were used as control. The

cells were then washed with 0.2% (v/v) saline, diluted to an OD600 of 0.2 and immobilized on a

poly-L-lysine coated coverslip. The unbound cells were washed with sterile water and stained

with 1 µg/ml BODIPY-vancomycin for 5 min on the coverslip. The cells were observed as

described, using standard filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine

(540/575 nm).

Sytox Green assays

B. subtilis cells were grown to an OD600 of 0.2 and washed twice with 20 mM Na-phosphate,

30 mM NaCl buffer at pH 6.0 and centrifuged at 7000 x g for 1.5 min. The cells were

resuspended and diluted to an OD600 of 0.1 in 20 mM Na-phosphate, 30 mM buffer at pH 6.0

containing 1 µM Sytox Green dye (Life Technologies, USA). After incubation in the dark at RT for

15 min, 100 µl of the cell suspension was filled in wells of a 96-well plate and fluorescence

measurements were made using the Victor Wallac 1420 Spectrofluorimeter (PerkinElmer, USA).

At approximately 7 min after initiating and stabilizing fluorescence signal reading, 4xMIC

concentrations of antibiotic was added to the cell suspension, and the increase in Sytox Green

fluorescence was measured every 40 seconds for 45 min (excitation wavelength at 485 nm and

emission at 520 nm). Permeabilization of the cells with nisin (MIC: 40 ng/ml) was used as a

positive control. Vancomycin (MIC: 50 ng/ml) was used as a negative control. The MIC for nisin

and vancomycin against B. subtilis was determined in the lab by the microdilution broth method

as described 33. A blank control with cells incubated with Sytox Green but no antibiotic added

was also included in the study.

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69

For the cell stage dependency assay, a bacterial culture was divided in two sub-cultures and

grown independently to an OD600 of 0.15 and 1.2, respectively. Both culture were then diluted to

an OD600 of 0.1 and processed as described.

For microscopy, 100 µl of the cells incubated with Sytox Green were treated separately with

4xMIC of the corresponding antibiotic. At 10 min after treatment, cells were immobilized on a 1%

agarose pad for viewing under the microscope. The cells were observed under a 20x Zeiss

objective of an Axioscope2 Apotome microscope (Zeiss, Germany) and imaged using AxioCam

MR (Zeiss, Germany).

Double disk synergy test

A disk diffusion assay was performed by placing 2 sterile blank paper discs about 1 cm apart

and loading one disc with 30 µl of 750 µg/ml copsin and the other with 30 µl of 780 µg/ml of

lysozyme (HEWL). This synergy assay was also performed on an E. coli plate with 30 µl of a 0.1

and 0.5 M EDTA solution.

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Supplementary Information

1 ATG GAA CTC ACT GCA TCC TTC CTC TCT GCA GTA GCG GTC GCC TCC ACC TTC GTC GGA ACC

M E L T A S F L S A V A V A S T F V G T

>-------------------------------- Signal peptide ------------------------------

61 GCC CTC TCA GCG ACC ACC GTC CCC GGA TGC TAC GCC GAG TGC ATC GAG AAG GGT GCT GCA

A L S A T T V P G C Y A E C I E K G A A

----------< >-------------------- Pro peptide ---------------------------------

121 GCC GTC AAC TGC GCC GTC GAC GAC ATC GAC TGC CTC AGA CTC GCT TCC TCG CAG TTC ACC

A V N C A V D D I D C L R L A S S Q F T

-------------------------------------------------------------------------------

181 ACC ATC ACC CGC GAA TGC CTC GAC ACT AAC AAC TGT ACC AGT CTC ACC CCT GGT ACA CCC

T I T R E C L D T N N C T S L T P G T P

-------------------------------------------------------------------------------

241 GCC GAC GAA GCC TCC ATC ACT ACA ACC TTC AAC ATC CTC TCA GGC CTC GGC CTC ATC GAC

A D E A S I T T T F N I L S G L G L I D

-------------------------------------------------------------------------------

301 TCC TCC GAA GTC TTC AGC CTC GCC GAC GTC CTC CAA GTC CAC CAA CGC GAC CTC ACA GGC

S S E V F S L A D V L Q V H Q R D L T G

-------------------------------------------------------------------------------

361 CTC AGC CGC ATC CTA CCT ATC GAC AAA CGC CAA AGG TGC ATC GTC CGT CGC GCC ACA TGC

L S R I L P I D K R Q R C I V R R A T C

--------------------------------------< >--------------------------------------

421 GTC ACT TCA GGC TTG ACA GCA TGC GTA AAC CAC TGC ATC TCG TGC CAT CGC GGG AGT GGC

V T S G L T A C V N H C I S C H R G S G

--------------------------------- Mature peptide ------------------------------

481 AGC AGC AAC TGT GTC GAG TGC AGC GGG GGT CGG TGC ACA GGC ACT CTG GGC ACG ACG TGC

S S N C V E C S G G R C T G T L G T T C

-------------------------------------------------------------------------------

541 ACA TGT CAG AAC CCG TGT CGT AGT TGC TAA

T C Q N P C R S C -

----------------------------------<

Fig. S1. Prepro-protein CC82 (CC1G_08260). Prediction of the signal peptide was performed with

SignalP 4.0 34.

Chapter 3

73

1 ATG AAA TTG TCA ACA TCC CTT CTT GCT ATC GTC GCA GTC GCT TCC ACC TTT ATT GGT AAC

M K L S T S L L A I V A V A S T F I G N

>-------------------------------- Signal peptide ------------------------------

61 GCC TTG TCC GCT ACT ACA GTT CCA GGA TGT TTT GCT GAA TGC ATT GAT AAG GCT GCC GTT

A L S A T T V P G C F A E C I D K A A V

----------< >-------------------- Pro peptide ---------------------------------

121 GCT GTC AAC TGT GCA GCT GGA GAT ATT GAC TGC TTG CAA GCC TCT TCC CAG TTC GCA ACT

A V N C A A G D I D C L Q A S S Q F A T

-------------------------------------------------------------------------------

181 ATC GTT TCT GAG TGT GTC GCT ACT TCC GAC TGC ACA GCC CTT TCA CCA GGA TCA GCA AGT

I V S E C V A T S D C T A L S P G S A S

-------------------------------------------------------------------------------

241 GAT GCT GAC AGT ATT AAC AAG ACT TTT AAC ATC TTG TCT GGT CTT GGT TTT ATT GAT GAA

D A D S I N K T F N I L S G L G F I D E

-------------------------------------------------------------------------------

301 GCT GAC GCC TTC AGT GCC GCA GAT GTT CCT GAA GAG AGA GAC TTG ACA GGT CTT GGA AGA

A D A F S A A D V P E E R D L T G L G R

-------------------------------------------------------------------------------

361 GTT TTG CCA GTC GAG AAA AGA CAA AAT TGT CCT ACC AGA AGA GGT TTG TGT GTT ACC TCC

V L P V E K R Q N C P T R R G L C V T S

--------------------------< >--------------------------------------------------

421 GGA CTT ACT GCT TGT AGA AAC CAT TGT AGA TCA TGC CAC AGA GGA GAT GTT GGA TGT GTC

G L T A C R N H C R S C H R G D V G C V

--------------------------------- Mature copsin --------------------------------

481 AGA TGC TCT AAT GCT CAA TGT ACT GGT TTT CTT GGA ACT ACC TGC ACT TGT ATC AAT CCT

R C S N A Q C T G F L G T T C T C I N P

-------------------------------------------------------------------------------

541 TGT CCT AGA TGT TAG

C P R C -

--------------<

Fig. S2. Codon optimized prepro-protein of copsin.

Chapter 3

74

Chapter 4

Fungal lysozyme identified

in the secretome of C. cinerea

Contributions

Analysis and description of the C. cinerea secretome by ESI-MS/MS

Identification and characterization of CC49 and CC92

The heterologous expressions of CC49 and CC92 were performed by John Hintze during his

Master studies at the ETH Zürich under the supervision of Andreas Essig and Pauli Kallio.

75

Introduction

In 1909, P. Laschtschenko, a researcher of the University of Tomsk in Russia, first described

the inhibitory effect of hen egg white on bacteria 1. Thirteen years later, it was Sir Alexander

Fleming, known as the discoverer of penicillin (1928), who coined the word lysozyme, when

he discovered that nasal mucus of one of his patients strongly reduced the growth of

Micrococcus lysodeikticus 2. It took another forty years until the primary sequence of hen egg

white lysozyme (HEWL) was deciphered followed by the 3D structure 3,4. HEWL is a

milestone in enzymology as it was the first enzyme for which an X-ray structure was solved

and for which the enzymatic mechanism was described in detail 5,6. Lysozymes are

characterized by their ability to hydrolyze the β1,4-glycosidic bond between N-acetylmuramic

acid (MurNAc) and N-acetylglucosamine (GlcNAc) of peptidoglycan (PG), also called murein.

PG is a unique structural element of the bacterial cell wall, composed of linear chains of

alternating β1,4-linked MurNAc and GlcNAc residues interconnected by peptide bridges 7.

The disaccharide and the amino acid moieties are frequently modified and linked to other cell

wall compounds, leading to a high structural variation of PG in different bacterial species 8.

The cell wall and in particular PG provide the cell with stability and rigidity against turgor

pressure. Therefore, disruption of the integrity of the peptidoglycan layer through the action

of a lysozyme is leading to a rapid hypotonic lysis of a bacterial cell. Most lysozymes are

predominately active against Gram positive species, where the PG is freely accessible, in

comparison to Gram negative bacteria with a protective outer membrane.

Today, a wide variety of lysozymes is described in literature, originating from bacteria, plants,

animals, and viruses. Lysozymes are categorized according to their primary sequence and

biochemical properties, including the c-type (chicken-type) group in animals 9. C-type

lysozymes are produced by most vertebrates, for example, human lysozyme and HEWL. In

mammals, they can be identified in various body fluids, such as saliva, tears, or breast milk,

but also in the lysosomal granules of neutrophils and macrophages. As a part of the innate

immune defense, lysozymes act against bacteria and may possess immunomodulatory

functions 10. Additionally, some animals recruit lysozymes as digestive enzymes to gain

nutrients from bacteria 9. A different functionality of lysozymes is found in viruses and

bacteria. Bacteriophages exploit the action of lysozymes to partially digest the bacterial cell

wall before they enter the cell. At the end of the infection cycle, larger phages use lysozymes

and amidases, called endolysins, to induce the release from the host cell 11,12. The best

characterized examples are the member of the T4 phage group of endolysins 13. Bacteria

themselves express lysozymes or autolysins, which are required for an efficient degradation

and recycling of the cell wall during the cell division process 11. Cellosyl, a bacterial

muramidase from Streptomyces coelicolor, shows a high similarity to the only fungal

Chapter 4

76

lysozyme identified, so far 14. It was extracted from the secretome of Chalaropsis, a member

of the phylum of ascomycota and defines an own group of ch-type lysozymes 15,16. They are

characterized by the ability to cleave 6-O-acetylated peptidoglycan, a modification that

renders the cell wall of, for example, Staphylococcus aureus insensitive towards most other

types of lysozymes 8. However, little is known about the structures and enzymatic properties

of ch-type lysozymes.

Nowadays, lysozymes and especially HEWL are used extensively in research and as food

preservatives. Treatments of systemic bacterial infections are restricted mainly due to

immunogenic reactions, a short plasma half-life, and an increased cytokine production upon

release of cell debris 17.

Here, we characterized a novel fungal lysozyme secreted by the basidiomycete Coprinopsis

cinerea. It showed a distinct antibacterial profile and revealed a highly conserved

autolysin/endolysin domain.

Chapter 4

77

Results

Lysozyme identified in the secretome of C. cinerea

A mass spectrometry based analysis of the secretome of C. cinerea grown on glass beads

revealed in total 182 proteins (Table S1). A majority of these proteins had a predicted

enzymatic or digestive function, such as glycosidases or peptidases, adapted to the

saprophytic lifestyle of this mushroom. The list included two lysozyme-like enzymes,

CC1G_03049 and CC1G_03076. CC1G_03049, hereafter called CC49, was further

characterized. The locus encoded a protein of 268 amino acids with a predicted signal

peptide (position: 1-20) (Fig. S1). A Blastp search with the amino acid sequence of CC49

showed a conserved autolysin/endolysin domain commonly identified in bacterial and phage

lysozymes (Fig. 1). No significant similarity was found to HEWL and human lysozymes.

Enterobacteria phage 4 ISSNGITRLKREEG.[5].YSDSR GIPTIGVGHTGK.[9].M TITAEKSSELLKEDLQWV.[5].SLV 75

C. cinerea CC49 109 VNSRTVQEIKNSEG.[5].APDPI GLPTVGYGHLCK.[3].C.[7].PLTEAQATSLLMTDLKTF.[5].DQI 181

Pseudomonas phage 9 ALAAALAGLVALEG.[5].YRDIA GVPTICSGTTAG.[4].D KATPEQCYQMTIKDFQRF.[5].DAI 75

C. crescentus 7 VSRAAVDLIKRFEG.[5].AQLPD GRWTVGYGHTLT.[4].A SVSEKDAEALLLYDLISV.[5].EHT 73

Xanthomonas phage 68 LSAAGVVAISSHEG.[5].YPDPA.[3].APWTICYGHTGP.[5].L VVTQSQCDKWLAQDLSKA.[5].AVV 138

Fig. 1. Alignment of CC49 with phage and bacterial lysozymes. The conserved catalytic

autolysin/endolysin domain is indicated within yellow borders (Enterobacteria phage, gi138699;

Pseudomonas phage, gi33300856; Caulobacter crescentus, gi16126412; Xanthomonas phage,

gi32128440). The alignment was performed with the BlastP algorithm 18.

Further analysis of the secretome revealed that the open reading frame of CC1G_06692,

hereafter called CC92, was highly similar to the N-terminal region of CC49 (Fig. 2). The

genetic locus of CC92 encoded 104 amino acids, consisting of a predicted signal peptide

(position: 1- 19) and a mature polypeptide (position: 20–104) (Fig. S2). Comparisons on the

AMP database exhibited sequence similarities of 25–30% to defensins and bacteriocins,

such as the human beta defensin 2 (AP00524) and salivaricin P produced by Lactobacillus

salivarius (AP01173) 19. The result of an alignment of CC92 with the N-terminal region of

CC49 is illustrated in figure 2, which exhibited a highly conserved pattern of twelve cysteine

residues, also found in copsin.

Chapter 4

78

Fig. 2. Alignment of CC92, CC49, and copsin. A schematic alignment of CC92 and CC49 precursor

protein is illustrated. The corresponding amino acid positions are indicated. A sequence alignment is

shown for CC92, CC49, and copsin. The Kex2 recognition site is marked in green and the cysteine

residues are marked in red. The sequence alignment was performed with the ClustalW algorithm and

visualized by Jalview 20,21.

Recombinant expression of CC92 and CC49

CC92 and CC49 were recombinantly produced in Pichia pastoris, an efficient expression

system for secreted fungal proteins, as demonstrated for copsin. Both proteins potentially

undergo cleavage of a pro-peptide, due to a Kex2 recognition site identified (Fig. 2) 22.

Antibacterial activities of the culture supernatants were tested against B. subtilis (Fig. 3A).

CC49 exhibited a clear inhibition zone in a disk diffusion assay, in contrast to CC92 that was

weakly active when expressed with a C-terminal polyhistidine (His) tag and its native signal

sequence (CC92-His). This construct showed a molecular mass corresponding to the

uncleaved pro-protein in an immunoblot (Fig. 3B). When the native signal sequence of CC92

was replaced by the α-factor secretion signal of S. cerevisiae, the polypeptide exhibited an

increased molecular mass on the blot, most likely due to an uncleaved pro-peptide of the α-

factor. CC49 displayed multiple bands including the molecular mass for the processed and

unprocessed pro-protein. Therefore, it could not be concluded, whether a pro-peptide is

cleaved off. The assignments of the protein bands were additionally confirmed by tandem

mass spectrometry measurements of in-gel proteolytic digests.

Chapter 4

79

Fig. 3. Antibacterial activity of recombinantly expressed CC92 and CC49. The predicted signal

peptide of CC92 and CC49 was replaced by the α-factor secretion signal of S. cerevisiae and a

polyhistidine tag was fused to the C-terminus (αCC92-His; αCC49-His). The protein production in P.

pastoris was performed in shake flask cultures. CC92 was additionally expressed without the His-tag

(αCC92) and with the native signal sequence (CC92-His). (A) The culture supernatant was

concentrated and spotted on B. subtilis. The disk assay showed a clear halo for the lysozyme-like

protein CC49 and a weak inhibition zone for CC92-His. Copsin was used as a positive control. (B) The

concentrated culture supernatant was separated on a SDS-PAGE followed by immunoblotting with a

histidine specific antibody. CC92-His displayed exclusively the molecular mass corresponding the pro-

protein at 8.5 kDa. CC49 showed multiple bands with the molecular masses of the processed

(21.7 kDa) and unprocessed (26.2 kDa) pro-protein indicated in red. M indicates the marker lane.

Expression and purification of CC49

The histidine tagged construct of CC49 was expressed in P. pastoris shake flask cultures.

After concentrating the culture supernatant, a one-step purification was performed on Ni-NTA

beads. The different fractions were analyzed on an SDS-PAGE and the antibacterial activity

determined in a disk assay on B. subtilis cells (Fig. 4). The elution step exhibited multiple

bands consistent with the immunodetection assay and it was the only fraction that showed an

antibacterial activity.

Both, the immunoblot and the Coomassie stained SDS-PAGE, displayed proteins with a

molecular mass higher than expected (Fig. 3B and 4A). CC49 harbors three potential N-

glycan sites (Fig. S2; N85, N208, N544), which could result in this mass shift. To investigate,

whether CC49 is expressed as a glycoprotein, we treated purified CC49 with two specific

endoglycosidases 23. The Peptide-N-Glycosidase F (PNGaseF) cleaves at the N-

acetylglucosamine (GlcNAc) and asparagine residue of high-mannose, hybrid, and complex

N-glycans. The Endoglycosidase H (EndoH) specifically cleaves between the two innermost

GlcNAc residues of high-mannose N-glycan structures. Additionally, we analyzed the thermal

stability of CC49 and the sensitivity towards proteinase K.

Chapter 4

80

Fig. 4. Recombinant expression and purification of CC49. CC49, including the α-factor secretion

signal of S. cerevisiae and a C-terminal polyhistidine tag, was expressed in P. pastoris. The culture

supernatant was concentrated and dialyzed against PBS. A separation was performed on Ni-NTA

beads with a one-step elution of 400 mM imidazole. (A and B) Flow through (FT), wash (W), and

elution fraction (E) were analyzed on a SDS-PAGE and spotted on a disk diffusion assay against

B. subtilis. The elution step was the only fraction that displayed Coomassie stainable bands and an

inhibition of bacterial growth.

CC49 was completely degraded when simultaneously exposed to heat and proteinase K

(Fig. 5). Protein corresponding to the potential glycoform (>26,2 kDa) was not detectable

anymore upon treatment with PNGaseF or EndoH. This result indicated that CC49 is indeed

carrying N-glycans, most likely of the high-mannose type, a structure often found on secreted

fungal proteins.

Fig. 5. Stability and glycosylation of CC49. Purified recombinant CC49 was subjected to a heat

treatment (60 °C) with and without proteinase K (100 ng/µl) for 2 h and it was exposed to PNGaseF

and EndoH according to the manufacturers protocol. The proteins were separated and visualized on a

Coomassie stained SDS-PAGE. Proteinase K degraded CC49 and the endoglycosidases led to a shift

of the upper bands (>26.2 kDa), assumed to be the glycoform.

Chapter 4

81

Optimization of CC49 expression in P. pastoris and purification

The degradation of CC49 most likely resulted of the action of endogenous proteases of P.

pastoris secreted during cultivation. To reduce the level of these proteases, we combined

three commonly known strategies 22. First, the expression was performed in the protease A

deficient strain SMD 1168H (pep4). Second, we reduced the pH of the culture medium, as it

is known that P. pastoris has the ability to grow at lower pH, while the activity of secreted

proteases is strongly reduced. Third, protease inhibitor was added to the concentrated

culture supernatant before the pH was increased again. Additionally, we used a CC49

construct without a His-tag. The histidine residues potentially interfere with the antibacterial

activity. Due to the high number of arginine and lysine residues identified in CC49, we

performed the purification on a cation exchange column (Fig. 6). The combined strategies

strongly reduced degradation of CC49 and its glycoform. The antibacterial activity was

retained without the His-tag, verified in a disk diffusion assay on B. subtilis.

Lysozyme activity of CC49 and the antibacterial profile

CC49 possessed a predicted endolysin/autolysin domain, as shown in the initial alignments

with known hydrolases (Fig. 2). Therefore, we compared the enzymatic activity of CC49 with

HEWL and bovine serum albumin (BSA), using a commercial lysozyme assay kit. Increasing

amounts of CC49, HEWL, and BSA were incubated with fluorescein labeled Micrococcus

lysodeikticus cell wall and fluorescence was measured after 30 and 60 min. Both, CC49 and

the HEWL positive control, showed a clear increase in fluorescence with an increasing

concentration of protein (Fig. 7). These results confirmed that CC49 exerts an enzymatic

action on peptidoglycan, hydrolyzing the β1,4-glycosidic linkage between MurNAc and

GlcNAc.

Chapter 4

82

Fig. 6. Optimized expression of CC49 and purification on an ion exchange column. The

expression of CC49 was performed in medium buffered at pH 4 with a protease A deficient P. pastoris

strain. The culture supernatant was concentrated, dialyzed, and loaded on a 5 ml self-made cation

exchange sephadex column. (A) The elution was done at 200 mM NaCl over 5 column volumes and

the eluent was monitored at 280 nm. (B) Collected fractions were concentrated and analyzed by SDS-

PAGE and silver-staining. The major fraction of CC49 (26.2 kDa) eluted at 200 mM NaCl (96 ml

elution volume) and showed a clearly reduced degradation.

Chapter 4

83

Fig. 7. Lysozyme activity of CC49. The EnzCheck lysozyme assay kit (Life technologies) was used

for determining the enzymatic activity of CC49. The assay measures lysozyme activity on Micrococcus

lysodeikticus cell wall, which is labeled with fluorescein. The initially quenched fluorescence is

released upon enzymatic treatment proportional to lysozyme activity. The final concentration of CC49

and BSA in the reactions were 3.9, 7.9, 15.8, 31.3, 63, 125, 250 µg/ml. HEWL was prepared according

to the manufacturers protocol. The excitation and emission wavelength was 485 and 535 nm,

respectively, and was measured after 30 and 60 min. Mean and standard deviation was calculated

from three replicates performed for HEWL and CC49. Two replicates were made for the negative

control BSA.

Chapter 4

84

For determining the antibacterial profile of CC49, it was spotted on B. subtilis, S. carnosus,

E. coli, and S. aureus cells and the activity was compared to HEWL (Fig. 8). CC49 and

HEWL displayed a comparable activity against B. subtilis and E. coli and no inhibitory effect

on S. aureus cells. HEWL was additionally active on S. carnosus, in comparison to CC49

that did not exhibit an inhibition against this bacterial species.

Fig. 8. Antibacterial activity of CC49. 130 µg of purified CC49 and HEWL dissolved in sodium

phosphate buffer pH 6.5 supplemented with 50 mM NaCl were spotted on Bacillus subtilis (strain 168),

Staphylococcus carnosus (strain 361), Escherichia coli (strain BL21), and Staphylococcus aureus

(strain 113) cells.

Chapter 4

85

Discussion

Analysis of the C. cinerea secretome revealed that this basidiomycete encodes a lysozyme,

CC49, active against Gram positive and Gram negative bacteria.

CC49 and CC92 were recombinantly produced in Pichia pastoris and an inhibition zone was

shown on B. subtilis for both proteins. It was a unique finding that a fungus encodes the N-

terminal region of a lysozyme in an independent genetic locus with an antimicrobial function.

Known is that cleavage of lysozymes by specific proteases can lead to a release of AMPs.

One example is processing of the N-terminal region of human breast milk lysozymes by

pepsin and subsequent release of AMPs. It is proposed that this process is relevant in

stomach of newborns, protecting the gastrointestinal tract against bacterial infections 24,25.

However, the rather low expression levels of CC92 could not be reproduced and therefore,

further optimization of the expression system is needed to conclusively demonstrate that

CC92 acts antibacterial.

The expression of CC49 exemplarily exhibited the advantages and disadvantages of the

Pichia system 22. P. pastoris can easily be genetically modified and yields high expression

levels of heterologous proteins in shake flasks and fermenters with an inexpensive basal

salts medium. Furthermore, P. pastoris is capable of efficiently secrete recombinant proteins

with disulfide connectivities and modifications as N- an O-glycosylations, with a very low level

of endogenously produced and secreted proteins. We identified initially an N-glycosylated

version of recombinant CC49, verified by an endoglycosidase treatment. However,

hyperglycosylation by N-linked high-mannose structures and O-mannoses is a known

problem of secreted proteins in yeast 26. Therefore, further investigations are required to

show, whether the protein of C. cinerea is indeed glycosylated and what the impact of these

modifications are on the structure and activity. Unexpectedly, this glycoform disappeared

upon pH reduction of the culture medium, a phenomenon that we could not confirm in

literature. Another disadvantage of Pichia is the endogenous expression of intra- and extra-

cellular proteases, which led to a degradation of CC49. A reduction of this proteolytic

cleavage was achieved by using a protease deficient strain in combination with a lowered pH

of the culture medium. However, the impact of the protease deficient strain was negligible, as

determined in an independent expression. Because these mutant strains often show reduced

viability and expression levels, an important fact for further optimization of the expression

system 22. CC49 harbors a Kex cleavage site, which could potentially lead to processing of a

pro-peptide. Based on the molecular masses detected and the variability of Kex recognition

sites in different organisms, it was not possible to conclude, whether CC49 is indeed

encoding a pro-sequence 27.

Chapter 4

86

Comparisons at the sequence level revealed that CC49 has a highly conserved cysteine

pattern identified in several homologous proteins in C. cinerea (e.g. CC1G_03042, 03040,

03076) and other fungi, such as Laccaria bicolor (S238N-H82). Similar to copsin, the

potentially high number of disulfide bonds could serve as a scaffold to stabilize secreted

lysozymes and other proteins in the harsh environment of dung, as the natural habitat of C.

cinerea. Furthermore, sequence alignments demonstrated a similarity to autolysins and

endolysins with a highly conserved catalytic domain. Based on these findings, it can be

speculated about the biological function of CC49. Besides directly killing and potentially

acquiring nutrients from bacteria, it is possible that CC49 is involved in remodeling of the

fungal cell wall. Especially invertebrate type (i-type) lysozymes often possess a muramidase

and chitinase activity, cleaving the β1,4-linked backbone of PG and of the GlcNAc

homopolymer chitin 28,29.

Different modifications of the glycan strand of PG can contribute to a lysozyme resistance 8.

The O-acetylation at the C6 position of MurNAc, linkage of PG to teichoic acid, or a high

degree of cross-linking are three examples, which render PG of S. aureus insensitive to

HEWL 30,31. In contrast, S. carnosus that lacks O-acetylated MurNAc is killed upon exposure

to HEWL. CC49 did not display an inhibition zone on S. carnosus and S. aureus cells, which

indicated that binding properties and enzymatic activity are different to c-type and ch-type

lysozymes.

Due to its low similarity to other lysozymes and the specific antibacterial profile, CC49 can be

considered as a novel class of lysozymes. However, further investigations at the structural

level and biochemical assays are needed to accurately categorize this enzyme.

Chapter 4

87

Materials and Methods

Secretome analysis of C. cinerea

The cultivation and extraction was performed twice (A and B) with two technical replicates

each time. C. cinerea was grown on 40 g glass beads in CCMM (A: 10 ml; B: 15 ml) for 4 (A)

and 5 (B) days, respectively. Afterwards, the medium was extracted, centrifuged (3800 x g,

15 min), and the supernatant concentrated to 1 ml by lyophilization. 200 µl of a 100% TCA

solution was added and the proteins precipitated at 4 °C for 15 min. After centrifugation

(16000 x g, 15 min), the pellet was washed twice with 100% acetone and dissolved in

denaturing buffer (8 M Urea, 100 mM ammonium bicarbonate buffer pH 8). After 30 min (60

°C, 900 rpm), DTT was added to 10 mM and incubated at 37 °C for 45 min. Then, the

proteins were incubated with 30 mM IAM for 45 min at 30 °C in the dark. The proteins were

digested by 500 ng trypsin in 250 µl ammonium bicarbonate buffer (100 mM, pH 8) at 37 °C

for 16 h. The peptides were desalted on C18 ZipTip columns (Millipore, USA). The MS

analyses were performed on a hybrid Velos LTQ Orbitrap mass spectrometer (Thermo

Scientific, USA) coupled to an Eksigent-nano-HPLC system (Eksigent Technologies, USA).

Separation of peptides was done on a self-made column (75 µm x 80 mm) packed with C18

AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were eluted with a linear gradient from

2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250 nl/min. MS and MS/MS spectra

were acquired in the data dependent mode with up to 20 collision induced dissociation (CID)

spectra recorded in the ion trap using the most intense ions. All MS/MS spectra were

searched against the p354_filteredMod_d C. cinerea database using the Mascot search

algorithm v2.3 (Matrix Science Inc. , USA) with oxidation (M) as variable modification and

carbamidomethyl as fixed modification. Further statistical validation was performed with

Scaffold 4.0 (Proteome Software, USA) with a minimum protein probability of 90% and a

minimum peptide probability of 50%. This program was also used for determining the total

non-normalized spectral counts for each protein identified in the fungal secretome.

Cloning of CC92 and CC49

The coding sequence of CC92 and CC49 precursor protein was amplified from C. cinerea

(strain AmutBmut (A43mut B43mut pab1.2)) vegetative mycelium cDNA library by PCR with

the Phusion high-fidelity DNA polymerase (Thermo Scientific, USA) according to standard

protocols (Sambrook J, Russell D, 2001, Molecular cloning, 3rd edition) using the following

primers:

Chapter 4

88

Table 1. Primers, restriction enzymes and plasmids used for cloning in E. coli DH5α.

Construct Primer (5' to 3') Restriction enzyme Plasmid

αCC92-His FP: ATTTGAATTCGCCCTCAACGGCCCCTG EcoRI pPICZαA

RP: ATTTGTCGACTGATGGCAAGCAGCACCTAA SalI

CC92-His FP: ATTTGAATTCATGAAGTTTACCACATCCCTCTT EcoRI pPICZA

RP: ATTTGTCGACTGATGGCAAGCAGCACCTAA SalI

αCC92 FP: ATTTGAATTCGCCCTCAACGGCCCCTG EcoRI pPICZαA

RP: ATTTTCTAGATCTAGATTATGATGGCAAGCAGC Xbal

αCC49-His FP: ATTTGAATTCGCCATCAACGATCCCTGCTC EcoRI pPICZαA

RP: ATTTGTCGACGCACCTGGGAGGATGCC SalI

αCC49 FP: ATTTGAATTCGCCATCAACGATCCCTGCTC EcoRI pPICZαA

RP: ATTTGTCGACCTAGCACCTGGGAGGATGCC SalI

The PCR product was cloned into the corresponding expression plasmid (Life Technologies,

USA) containing a zeocin resistance gene using the corresponding restriction enzymes

(Fermentas GmbH, Switzerland) (Table 1). The affinity tag was composed of six histidine

residues encoded on the plasmid. The resulting plasmid was linearized by the SacI restriction

enzyme (Thermo Scientific, USA) and transformed into the P. pastoris strain NRRLY11430

by electroporation with 1.2 kV of charging voltage, 25 µF of capacitance and 129 Ω

resistance 32. Positive clones were selected on YPD plates (1% (w/v) yeast extract, 2% (w/v)

peptone, 2% (w/v) glucose, 2% (w/v) agar) containing 100 µg/ml zeocin (LabForce,

Switzerland).

Expression of CC92 and CC49 in P. pastoris

P. pastoris transformants were cultured in BMGY medium (1% (w/v) yeast extract, 2% (w/v)

peptone, 1.3% (w/v) YNB w/o amino acids (Becton Dickinson, USA), 100 mM K-phosphate

buffer pH 6, 1% (v/v) glycerol) supplemented with 100 µg/ml of zeocin at 30 °C, 200 rpm, for

48 h. Maximum 10 % of the baffled Erlenmeyer flask volume was used to provide sufficient

aeration for glycerol and methanol metabolism. Cells were harvested at 3800 x g for 5 min at

room temperature and resuspended in the same volume of minimal medium (1.3% (w/v) YNB

w/o amino acids, 100 mM K-phosphate buffer pH 6, 0.4 µg/ml biotin, 0.5% (w/v) NH4Cl, 1%

(v/v) MeOH) for production of recombinant protein using methanol induction. Expression

Chapter 4

89

cultures were incubated for 48 h at 30 °C, 200 rpm. Methanol was added to 1% (v/v) in a time

interval of 12 h and NH4Cl was added to 0.5% (w/v) in a time interval of 24 h.

For the optimized expression of CC49, the P. pastoris strain SMD 1168H (pep4; Life

technologies, USA) was used and the pH of the minimal medium was adjusted to 4.

Purification of CC49 on cation exchange column

The post-fermentation broth was centrifuged at 3000 x g for 10 min and the supernatant

concentrated in a 3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc.,

USA) by a treatment with polyethylene glycol 6000 at 4 °C. For the optimized expression of

CC49, the concentrated supernatant was then incubated with 1 mM PMSF and protease

inhibitor cocktail (Roche, Germany) for 1 h at 4 °C. After dialysis against 20 mM Na-

phosphate, 50 mM NaCl buffer pH 6.5 (buffer A) at 4 °C for 24 h, the protein solution was

sterile-filtered and loaded on a self-made SP sephadex cation exchange column equilibrated

with buffer A. The column was washed with buffer A and CC49 eluted at 200 mM NaCl in

20 mM Na-phosphate buffer pH 6.5. The effluent was monitored by absorbance at 210, 254,

and 280 nm.

Ni-NTA purification

0.5 ml of Ni-NTA beads (Macherey-Nagel, Germany) were washed and equilibrated in a total

of 50 ml PBS pH 7.4 and transferred to a 10 ml drop column (Macherey-Nagel, Germany).

Conditioned expression supernatant was loaded, the column washed with 50 CV 50 mM

imidazole in PBS and the elution performed with 8 CV 400 mM imidazole in PBS. Elution

fractions were concentrated and dialyzed against PBS pH 7.4.

Protein separation on polyacrylamide gel and detection

The protein solution was mixed with Lämmli buffer and boiled at 95 °C for 20 min. The

denatured proteins were separated on a 15% polyacrylamide gel. Detection of proteins was

performed in three different ways:

Proteins were stained with Coomassie Brilliant Blue R.

Immunoblotting was performed with a primary mouse anti-His antibody (dilution 1:2000;

Qiagen, Germany) and with a goat anti-mouse secondary antibody (dilution 1:2000)

according to standard protocols.

Silver staining was performed according to a standard protocol (Blum et al, (1987),

Electrophoresis 8, 93)

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90

Lysozyme activity assay

The lysozyme assay kit EnzCheck (Life technologies, USA) was used according to the

suppliers protocol. The assay is based on Micrococcus luteus cell wall being labeled with

fluorescein in such a manner that fluorescence is quenched. When the cell wall is cleaved by

lysozyme the quenching is relieved and increased fluorescence can be detected. The

reactions were performed with a final concentration of 3.9, 7.9, 15.8, 31.3, 63, 125, and

250 µg/ml CC49. BSA was prepared in the same manner as negative control. Fluorescence

was measured using the Victor3 plate reader (PerkinElmer, USA) with an excitation

wavelength of 485 nm and emission wavelength of 535 nm. Reactions for the positive control

HEWL and CC49 were performed in triplicates and the negative control BSA in duplicates.

Chapter 4

91

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11. Lysozymes: Model Enzymes in Biochemistry and Biology. (Birkhäuser, 1996).

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14. Rau, A., Hogg, T., Marquardt, R. & Hilgenfeld, R. A new lysozyme fold. Crystal structure of the muramidase from Streptomyces coelicolor at 1.65 A resolution. J. Biol. Chem. 276, 31994–9 (2001).

15. Hash, J. H. Purification and Properties of Staphylolytic Enzymes from Chalaropsis sp. Arch.

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17. Fischetti, V. A. Bacteriophage endolysins: a novel anti-infective to control Gram-positive pathogens. Int. J. Med. Microbiol. 300, 357–62 (2010).

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21. Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–91 (2009).

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24. Ibrahim, H. R., Imazato, K. & Ono, H. Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration. J. Agric. Food Chem. 59, 10336–45 (2011).

25. Ibrahim, H. R., Inazaki, D., Abdou, A., Aoki, T. & Kim, M. Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach. Biochim. Biophys. Acta 1726, 102–14 (2005).

26. Brierley, R. A. in Methods Mol. Biol. 103, 149–177

27. Bader, O., Krauke, Y. & Hube, B. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiol. 8, 116 (2008).

28. Herreweghe, J. M. & Michiels, C. W. Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function. J. Biosci. 37, 327–348 (2012).

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30. Bera, A., Herbert, S., Jakob, A., Vollmer, W. & Götz, F. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol. Microbiol. 55, 778–87 (2005).

31. Bera, A. et al. Influence of wall teichoic acid on lysozyme resistance in Staphylococcus aureus. J. Bacteriol. 189, 280–3 (2007).

32. Wu, S. & Letchworth, G. J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152–4 (2004).

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Supplementary Information

Table S1. Secretome of unchallenged C. cinerea. The cultivation and extraction was performed

twice (A and B) with two technical replicates. Signal peptides were predicted by the software SignalP

4.0 33. Shown are the predicted biological function and the non-normalized spectral counts for each

protein and replicate.

Accession Protein function A1 A2 B1 B2

CC1G_00158 Hypothetical protein 2 2 CC1G_00306 Chitin deacetylase 0 1 CC1G_00332 Endopeptidase 6 8 19 21 CC1G_00344 Disulphide isomerase 0 5 3 2 CC1G_00471 Hypothetical protein 4 4 11 7 CC1G_00563 Glycosyl hydrolase 53 domain-containing protein 1 1 CC1G_00916 Predicted protein 7 8 9 13 CC1G_00929 Hypothetical protein 2 2 CC1G_01102 Crystal protein 0 3 5 4 CC1G_01107 Exocellobiohydrolase 0 3 14 12 CC1G_01219 Hypothetical protein 1 1 CC1G_01248 Peptidase 2 1 CC1G_01253 Hypothetical protein 2 3 CC1G_01315 Endochitinase 0 1 CC1G_01443 Hypothetical protein 0 2 CC1G_01543 Aminopeptidase 6 4 21 25 CC1G_01658 Aryl-alcohol oxidase 2 2 3 6 CC1G_01797 Hypothetical protein 1 1 3 3 CC1G_02104 Peroxidase 14 13 113 92 CC1G_02174 Hydrophobin 6 5 CC1G_02182 Hydrophobin 1 1 4 3 CC1G_02286 Peptidase 14 15 32 29 CC1G_02351 Hypothetical protein 2 1 CC1G_02845 Sphingomyelin phosphodiesterase 1 1 CC1G_02862 Predicted protein 0 2 CC1G_03046 Predicted protein 3 3 CC1G_03049 Lysozyme 6 5

CC1G_03076 Lysozyme 5 2 CC1G_03149 Hypothetical protein 4 2 CC1G_03154 Hypothetical protein 1 1 4 4 CC1G_03158 Hypothetical protein 0 1 CC1G_03180 Hypothetical protein 4 5 CC1G_03223 Peptidylprolyl isomerase B 0 1 3 4 CC1G_03329 Copper radical oxidase 0 1 3 0 CC1G_03339 Hypothetical protein 4 4 CC1G_03340 Hypothetical protein 2 0 CC1G_03354 DJ-1/PfpI family protein 1 1 5 10 CC1G_03407 Trehalase 2 5 10 10 CC1G_03420 Beta-hexosaminidase 4 9 19 41 CC1G_03425 Beta-hexosaminidase 1 2 CC1G_03442 Endonuclease/exonuclease/phosphatase 3 1 CC1G_03477 Hypothetical protein 12 11 CC1G_03541 Predicted protein 2 2 CC1G_03653 Aminopeptidase 5 3 3 1 CC1G_03680 Predicted protein 0 2 14 10 CC1G_03940 Laccase 6 11 0 3 CC1G_04051 Glycosyl hydrolase family 16 3 4 7 7 CC1G_04162 Hypothetical protein 2 1 5 1 CC1G_04169 Hypothetical protein 1 1 CC1G_04257 Beta-mannase 1 0 CC1G_04305 Hypothetical protein 1 1 2 1 CC1G_04336 Predicted protein 3 6 CC1G_04337 Predicted protein 4 3 8 12 CC1G_04470 Serine-type endopeptidase 2 5 13 20 CC1G_04562 Serine protease 21 22 213 256 CC1G_04712 Copper radical oxidase 64 70 487 600 CC1G_04843 Hypothetical protein 0 0 CC1G_04844 5'-nucleotidase 3 7 14 19 CC1G_04876 alpha-glucosidase 31 38 60 77 CC1G_04923 FAD/FMN-containing protein 3 3 CC1G_04928 leucine aminopeptidase 1 10 10 5 8 CC1G_04948 Aminopeptidase 1 2 1 2 CC1G_04997 Glucoamylase precursor 6 8 33 32

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94

CC1G_05039 Predicted protein 5 4 7 5 CC1G_05043 Predicted protein 1 1 5 2 CC1G_05139 Predicted protein 1 0 6 6 CC1G_05192 Fasciclin domain family 0 0 CC1G_05246 Hypothetical protein 4 7 22 22 CC1G_05285 Endochitinase 1 0 CC1G_05355 Hypothetical protein 3 2 1 5 CC1G_05418 Aryl-alcohol oxidase 11 12 25 21 CC1G_05573 Hypothetical protein 1 1 CC1G_05600 Predicted protein 2 1 CC1G_05607 Hypothetical protein 3 3 7 1 CC1G_05612 Hypothetical protein 3 2 2 3 CC1G_05638 Hypothetical protein 5 5 CC1G_05778 ycaC 3 2 CC1G_05798 Hypothetical protein 0 2 0 2 CC1G_05860 Hypothetical protein 1 0 2 2 CC1G_05883 Aminopeptidase 3 1 CC1G_05917 Aminopeptidase 1 1 7 6 CC1G_05923 Aminopeptidase 3 0 3 5 CC1G_05954 Hypothetical protein 1 1 CC1G_06012 Predicted protein 2 2 CC1G_06019 Laccase 5 1 0 CC1G_06068 Alginate lyase 0 3 CC1G_06086 Predicted protein 3 4 CC1G_06262 Hypothetical protein 1 3 11 6 CC1G_06324 Endoglucanase 2 2 CC1G_06563 Exo-beta-1,3-glucanase 3 2 9 6 CC1G_06564 Exo-beta-1,3-glucanase 6 5 30 23 CC1G_06692 Predicted protein 18 15

CC1G_06696 Predicted protein 1 1 CC1G_06703 Predicted protein 1 1 5 3 CC1G_06773 Vacuole protein 9 10 CC1G_06848 Hypothetical protein 6 4 CC1G_06885 Predicted protein 3 3 4 5 CC1G_06891 Metalloprotease 3 3 0 0 CC1G_06959 Thaumatin-like protein 4 4 CC1G_07128 Predicted protein 3 3 10 8 CC1G_07167 Cu/Zn superoxide dismutase 3 2 0 0 CC1G_07201 Glucoamylase 10 7 9 6 CC1G_07205 Predicted protein 4 2 CC1G_07631 Predicted protein 2 2 CC1G_07990 Elastinolytic metalloproteinase 4 3 CC1G_08049 Predicted protein 2 2 20 15 CC1G_08051 Predicted protein 1 1 7 5 CC1G_08056 Secreted protein 15 13 CC1G_08067 Macrophage activating glycoprotein 2 1 2 3 CC1G_08068 Predicted protein 7 5 CC1G_08239 Predicted protein 4 3 0 2 CC1G_08260 Predicted protein 2 2 CC1G_08277 Cellobiohydrolase II-I 1 0 10 8 CC1G_08310 Predicted protein 4 4 CC1G_08358 Metalloprotease 12 11 13 9 CC1G_08391 Predicted protein 2 2 10 7 CC1G_08427 Hypothetical protein 1 4 0 0 CC1G_08561 Predicted protein 2 2 CC1G_08598 G-X-X-X-Q-X-W domain-containing protein 2 2 8 6 CC1G_08717 Carboxylesterase 3 3 CC1G_08822 Putative fungistatic metabolite 7 5 90 105 CC1G_08921 Hypothetical protein 5 1 CC1G_08950 Metalloprotease 4 6 4 3 CC1G_09057 Hypothetical protein 2 4 14 11 CC1G_09088 Mannoprotein 6 9 7 6 CC1G_09149 Hypothetical protein 0 0 CC1G_09154 Snodprot1 2 3 CC1G_09155 Snodprot1 3 2 7 5 CC1G_09180 Carboxypeptidase C 0 2 CC1G_09189 Hydrophobin-315 1 0 0 1 CC1G_09291 Hypothetical protein 13 15 23 24 CC1G_09292 Glucooligosaccharide oxidase 1 1 CC1G_09340 CEL4b mannanase 2 2 1 1 CC1G_09406 Hypothetical protein 0 1 CC1G_09442 Hypothetical protein 0 0 CC1G_09595 Hypothetical protein 5 3 13 11 CC1G_09604 Hypothetical protein 4 3 CC1G_09659 Hypothetical protein 3 4 CC1G_09727 Hypothetical protein 2 3 0 1

Chapter 4

95

CC1G_09770 Mala s 12 allergen 1 1 4 4 CC1G_09868 Lipase 2 3 5 6 CC1G_09883 Lipase 4 3 12 16 CC1G_09921 Class V chitinase ChiB1 0 0 CC1G_09938 Hypothetical protein 2 1 CC1G_09992 Predicted protein 1 1 CC1G_10038 B-(1-6) glucan synthase 4 6 1 1 CC1G_10047 WSC domain-containing protein 3 5 CC1G_10245 Hypothetical protein 10 15 60 31 CC1G_10446 Mala s 12 allergen 2 6 39 34 CC1G_10592 Serine protease 1 4 16 24 CC1G_10603 G-X-X-X-Q-X-W domain-containing protein 2 2 11 7 CC1G_10606 Hypothetical protein 6 6 CC1G_10796 Hypothetical protein 12 8 CC1G_10812 B2-aldehyde-forming enzyme 1 1 CC1G_10860 Predicted protein 0 0 CC1G_10969 Hypothetical protein 0 1 2 2 CC1G_11193 Hypothetical protein 5 4 CC1G_11195 Hypothetical protein 1 2 CC1G_11246 Ricin B lectin 0 1 CC1G_11283 Predicted protein 7 9 6 3 CC1G_11355 FAD binding domain-containing protein 6 6 21 15 CC1G_11511 Glucuronyl hydrolase 9 12 52 46 CC1G_11527 Hypothetical protein 6 5 CC1G_11695 Glucoamylase 19 22 49 48 CC1G_11771 Metalloprotease 8 11 CC1G_12036 Predicted protein 3 2 6 5 CC1G_12142 Predicted protein 9 8 22 17 CC1G_12227 1,3-beta-glucanosyltransferase 2 2 5 0 CC1G_12269 Hypothetical protein 3 3 CC1G_12415 Hypothetical protein 1 1 CC1G_12425 Hypothetical protein 0 1 3 3 CC1G_12510 Carotenoid ester lipase 12 11 51 57 CC1G_13219 Predicted protein 2 3 0 0 CC1G_13656 Riboflavin aldehyde-forming enzyme 4 4 3 3 CC1G_13671 Cellulose-binding beta-glucosidase 16 18 CC1G_13682 1,3-beta-glucanosyltransferase 3 3 6 7 CC1G_13742 Calnexin 0 1 0 0 CC1G_13813 Copsin 3 3 37 43

CC1G_14064 Hypothetical protein 2 1 CC1G_15015 Hypothetical protein 3 2 13 11 CC1G_15645 Predicted protein 3 3 3 5 CC1G_15739 Macrophage activating glycoprotein 5 5 10 8

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96

1 ATG AAG CCC ATT GCT CTC CTC TCC ACC ATC GTC TTC GCC TTG ACC GTC TCG GTC CAA GGC

M K P I A L L S T I V F A L T V S V Q G

>---------------------------- Signal peptide ---------------------------------<

61 GCC ATC AAC GAT CCC TGC TCA GTC AAC GGC ACG CCC GGT ATT TGC ATC ACC ACG ACG GCT

A I N D P C S V N G T P G I C I T T T A

>------------------------------------------------------------------------------

121 TGT GCC AAC GCT GGT GGC ACC AAC GCT GTC GGT TTC TGC CCC AAC GAT CCT GCC AAC GTC

C A N A G G T N A V G F C P N D P A N V

-------------------------------------------------------------------------------

181 CGT TGC TGC ACC AAG AAG TGC AGT ACC AAC GGC ACC TGC CGT TTC ACC AAC ACC TGT TCT

R C C T K K C S T N G T C R F T N T C S

-------------------------------------------------------------------------------

241 AGC GGC AAC GTC CTC GTT GGC CTT TGC CCC GGT CCC TCC AAC TTC AGG TGT TGC ATC CCC

S G N V L V G L C P G P S N F R C C I P

-------------------------------------------------------------------------------

301 TCT AGC AGC TGC GCC TAC AGC CCG GTC AAT TCC CGC ACC GTC CAG GAA ATC AAG AAC TCC

S S S C A Y S P V N S R T V Q E I K N S

-------------------------------------------------------------------------------

361 GAA GGA TTC GTC AGG TCC CCT GCA CCC GAT CCA ATC GGT CTC CCC ACG GTC GGA TAC GGC

E G F V R S P A P D P I G L P T V G Y G

---------------------------- Mature lysozyme ----------------------------------

421 CAC CTC TGC AAG AAT AAG GGA TGC AGC GAG GTC CCA TAC AGT TTC CCC CTG ACC GAA GCG

H L C K N K G C S E V P Y S F P L T E A

-------------------------------------------------------------------------------

481 CAG GCC ACC TCT CTC CTC ATG ACC GAC TTG AAG ACC TTC CAG AAG TGC ATC TCC GAC CAA

Q A T S L L M T D L K T F Q K C I S D Q

-------------------------------------------------------------------------------

541 ATC AAC GAT TCC ATC AGG CTG AAC GAG AAC CAG TAC GGT GCC TTG GTT TCG TGG GCC TTC

I N D S I R L N E N Q Y G A L V S W A F

-------------------------------------------------------------------------------

601 AAC GTT GGC TGC GGT AAC ACC GCC TCT TCT GCC TTG ATC TCG CGA CTC AAC AAG GGA GAG

N V G C G N T A S S A L I S R L N K G E

-------------------------------------------------------------------------------

661 AGC CCG AAC AAG GTT GCG GAG GAG GAG CTT CCT CGA TGG AAG TAC GCC GGT GGC CAG GTT

S P N K V A E E E L P R W K Y A G G Q V

-------------------------------------------------------------------------------

721 CTG CCC GGC TTG GTT GCT CGT AGG AAC AGG GAG ATC GCA TTG TTC AAG ACT GCT TCG AGC

L P G L V A R R N R E I A L F K T A S S

-------------------------------------------------------------------------------

781 GTC GTC GGG CAT CCT CCC AGG TGC TAG

V V G H P P R C ---

------------------------------<

Fig. S1. Precursor protein CC1G_03049 (CC49). Prediction of the signal peptide was performed with

SignalP 4.0 33. The potential Kex cleavage site and N-glycan sites are indicated in bold.

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97

1 ATG AAG TTT ACC ACA TCC CTC TTC GCC ATC TTC TTG ACC CTC GGT GTC GCC CAA GCC GCC

M K F T T S L F A I F L T L G V A Q A A

>------------------------------ Signal peptide ---------------------------< >--

61 CTC AAC GGC CCC TGC AAC ATC CCC GGC GTC GGT CCT GGT ACC TGC CTC CAC ACC TCC ACC

L N G P C N I P G V G P G T C L H T S T

-------------------------------------------------------------------------------

121 TGC GCC AAC GGC GGT GGC GGT TCT TTC TCT GGC TAC TGC CCG AAC GAC CCC GCA GAC GTC

C A N G G G G S F S G Y C P N D P A D V

------------------------------- Mature peptide --------------------------------

181 CGG TGC TGC TTC AAG CGC TGC CCC ACA TCT CTT GGC AGT GGC AGA TGC CGC CCT GTT GCC

R C C F K R C P T S L G S G R C R P V A

-------------------------------------------------------------------------------

241 TCG TGC CCC AGC GGC AGA ACC CTC ACA GGA TAC TGC CCT GGA CCC GCC ACG GTT AGG TGC

S C P S G R T L T G Y C P G P A T V R C

-------------------------------------------------------------------------------

301 TGC TTG CCA TCA TAA

C L P S ---

--------------<

Fig. S2. Precursor protein CC1G_06692 (CC92). Prediction of the signal peptide was performed with

SignalP 4.0 33. The potential Kex2 cleavage site is indicated in bold.

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98

Chapter 5

O-mannosylated cell wall proteins

of S. cerevisiae

Andreas Essiga, Paolo Nannib, Markus Aebia

a: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland

b: Functional Genomics Center Zurich, CH-8057 Zurich, Switzerland

Contributions

Extraction of yeast cell wall proteins

MS sample preparation

MS measurements Orbitrap Velos

MS data analysis

99

Abstract

The cell wall of fungi is composed of a unique polysaccharide scaffold interconnected to

structural and enzymatic proteins. The majority of cell wall proteins (CWPs) are extensively

decorated with N- and O-glycans influencing their stability, function, and localization. O-

mannosylation is one type of glycan found on CWPs, an ubiquitously known protein modification

up to humans. In yeast, O-mannose (O-Man) glycans are generated in the endoplasmic

reticulum by the transfer of a single mannose to serine and threonine residues catalyzed by

protein O-mannosyltransferases (PMTs). However, little is known about which proteins are

targeted by PMTs and about the actual site localization of O-Man glycans. Here, we present a

novel workflow for the detection of O-mannosylations on covalently linked cell wall proteins of

Saccharomyces cerevisiae, which combines an α-mannosidase treatment of extracted CWPs

with a liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement. Based on

this strategy, we described a comprehensive yeast cell wall O-Man glycoproteome with 24

unique O-mannosylated CWPs identified. Analysis of the acquired spectra revealed an

extremely high complexity and heterogeneity of this type of O-glycosylation. Further, we could

successfully implement SILAC (stable isotope labeling by amino acids in cell culture) for relative

quantification of O-mannosylated peptides, providing new opportunities for studying O-

mannosylated proteins in yeast and other organisms.

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100

Introduction

The cell wall of S. cerevisiae is a dynamic and unique network of proteins and polysaccharides,

which represents 15-30% of the dry weight of vegetative cells 1. This extracellular matrix

possesses important biological functions, such as maintenance of the cell shape, interaction with

the environment, or acting as barrier and protective line for biological molecules and competing

microbes 2,3,4. The major compounds of the cell wall are β-glucans, chitin, and glycoproteins.

β1,3- and β1,6-linked glucose (Glc) polymers are the two predominant types of glucan found in

the cell wall of S. cerevisiae with more than 50% of the total cell wall dry weight. β1,3-glucan

chains are highly branched and can adopt helix-like structures, which provide the cell wall with

elasticity. β1,6-glucan chains are shorter than the ones with a β1,3 linkage and have a more

amorphous structure. Both types of glucan are essential for a proper cell wall assembly, as they

are highly interconnected to other cell wall components like chitin and glycoproteins 3. Chitin is a

linear polymer of β1,4-linked N-acetylglucosamine (GlcNAc), which occurs as free chitin or linked

to β-glucan 5. These polymer chains of more than 100 residues are able to form microfibrils with

an enormous tensile strength that confer a high rigidity to the cell wall. Major deposits of chitin

are found at the bud neck and septum of dividing cells and in a lesser extent also in the lateral

wall near the plasma membrane 1,4. In total, ~180 proteins are covalently linked or associated to

the polysaccharide scaffold 4. They execute a diversity of biological functions, such as enzymatic

activities like cross-linking and remodeling of cell wall components, mediating adhesion,

regulating permeability, or transmitting signals from environmental stimuli 6. Many of the

covalently linked cell wall proteins (CWPs) are synthesized with a glycosylphosphatidylinositol

(GPI) anchor that localizes the protein to the outer leaflet of the plasma membrane, for example,

the Tip1, Gas, and Sed1-Spi1 protein families 7,8. GPI-CWPs can be further processed and

released from the lipid carrier by a cleavage between the glucosamine (GlcN) and Man residue

of the GPI anchor. The reducing end of the GPI remnant is then linked to a glucose residue of a

non-reducing end of β1,6-glucan 9. PIR (proteins with internal repeats) proteins are a group of

covalently attached CWPs that are characterized by the repeating sequence unit

SQ[I/V][S/T/G]DGQ[I/V]Q[A][S/T/A] and can be released from the cell wall by a mild alkali

treatment 4. The connection of PIR proteins to other cell wall components is mediated most likely

via linkages between their amino acids or glycan structures and the glucose residues of β1,3-

glucan 10. Treatment of the cell wall with reducing agents releases PIR proteins, indicating that

they can also be linked through a disulfide bond to other CWPs 11.

Most of these cell wall associated proteins are decorated with N- and O-linked glycans after

passing the secretory pathway. N-linked glycosylation is an essential protein modification in

eukaryotes, which is involved in numerous biological processes, such as protein folding and

quality control, solubility and activity of a protein, or mediating interactions with other biological

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molecules 12,13,14. N-glycans are initiated in the endoplasmic reticulum (ER) through the action of

an oligosaccharyltransferase complex (OST), which transfers the core oligosaccharide

Glc3Man9GlcNAc2 from a dolichylpyrophosphate lipid carrier to an asparagine site chain of a

nascent polypeptide chain 15,16,17. After trimming to a Man8GlcNAc2 structure, the glycoprotein is

moved to the Golgi complex, where the N-glycan structures are further processed. In baker's

yeast, a frequent elaboration of the core N-glycan is the addition of a mannose-rich

oligosaccharide with up to 150-200 mannoses (Man) residues. The so-called mannan structures

include the addition of chains of α1,6-linked Man, extensively branched with α1,2-Man

terminating in an α1,3-Man or a Man-1-P (mannose phosphate) 3,4. The negatively charged

phosphate groups are most likely involved in regulation of the cell wall permeability and in

retaining water molecules 2,4.

In comparison to N-glycans, O-mannosylations in S. cerevisiae are short with a chain length of

up to five mannoses. The chain is α1-linked to hydroxyl-groups of serine and threonine residues 18. The first mannose is attached in the luminal part of the ER by protein O-mannosyltransferases

(PMTs), which use dolichol phosphate β-D-mannose as their mannosyl donor 19. PMTs are

evolutionarily conserved ER transmembrane proteins that are divided in three subfamilies

referred to as PMT1, PMT2, and PMT4 20,21. In S. cerevisiae, six PMTs have been identified

assigned to the three subfamilies, PMT1 (Pmt1, Pmt5), PMT2 (Pmt2, Pmt3), and PMT4 (Pmt4).

To achieve highest activity, Pmts of the subfamilies PMT1 and PMT2 form heterodimers,

whereas Pmt1-Pmt2 and Pmt5-Pmt3 complexes are most prevalent 22. Pmt4, the only member of

subfamily PMT4, enhances its activity by forming homodimers. It was shown that PMTs act on

specific sets of proteins: the Pmt4 dimer exclusively O-mannosylates membrane associated

proteins 23. Substrates of the Pmt1-Pmt2 complex are soluble or membrane associated proteins

and also misfolded proteins in the ER. Different studies demonstrated that O-mannosylations are

involved in the unfolded protein response (UPR) and the ER-associated protein degradation

(ERAD) pathway 24,25,26. Further processing of O-mannosylations takes place in the Golgi

apparatus by different mannosyltransferases using GDP-Man as sugar donor. The second and

third mannose is added in α1,2-linkage by three KTR family members (Kre2, Ktr1, Ktr3) 27.

Members of the MNN1 family (Mnn1, Mnt2, Mnt3) catalyze the attachment of the two terminal

α1,3-linked mannoses. The linear mannose chain can additionally be modified by Man-1-P, the

same structural element identified on N-linked mannans 28. The analysis of viable pmt∆ mutants

showed that O-mannosylations affect the stability, localization, and functionality of CWPs and

consequently also the cell wall integrity 18,29. Furthermore, inhibition of PMTs by the rhodanine-3-

acetic acid derivative OGT2468 revealed an activation of the cell wall integrity (CWI) pathway,

which is involved in maintenance and repair of the cell wall 30. The biological significance of O-

mannosylations could also be shown in humans, where the structural composition and diversity

of glycans is much more complex than in yeast 31. One of the few well studied O-mannosylated

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proteins is α-dystroglycan, a component of the dystrophin-glycoprotein complex (DGC). Within

the DGC complex, α-dystroglycan interacts with components of the extracellular matrix,

mediated among other glycan types by O-mannosylations. A non-functional O-Man structure

leads to a detachment of the extracellular matrix with a strong impact in muscular tissue, where it

can cause severe forms of congenital muscular dystrophy 31,32.

Despite efforts done in the past, the localization and stoichiometry of O-Man glycans on CWPs is

largely unknown. Reasons for this lack of knowledge are technical challenges to identify O-

mannosylated peptides, such as the instability of O-glycans in standard collision induced

dissociation (CID) MS/MS and the fact that there is no consensus sequence known for O-Man

glycan sites as for N-glycans. In these studies, we present a unique workflow for the detection

and quantification of O-mannosylations on S. cerevisiae CWPs based on electron transfer

dissociation (ETD) MS/MS 33,34. ETD has been proven to be a powerful tool for the identification

of labile post-translation modifications (PTMs), as it preserves PTMs during the fragmentation

process 35. In combination with higher energy collisional dissociation (HCD) MS/MS, 24 unique

O-mannosylated CWPs were identified and quantified by the implementation of SILAC (stable

isotope labeling by amino acids in cell culture) 36,37. The detected O-mannosylated peptides

exhibited a highly complex pattern of O-Man modifications giving new insights in the diverse and

dynamic nature of this O-glycosylation.

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Results

Identification of O-mannosylated cell wall proteins of yeast

Previous studies indicated that O-Man glycans are highly clustered in serine and threonine rich

regions and appear in various lengths with up to five mannose residues connected. This

heterogeneity and the lability of O-glycosidic bonds were taken into account, when we developed

a LC-MS/MS based workflow for the identification of O-Man glycans on covalently linked CWPs

of yeast. An analytical method that had previously been established in our lab for the analysis of

N-linked glycans on CWPs, was further developed and adapted for ETD measurements of O-

mannosylated peptides 34. In brief, yeast cells grown to the midlog phase were lyzed and the cell

wall material separated and enriched. To achieve an optimal coverage of the amino acid

sequences, the proteolytic cleavages were performed with three different proteases ideal for

ETD-MS/MS measurements, including the lysyl-endopeptidase (LysC), the peptidyl-Lys

metalloendopeptidase (LysN), and the endoproteinase AspN 38,39. To simplify the data analysis,

proteolytic peptides released from the cell wall were incubated with the α-mannosidase from

Jack bean to trim down O-Man glycans to a single α1-linked mannose 40. For each digest, an

ETD-MS/MS measurement was performed and the acquired data analyzed with a hexose as

variable modification on serine and threonine residues. To verify the ETD output and to cover

more optimally doubly charged peptides, an HCD-MS/MS measurement was executed

additionally for the LysC and LysN digest. As we determined a low stability of O-Man glycans in

HCD fragmentations, the subsequent data analysis was performed under the assumption of a

neutral loss of a single hexose from serine and threonine residues.

The ETD and HCD measurements resulted in the identification of in total 24 unique O-

mannosylated CWPs, most of them yet not experimentally shown to be modified by O-Man

glycans (Supplementary table 1). The majority of the identified proteins are connected to the

cell wall scaffold via a GPI anchor or remnant (e.g. Aga1, Ecm33, Gas1, Sag1) or by an alkali-

sensitive linkage (ASL), including PIR proteins (e.g. Hsp150, Cis3). Uth1, a SUN protein family

member, was shown to be released of the cell wall after a dithiothreitol (DTT) treatment 41.

Ym122 is uncharacterized concerning the type of linkage to the cell wall and was so far not

described as cell wall protein.

Considering the peptide level, 107 O-mannosylated peptides were identified by ETD- and HCD-

MS/MS with an overlap of 23 peptides between the two fragmentation techniques

(Supplementary table 1). Even though ETD was applied, only for a few peptides it was possible

to unambiguously localize the modifications (Table 1). In addition to an insufficient peptide

fragment coverage, the major reason for this failure was that often several isobaric versions of

an O-mannosylated peptide existed with the modifications located at different positions. The

peptide AAAVSQIGDGQIQAT*T*K containing a PIR repeat is illustrated as an example, where

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two hexoses could be localized by ETD and the peptide sequence be verified by HCD with a

neutral loss of two hexoses (Figure 1). ETD was highly efficient in sequencing of peptides with

multiple mannoses, for example, the triply charged peptide DGQIQAT*T*KT*KAAAVS*QIG with

four single mannoses attached and a mass of 2535.22 Da (Figure 2A). Another peptide is

shown in figure 2B with the amino acid sequence DSIKKIT*G originating from the CWP Ecm33.

Table 1. Peptides with localized O-Man glycans. O-mannosylated peptides, where the mannoses could

be localized on specific serine and threonine residues based on ETD-MS/MS spectra. Modified sites are in

bold and underlined. Peptides, which could not be assigned to a specific PIR protein are labeled with PIR

proteins.

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Fig. 1. ETD- and HCD-MS/MS of an O-mannosylated peptide. Spectra of the peptide

AAAVSQIGDGQIQAT*T*K containing a PIR repeat acquired by ETD with the precursor ion [M + 3H]3+ at

661.7 m/z (calculated: 661.7 m/z) and by HCD with the precursor ion [M + 2H]2+ at 991.99 m/z (calculated:

991.99 m/z). Two O-Man glycans were localized on threonine residues by ETD. The HCD analysis was

performed with a neutral loss of hexoses (162.0528 Da). Due to low peak intensities, certain areas were

multiplied indicated above the ETD spectrum.

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Fig. 2. Localization of single hexoses on serine and threonine residues by ETD-MS/MS. (A)

Fragmentation of the O-mannosylated peptide DGQIQAT*T*KT*KAAAVS*QIG originating from Pir1 with

the precursor ion [M + 3H]3+ at 846.5 m/z (calculated: 846.1 m/z). (B) Spectrum of the singly O-

mannosylated peptide DSIKKIT*G originating from the protein Ecm33 with the precursor ion [M + 2H]2+ at

512.5 m/z (calculated: 512.3 m/z). (C) The O-mannosylated peptide GT*T*T*KET*GVT*T*K originating

from the protein Sed1 with the precursor ion [M + 3H]3+ at 733.1 m/z (calculated: 732.7 m/z).

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Next, we analyzed peptides with different numbers of mannoses attached. Three examples are

described in table 2 (TSTNATSSSCATPSLK of Cis3, GTTTKETGVTTK of Sed1, and

ASSTSTSASASSSIK of Ym122).

Table 2. Heterogeneity of O-mannosylated peptides. For each peptide, all possible numbers of O-Man

sites (# Man) are shown originating from ETD-MS/MS measurements. As an indicator for a correct

assignment of a peptide version, the retention time (RT) is displayed. The area under the curve (AUC) was

determined manually from an extracted ion chromatogram (XIC) of the corresponding m/z. The relative

quantity of a peptide was calculated by dividing the AUC of the corresponding m/z by the sum of AUCs of

all peptide versions. The most abundant one is indicated in bold. No MS1 means that there was no MS1

trace detectable. No MS2 means that there was no MS/MS performed for the corresponding m/z.

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For each peptide only a certain range of number of modifications was detectable at the MS1

level. By determining manually the relative abundance of the different versions, it was shown that

within these ranges there is a maximum abundance as for the Cis3 peptide

TSTNATSSSCATPSLK at two amino acids modified. For none of the peptides it was possible to

clearly localize the modifications with the exception of the Sed1 peptide GT*T*T*KET*GVT*T*K,

where all threonine residues were modified (Figure 2C).

Relative quantification of O-mannosylated peptides

The site localization experiments revealed a high complexity of O-mannosylated peptides as

illustrated in table 2. To address this diversity in more detail and to get an idea how reproducible

these findings are, a quantitative assay was needed. Since a SILAC-MS/MS approach was

already implemented previously in the CWP extraction workflow, we adapted this strategy for the

relative quantification of O-mannosylated peptides by ETD- and HCD-MS/MS 34. A S. cerevisiae

wild type strain was grown independently in heavy and light labeled minimal medium to an

optical density (OD600) of 1.0. The extraction was performed as described above and in the

methods with a LysC proteolytic digest, applying the HCD and ETD fragmentation techniques for

the subsequent MS measurements.

Through this method, 14 O-mannosylated CWPs were identified combining the ETD and HCD

measurements (Supplementary table 2). All of them were reproducibly described in the site

localization experiments of the LysC digest (Supplementary table 1). A relative quantification

was performed for 36 O-mannosylated peptide pairs including the different versions of number of

sites modified. The light to heavy ratios were in a range of 0.7 to 1.5 with an average ratio of 1.1

for the ETD-MS/MS output and an average ratio of 1.0 for the HCD-MS/MS acquired spectra. An

exception was Hpf1 where the peptide ASSAISTYSK showed a ratio of 2.0 for the ETD

measurement and a ratio of 2.1 for the HCD measurement. In more detail, the result is shown for

the three peptides ISPTSANTK of Gas1, SEAPESSVPVTESKGTTTK of Sed1, and

ASSTSTSASASSSIK of Ym122 (Table 3). For each peptide a range of number of sites modified

was detected with a maximum of abundance. The peptide ASSTSTSASASSSIK of Ym122, also

shown in table 2, displayed reproducibly a detectable MS1 trace from 0 to 5 mannoses attached

with a slightly shifted maximum of abundance of 3 to 4 serine and threonine residues modified.

With the results obtained, it was shown that SILAC in combination with ETD- and HCD-MS/MS is

a valuable tool for the quantification of O-mannosylated peptides and proteins.

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Table 3. Relative quantification of O-mannosylated peptides. Wild type yeast cells were grown in light

and heavy medium to an OD600 of 1.0, combined, and the CWPs proteolytically digested with LysC. All

identifications shown here were acquired by HCD-MS/MS. The area under the curve (AUC) was

determined manually from a XIC chromatogram of the corresponding m/z. Light (L) to heavy (H) ratios

were calculated manually from the extracted AUCs. No MS1 means that there was no MS1 trace

detectable. No MS2 means that there was no MS/MS performed for the corresponding m/z.

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Discussion

The critical impact of O-mannosylations on yeast cell viability and especially on the cell wall

stability is demonstrated by the fact that Pmt double mutants exhibit an osmolabile phenotype

and the Pmt triple mutants pmt2∆ pmt3∆ pmt4∆ and pmt1∆ pmt2∆ pmt4∆ are even completely

inviable 4,42. To study this type of O-glycosylation in a high throughput manner, we developed a

novel analytical workflow for the identification of O-mannosylated peptides, based on an α-

mannosidase treatment of extracted and proteolytically digested CWPs followed by a LC-MS/MS

measurement. Using ETD and HCD as fragmentation techniques, this strategy revealed 24 O-

mannosylated and covalently linked CWPs, with a high reproducibility at the protein level.

However, O-mannosylated peptides identified in different runs exhibited a rather low overlap,

which is most likely due to experimental limitations or differences in the expression profile of O-

Man glycans on individual CWPs.

As expected, HCD preferably identified doubly charged peptides (87%) and ETD had a slight

bias toward charge states of three and higher. ETD preserved mannoses on serine and

threonine residues during the fragmentation procedure with up to eight single mannoses

identified on a peptide. However, based on ETD spectra it was often not possible to

unambiguously localize the modifications, because of several isobaric versions of a modified

peptide fragmenting together in the linear ion trap. Even though technically very challenging,

strategies originally developed for isobaric phosphopeptides could be implemented to separately

quantify these O-Man peptides 43,44. The α1 linkage of O-mannosylations to serine and threonine

residues was not stable in HCD fragmentations. Therefore, we applied a neutral loss scan for the

detection of O-mannosylated peptides by HCD, which revealed a set of peptides with a high

overlap to the ETD output. Taken together, both fragmentation techniques are capable of

conclusively identify O-mannosylated peptides.

The heterogeneity of O-mannosylated peptides turned out to be extremely high, due to the

existents of peptides that were modified with different numbers of mannoses. The number of

sites modified was restricted to a specific range with a maximum of attached mannoses.

However, differences in the ionization efficiency of glycopeptides can have an impact on these

distributions and they are difficult to predict. Further validation of these results was performed by

implementing SILAC in the CWP extraction workflow in combination with ETD- and HCD-

MS/MS. A relative quantification was done with a wild type yeast strain, which exhibited

differences of close to zero in quantities of O-mannosylated peptides. These findings

demonstrated that yeast cells in their exponential growth phase express a very similar profile of

O-Man glycans on their covalently linked cell wall proteins. The possibility of using SILAC for the

quantification of O-mannosylated peptides is opening new opportunities for studying differences

in O-Man glycoprofiles in yeast and other organisms, where SILAC is applicable.

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O-Man glycans are predominantly localized in protein regions with a high content of serine and

threonine residues, a feature of O-mannosylations described in literature, which we could also

confirm in our studies 18. A prominent class of identified CWPs were the PIR proteins with the

internal repeat SQ[I/V][S/T/G]DGQ[I/V]Q[A][S/T/A], such as Pir1, Hsp150, Pir3, and Cis3 45. PIR

proteins are thought to be involved in maintenance of the cell wall integrity and the regulation of

permeability 3. How PIR proteins are interconnected with the other cell wall components is not

fully understood. Besides the two possibilities mentioned in the introduction, O-mannosylations

could play an important role in linking PIR proteins and other CWPs to the cell wall scaffold. GAS

protein family members and especially Gas1 were another group of CWPs, which was found to

be extensively modified by O-mannosylations. Gas1 is a putative β1,3-glucanosyltransferase

involved in the formation of β1,3-glucan 46. The functionality of most other O-mannosylated GPI

proteins identified is not fully understood. They are often described as structurally relevant for the

cell wall, such as the proteins Ecm33, Tip1, Ccw12, and Sed1 4.

The complex pattern of O-Man glycans described in our studies is pointing on the biological

function of this protein modification. A dynamic localization and stoichiometry of O-

mannosylations can be correlated to selective regulation of cell wall permeability or to CWP

linkages within the polysaccharide scaffold. Our workflow developed for quantitative studies of

O-mannosylated proteins, provides a powerful tool to uncover more details about how O-Man

glycans are involved in the formation and maintenance of the fungal cell wall.

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Materials and Methods

Chemicals and Yeast strains

All experiments were performed with the S. cerevisiae strain SS328 (ade2-101; his3Δ200; lys2-

801; ura3-52). For SILAC experiments arg4 deficient cells were used (ade2-101; his3Δ200; lys2-

801; ura3-52; delta arg4 : NAT).

All chemicals, if not otherwise mentioned, were bought at the highest available purity from

Sigma-Aldrich, USA.

S. cerevisiae cell wall protein preparation for MS analysis

The protocol is adapted from Schulz and Aebi 2009 and optimized for ETD-MS/MS

measurements of O-mannosylated cell wall proteins 34.

Yeast cells were grown in YPD medium (1% (w/v) yeast extract (Oxoid AG, England), 2% (w/v)

peptone (Oxoid AG, England), 2% (w/v) glucose) at 30 °C to an optical density (OD600) of 1 to 1.2

and 50 OD equivalent of cells (~5 x 108 cells). Cells were then harvested by centrifugation at

2500g at 4 °C for 10 min and resuspended in extraction buffer (10mM TrisHCl pH 7.5, 1x

protease inhibitor cocktail (Roche Applied Science, Germany), 2 mM PMSF). After lysis of the

cells using glass beads (0.5 mm; BioSpec, USA) at 4 °C, covalently linked cell wall material was

pelleted by centrifugation at 16000g for 1 min and resuspended in denaturing buffer (50 mM

TrisHCl pH 7.5, 2 M thiourea, 7 M urea, 2% (w/v) SDS). Cell wall proteins were reduced by the

addition of DTT (Axon Lab, Switzerland) to 10 mM and an incubation at 30 °C for 2 h. After an

alkylation with 25 mM acrylamide at 30 °C for 1 h in the dark, the cell wall pellet was five times

washed with denaturing buffer and subsequently six times with 2% (w/v) SDS. The pellet was

then dissolved in 2% SDS and the N-glycans trimmed down to a single HexNAc by the endo-β-

N-acetylglucosaminidase H for 16 h according to the manufacturer's protocol (EndoH; New

England Biolabs, USA). After washing and dissolving the pellet in 50 mM Na4HCO3 (pH 8), the

cell wall proteins were digested and released with either AspN (2 µg/ml; Merck, Germany), LysC

(20 µg/ml; Wako, Japan), or LysN (6 µg/ml; U-Protein Express BV, Netherlands) at 37 °C for 16

h. The remaining cell wall material was separated from soluble peptides by centrifugation at

16000 x g for 1 min and the supernatant reduced to complete dryness in a centrifugal

evaporator. The peptides were dissolved in 100 mM sodium acetate buffer (pH 4.6) and treated

with α-mannosidase from Jack beans at 37 °C for 16 h. Afterwards, the peptide solution was

acidified with formic acid to pH 2-3 and desalted using C18 ZipTip pipette tips (Merck Millipore,

Germany).

For both fragmentation techniques, the sample preparation was performed according to the

described procedure. For the LysN digest, HCD-MS/MS was performed with the same extract as

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prepared for ETD. For the LysC digest, the ETD and HCD measurement was performed with two

independently prepared extracts. ETD was solely used for the extract digested with AspN.

S. cerevisiae cell wall protein preparation for SILAC analysis

The lysine/arginine auxotrophic yeast strain was grown at 30 °C in minimal medium (0.67% (w/v)

Yeast Nitrogen Base w/o amino acids (Becton Dickinson, USA), 2% (w/v) glucose)

supplemented either with 20 mg/l of the light version of L-lysine [12C6/14N2] and L-arginine [12C6]

or the heavy version of L-lysine [13C6/15N2] and L-arginine [13C6] (Cambridge Isotope

Laboratories, USA). The cells of the light and heavy culture were grown both to an OD600 of 1.0,

mixed 1:1 (v:v), and harvested by centrifugation at 2500 x g at 4 °C. Afterwards, the cell wall

proteins were prepared as described with a LysC proteolytic digest using the same extract for

the ETD and HCD measurement.

MS analysis

All MS analyses were performed on a hybrid Velos LTQ Orbitrap mass spectrometer (Thermo

Scientific, USA) equipped with an ETD unit and coupled to an Eksigent-nano-HPLC system

(Eksigent Technologies, USA). Separation of peptides was done on a self-made column (75 µm

x 80 mm) packed with C18 AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were eluted with

a linear gradient from 2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250 nl/min.

For the ETD measurements, full MS data were measured in the Orbitrap unit in a mass range of

300 – 1700 m/z, with an automatic gain control (AGC) setting of 1 x 106, at a resolution of 60000

at 400 m/z. MS/MS spectra were acquired in the data dependent mode with up to 10 ETD

spectra recorded in the linear ion trap using the most intense ions. Supplemental activation

energy was activated and the AGC value was set at 5 x 104. Fluoranthene was used as anion

with an AGC value of 1 x 105 and a reaction time of 100 ms.

For HCD measurements, full MS sans were done with a resolution of 30000 at 400 m/z. A

maximum of 10 HCD MS/MS scans were acquired with stepped normalized collision energy

starting at 15% using three steps with a collision energy width of 15. Fragment ions were

detected in the Orbitrap at a resolution of 7500.

Data analysis

For all measurements, MS/MS spectra were searched against the S. cerevisiae protein database

including common contaminants (fgcz_4932) using the Mascot search algorithm v2.4 (Matrix

Science Inc., USA) with the following parameters: Propionamide (C) as fixed modification;

oxidation (M), hexose (S), hexose (T) as variable modifications; 5 ppm peptide tolerance and

0.8 Da fragment ion tolerance; LysC, LysN, or AspN set as proteases with a maximum of 3

missed cleavages. For HCD, a neutral loss of hexose (162.0528 Da) from serine and threonine

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114

residues was set as variable modification in addition to oxidation of methionine. The maximum

false discovery rate was set at 1% and peptides with an e-value of below 0.05 were rejected. All

acquired data were manually verified using the Mascot search output and the Xcalibur software

(Thermo Scientific, USA). Peptide abundances were determined manually from extracted ion

chromatograms (XIC) by calculating the area under the curve (AUC) using the Xcalibur software.

For SILAC experiments, the fold abundance ratio was determined by dividing the “light” by the

“heavy” AUC of a XIC of the corresponding m/z.

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Chapter 5

118

Supplementary Information

Chapter 5

119

Tab

le S

1.

O-m

an

no

syla

ted

pep

tid

es a

nd

pro

tein

s i

den

tifi

ed

by E

TD

- an

d H

CD

-MS

/MS

. Pr

otei

ns w

ere

cate

goriz

ed a

ccor

ding

to th

eir

linka

ge to

the

cell

wal

l sca

ffold

. Pep

tides

orig

inat

ing

from

a P

IR re

peat

, whi

ch c

ould

not

be

assi

gned

to a

spe

cific

pro

tein

are

indi

cate

d. A

n ox

idat

ion

of m

ethi

onin

e is

indi

cate

d

by a

n M

nex

t to

the

num

ber o

f man

nose

s (#

Man

) atta

ched

to th

e pe

ptid

e. T

he s

core

and

exp

ecta

tion

valu

e ar

e fro

m th

e M

asco

t out

put.

Ac

ces

sio

n

Pro

tein

P

rote

ase

Seq

ue

nce

# M

an

E

TD

H

CD

Ob

serv

ed

(m

/z)

z

Sco

re

Exp

ect

Ob

serv

ed

(m

/z)

z

Sco

re

Exp

ect

GP

I-C

WP

s

P323

23

Aga1

Ly

sC

SWVS

SMTT

SDED

FNK

1 94

8.39

52

2 70

.35

9.2E

-08

SW

VSSM

TTSD

EDFN

K 2

1029

.422

3 2

40.6

7 8.

6E-0

5 10

29.4

217

2 98

.38

1.5E

-10

Q12

127

Ccw

12

AspN

D

HVC

SETV

SPAL

VSTA

TVTV

1

1125

.044

7 2

21.2

2 0.

018

D

HVC

SETV

SPAL

VSTA

TVTV

D

1 78

8.70

83

3 29

.44

0.00

29

D

HVC

SETV

SPAL

VSTA

TVTV

D

2 84

2.72

55

3 22

.27

0.01

8

DH

VCSE

TVSP

ALVS

TATV

TVD

3

896.

7426

3

18.8

8 0.

041

LysC

N

GTS

TAAP

VTST

EAPK

1

847.

4109

2

48.4

5 2.

1E-0

5

NG

TSTA

APVT

STEA

PK

2 92

8.43

68

2 20

.65

0.00

99

N

GTS

TAAP

VTST

EAPK

3

1009

.461

8 2

17.2

2 0.

023

LysN

KN

GTS

TAAP

VTST

EAP

2

92

8.43

52

2 35

.54

0.00

043

KN

GTS

TAAP

VTST

EAP

3

10

09.4

601

2 54

.39

4.4E

-06

O

1354

7 C

cw14

Ly

sC

APSS

EESS

STYV

SSSK

1

897.

8911

2

45.6

5 2.

7E-0

5

ASSS

SASS

STKA

SSSS

AAPS

SSK

1 73

7.00

28

3 72

.65

8.1E

-08

AS

SSTK

ASSS

SASS

STK

2 62

8.95

16

3 19

.71

0.01

4

P533

01

Crh

1 As

pN

DST

TAAS

STAS

CN

PLKT

TGC

TP

1 12

09.5

325

2 29

.51

0.00

15

D

STTA

ASST

ASC

NPL

KTTG

CTP

2

860.

7094

3

15.6

0.

045

LysC

G

DTT

TYD

RG

EFH

GVD

TPTD

K 1

594.

2631

4

24.4

1 0.

0036

TLAS

SSVT

TSSS

ISSF

EK

1 99

0.97

67

2 45

.26

8.2E

-05

TL

ASSS

VTTS

SSIS

SFEK

3

1153

.029

9 2

28.4

3 0.

0038

TPAT

VSST

TRST

VAPT

TQQ

SSVS

SDSP

VQD

K 1

1104

.867

5 3

30.1

7 0.

0058

TPAT

VSST

TRST

VAPT

TQQ

SSVS

SDSP

VQD

K 8

1112

.494

7 4

20.5

5 0.

026

TT

GC

TPD

TALA

TSFS

EDFS

SSSK

1

858.

3735

3

45.6

2 2.

7E-0

5 12

87.0

561

2 11

8.18

1.

5E-1

2

TTG

CTP

DTA

LATS

FSED

FSSS

SK

2 91

2.39

11

3 52

.31

5.9E

-06

912.

3906

3

55.6

9 2.

7E-0

6

TVAS

SSTS

ESIIS

STK

1 87

3.92

73

2 54

.85

8.5E

-06

TV

ASSS

TSES

IISST

K 2

954.

9537

2

40.1

5 0.

0002

5

TVAS

SSTS

ESIIS

STK

3 10

35.9

8 2

25.9

3 0.

0049

Ly

sN

KTTG

CTP

DTA

LATS

FSED

FSSS

S 1

858.

3732

3

47.1

8 1.

9E-0

5 12

87.0

546

2 41

.83

0.00

0066

Chapter 5

120

P283

19

Cw

p1

AspN

D

ATG

VAIR

PTSK

SGSV

AA

1 61

7.32

11

3 69

.19

1.7E

-07

D

DAT

GVA

IRPT

SKSG

SVAA

1

655.

6635

3

67.7

3.

2E-0

7

DG

SFKE

GSE

S 1

602.

7458

2

25.8

8 0.

0026

Ly

sC

DG

SSYI

FSSK

1

626.

7811

2

37.2

6 0.

0002

4

EGSE

SDAA

TGFS

IK

1 78

0.84

91

2 35

.79

0.00

026

LG

SGSG

SFEA

TITD

DG

K 1

902.

4094

2

31.1

1 0.

0008

5

SSSG

FYAI

K 1

561.

2714

2

17.0

4 0.

02

YA

VVN

EDG

SFK

1 69

5.82

15

2 22

.2

0.00

6

QSD

DAT

GVA

IRPT

SK

1

56

9.95

14

3 51

.02

0.00

0017

P382

48

Ecm

33

AspN

D

SIKK

ITG

1

512.

2811

2

17.7

4 0.

017

LysC

LS

STST

ESSK

2

675.

8035

2

33.3

2 0.

0004

9

P4

2835

Eg

t2

AspN

D

SAQ

YAEH

TNLV

AI

1 84

7.39

84

2 41

.02

0.00

014

LysC

LT

EATA

TDK

1 55

6.27

21

2 53

.2

4.8E

-06

TS

LSTE

ESVV

AGYS

TTVG

AAQ

YAQ

HTS

LVPV

STIK

1

1248

.958

6 3

33.1

5 0.

0021

TSLS

TEES

VVAG

YSTT

VGAA

QYA

QH

TSLV

PVST

IK

2 13

02.9

778

3 33

.82

0.00

47

TS

LSTE

ESVV

AGYS

TTVG

AAQ

YAQ

HTS

LVPV

STIK

3

1356

.993

9 3

34.8

1 0.

0041

TSLS

TEES

VVAG

YSTT

VDSA

QYA

EHTN

LVAI

DTL

K 5

1504

.360

5 3

33.3

0.

0035

TSTF

QK

1 43

7.21

36

2 21

.36

0.00

88

P2

2146

G

as1

AspN

D

DFN

NYS

SEIN

KISP

TSAN

TKSY

SATT

S 1

1068

.814

9 3

45.4

6 0.

0001

2

DD

FNN

YSSE

INKI

SPTS

ANTK

SYSA

TTS

2 11

22.8

321

3 32

.17

0.00

27

D

DFN

NYS

SEIN

KISP

TSAN

TKSY

SATT

S 3

1176

.850

1 3

25.9

9 0.

011

LysC

IS

PTSA

NTK

1

540.

7734

2

23.7

8 0.

008

540.

7744

2

54.8

2 6.

1E-0

6

ISPT

SAN

TK

2 62

1.80

08

2 15

.12

0.04

9 62

1.80

06

2 40

.95

0.00

013

IS

PTSA

NTK

3

702.

8272

2

21.6

2 0.

0069

SDC

SFSG

SATL

QTA

TTQ

ASC

SSAL

K 3

1027

.447

3

18.4

8 0.

014

SY

SATT

SDVA

CPA

TGK

1 89

6.40

07

2 34

.68

0.00

036

896.

3995

2

114.

38

3.6E

-12

SY

SATT

SDVA

CPA

TGK

2

97

7.42

67

2 10

7.44

1.

8E-1

1

TLD

DFN

NYS

SEIN

K 1

911.

4039

2

43.7

2 4.

2E-0

5 91

1.40

31

2 10

3.64

4.

3E-1

1

Ly

sN

KISP

TSAN

T 1

540.

7737

2

36.4

8 0.

0004

2 54

0.77

34

2 50

.08

0.00

0018

KISP

TSAN

T 2

621.

8 2

45.6

1 3.

7E-0

5 62

1.79

99

2 46

.86

0.00

0027

KSYS

ATTS

DVA

CPA

TG

1 89

6.39

98

2 63

.09

4.9E

-07

896.

4001

2

60.1

7 1.

1E-0

6

KSYS

ATTS

DVA

CPA

TG

2

97

7.42

4 2

54.4

8 3.

6E-0

6

KYG

LVSI

DG

ND

VKTL

DD

FNN

YSSE

IN

1 10

28.1

483

3 63

.65

1.9E

-06

1028

.148

3 3

63.6

1.

5E-0

6

Q03

655

Gas

3 Ly

sC

ANSL

NEL

DVT

ATTV

AK

1

90

4.95

91

2 10

9.63

2.

1E-1

1

Chapter 5

121

AN

SLN

ELD

VTAT

TVAK

2

985.

9845

2

26.3

8 0.

0051

98

5.98

42

2 66

.53

4.8E

-07

AN

SLN

ELD

VTAT

TVAK

3

1067

.011

8 2

33.3

1 0.

0009

8 10

67.0

113

2 10

0.17

2.

2E-1

0

Ly

sN

KAN

SLN

ELD

VTAT

TVA

3

10

67.0

112

2 34

.74

0.00

077

Q

0516

4 H

pf1

LysC

AS

SAIS

TYSK

1

588.

785

2 25

.24

0.00

37

588.

7851

2

69.4

1.

3E-0

7

ATSL

TTAI

SK

1 57

7.81

11

2 27

.56

0.00

22

577.

8115

2

64.6

9 3.

9E-0

7

ATSL

TTAI

SK

2 65

8.83

73

2 21

.45

0.01

2 65

8.83

69

2 53

.98

5.8E

-06

AT

SLTT

AISK

3

739.

8645

2

15.2

3 0.

048

739.

8634

2

47.2

5 0.

0000

28

AT

SLTT

AISK

4

820.

8894

2

17.6

9 0.

028

820.

8898

2

39.6

8 0.

0001

6

ETSE

TSET

SAAP

K 1

750.

3342

2

34.0

7 0.

0003

9

ETSE

TSET

SAAP

K 2

831.

3591

2

24.5

8 0.

0035

ETSE

TSET

SAAP

K 4

993.

4118

2

16.4

6 0.

023

ET

SETS

ETSA

APK

5 10

74.4

386

2 18

.38

0.01

5

IITSQ

IPEA

TSTV

TATS

ASPK

1

1133

.087

7 2

28.1

9 0.

0036

IITSQ

IPEA

TSTV

TATS

ASPK

2

809.

7456

3

23.1

8 0.

015

IIT

SQIP

EATS

TVTA

TSAS

PK

3 86

3.76

31

3 21

.21

0.03

3

IITSQ

IPEA

TSTV

TATS

ASPK

5

971.

7981

3

36

0.00

13

SY

TTVT

SEG

SK

1 66

1.30

4 2

49.2

7 1.

2E-0

5

SYTT

VTSE

GSK

2

742.

33

2 22

.93

0.00

51

742.

3294

2

50.8

2 8.

3E-0

6

SYTT

VTSE

GSK

3

823.

3564

2

17.6

8 0.

017

823.

3555

2

43.5

4 0.

0000

44

SY

TTVT

SEG

SK

4 90

4.38

34

2 13

.21

0.04

8

TVTS

EAPK

1

497.

7518

2

24.3

5 0.

0073

TVTS

EAPK

ETSE

TSET

SAAP

K 5

987.

7708

3

16.7

3 0.

044

LysN

KA

SSAI

STYS

1

588.

7851

2

37.2

6 0.

0002

3 58

8.78

4 2

47.1

2 0.

0000

23

KE

TSET

SETS

AAP

3 91

2.38

42

2 14

.2

0.03

8

KSYT

TVTS

EGSK

ATSL

TTAI

S 6

1035

.806

5 3

25.7

9 0.

0094

P4

7033

Pr

y3

AspN

D

PTD

NSA

SPTD

NAK

HTS

TYG

SSST

GAS

L 4

1139

.810

1 3

13.4

8 0.

047

D

PTD

NSA

SPTD

NAK

HTS

TYG

SSST

GAS

L 5

1193

.827

6 3

16.6

2 0.

022

D

PTD

NSA

SPTD

NAK

HTS

TYG

SSST

GAS

L 6

1247

.844

7 3

20.4

1 0.

0091

Ly

sC

STTI

NPA

K 1

497.

2585

2

38.1

2 0.

0002

6

Q

1235

5 Ps

t1

LysC

KF

TSG

DIK

1

529.

2738

2

23.9

5 0.

0066

P2

0840

Sa

g1

LysC

N

TGYF

EHTA

LTTS

SVG

LNSF

SETA

VSSQ

GTK

4

1290

.581

3 3

36.7

4 0.

0008

7

TLLS

TSFT

PSVP

TSN

TYIK

1

1110

.068

9 2

40.9

0.

0002

6 11

10.0

67

2 35

.33

0.00

079

Chapter 5

122

TL

LSTS

FTPS

VPTS

NTY

IK

2 11

91.0

949

2 19

.32

0.04

2 11

91.0

942

2 54

.23

0.00

0012

QPS

SPSS

YTSS

PLVS

SLSV

SK

1

11

44.0

619

2 87

.28

5.8E

-09

Q01

589

Sed1

Ly

sC

GTT

TKET

GVT

TK

2 51

6.58

88

3 42

.11

0.00

012

G

TTTK

ETG

VTTK

3

570.

6069

3

21.9

7 0.

01

G

TTTK

ETG

VTTK

4

624.

6242

3

53.1

8 7.

9E-0

6

GTT

TKET

GVT

TK

5 67

8.64

17

3 37

.28

0.00

034

G

TTTK

ETG

VTTK

6

732.

6589

3

27.8

6 0.

0028

SEAP

ESSV

PVTE

SK

1 80

4.87

66

2 30

.51

0.00

089

804.

8768

2

73.2

6 4.

7E-0

8

SEAP

ESSV

PVTE

SK

2 88

5.90

37

2 14

.26

0.03

7 88

5.90

33

2 54

.86

3.3E

-06

SE

APES

SVPV

TESK

GTT

TK

2 75

3.69

2 3

43.4

5 0.

0001

8

SEAP

ESSV

PVTE

SKG

TTTK

3

807.

7089

3

52.4

4 2.

4E-0

5

SEAP

ESSV

PVTE

SKG

TTTK

ETG

VTTK

4

825.

6383

4

45.9

4 0.

0001

3

SEAP

ESSV

PVTE

SKG

TTTK

ETG

VTTK

5

866.

1515

4

29.7

4 0.

0048

SEAP

ESSV

PVTE

SKG

TTTK

ETG

VTTK

6

906.

6654

4

28.9

3 0.

0053

SEAP

ESSV

PVTE

SKG

TTTK

ETG

VTTK

8

987.

6903

4

24.1

2 0.

0066

Ly

sN

KSEA

PESS

VPVT

ES

1 80

4.87

69

2 33

.99

0.00

04

804.

8773

2

49.9

6 0.

0000

1

P276

54

Tip1

As

pN

DAY

TTLF

SEL

1 66

1.30

59

2 40

.83

8.3E

-05

D

TSAA

ETAE

LQAI

IG

1 82

6.39

9 2

28.3

3 0.

0023

DVL

SVYQ

QVM

TYTD

1

912.

4163

2

31.9

1 0.

0007

7

Ly

sC

AASS

SEAT

SSAA

PSSS

AAPS

SSAA

PSSS

AESS

SK

1 10

50.4

615

3 39

.29

0.00

019

AA

SSSE

ATSS

AAPS

SSAA

PSSS

AAPS

SSAE

SSSK

3

1158

.496

3 3

25.5

7 0.

0028

LPW

YTTR

LSSE

IAAA

LASV

SPAS

SEAA

SSSE

AASS

SK

1 12

78.2

898

3 50

.97

9.8E

-05

1278

.289

6 3

77.7

7 1.

7E-0

7

LPW

YTTR

LSSE

IAAA

LASV

SPAS

SEAA

SSSE

AASS

SK

2 99

9.48

33

4 64

.87

4.3E

-06

1332

.306

9 3

86.6

2.

4E-0

8

P108

63

Tir1

Ly

sC

ITSA

APSS

TGAK

1

626.

8174

2

28.5

4 0.

0015

ITSA

APSS

TGAK

2

707.

8436

2

44.6

5 5.

5E-0

5

MLT

MVP

WYS

SRLE

PALK

1M

73

4.03

85

3 29

0.

005

SS

SAAP

SSTE

AK

1 64

2.79

32

2 16

.52

0.02

2

TSAI

SQIT

DG

QIQ

ATK

1 91

2.46

4 2

28.6

9 0.

0037

Ly

sN

KITS

AAPS

STG

A 1

626.

8166

2

42.5

1 8.

4E-0

5

Q07

990

Yl04

2 As

pN

DLN

TALG

QKV

QYT

FL

1 93

6.98

22

2 37

.46

0.00

018

Q05

777

Yl19

4 Ly

sC

EAQ

ESAS

TVVS

TGK

1 77

8.36

97

2 29

.39

0.00

21

EA

QES

ASTV

VSTG

K 2

859.

3971

2

17.8

7 0.

03

Chapter 5

123

PIR

pro

tein

s

(AS

L-C

WP

s)

Q03

178

Pir1

As

pN

DG

QIQ

ATTK

TKAA

AVSQ

IG

1 68

4.02

61

3 68

.86

2E-0

7

DG

QIQ

ATTK

TKAA

AVSQ

IG

2 73

8.04

38

3 78

.81

2.6E

-08

D

GQ

IQAT

TKTK

AAAV

SQIG

3

792.

0615

3

47.4

6 4.

4E-0

5

DG

QIQ

ATTK

TKAA

AVSQ

IG

4 84

6.07

93

3 18

.72

0.03

4

P3

2478

H

sp15

0 As

pN

DG

QIQ

ATTK

TTSA

KTTA

AAVS

QIS

5

1063

.837

6 3

26.2

9 0.

012

D

GQ

IQAT

TKTT

SAKT

TAAA

VSQ

IS

6 11

17.8

549

3 24

.24

0.01

9

DG

QIQ

ATTT

TLAP

KSTA

AAVS

QIG

2

885.

4459

3

64.0

9 9E

-07

D

GQ

IQAT

TTTL

APKS

TAAA

VSQ

IG

3 93

9.46

36

3 29

.29

0.00

31

D

GQ

VQAA

TTTA

SVST

KSTA

AAVS

QIG

2

925.

4494

3

24.1

1 0.

0093

DG

QVQ

AATT

TASV

STKS

TAAA

VSQ

IG

3 97

9.46

8 3

33.2

6 0.

0014

DG

QVQ

ATTK

TTAA

AVSQ

IG

2 72

4.35

74

3 80

.74

4E-0

8

DG

QVQ

ATTK

TTAA

AVSQ

IG

3 77

8.37

4 3

57.4

7 1.

1E-0

5

DG

QVQ

ATTK

TTAA

AVSQ

IG

4 83

2.39

11

3 41

.62

0.00

044

D

GQ

VQAT

TKTT

QAA

SQVS

1

991.

9783

2

30.7

5 0.

0022

DG

QVQ

ATTK

TTQ

AASQ

VS

2 10

73.0

048

2 21

.24

0.02

DG

QVQ

ATTK

TTQ

AASQ

VS

3 76

9.69

3

19.7

3 0.

029

D

GQ

VQAT

TKTT

QAA

SQVS

4

823.

7077

3

25.5

3 0.

0069

DG

QVQ

ATTT

TLAP

KSTA

AAVS

QIG

1

826.

7556

3

43.8

4 7.

4E-0

5

DG

QVQ

ATTT

TLAP

KSTA

AAVS

QIG

3

934.

7915

3

34.6

0.

0007

3

DG

QVQ

ATTT

TLAP

KSTA

AAVS

QIG

4

988.

8091

3

24.4

0.

0085

Ly

sC

STAA

AVSQ

IGD

GQ

IQAT

TK

1 10

05.0

033

2 42

.35

0.00

015

ST

AAAV

SQIG

DG

QIQ

ATTK

3

1167

.057

9 2

26.7

2 0.

0076

STAA

AVSQ

IGD

GQ

IQAT

TK

4 83

2.39

19

3 21

.63

0.02

4

STAA

AVSQ

IGD

GQ

VQAT

TK

1 99

7.99

68

2 22

.36

0.02

2

STAA

AVSQ

IGD

GQ

VQAT

TK

2 71

9.68

65

3 43

.48

0.00

016

ST

AAAV

SQIG

DG

QVQ

ATTK

3

773.

7025

3

62.1

4 1.

8E-0

6

STAA

AVSQ

IGD

GQ

VQAT

TK

4 82

7.71

95

3 54

.25

1.1E

-05

ST

AAAV

SQIG

DG

QVQ

ATTT

TLAP

K 1

826.

7555

3

42.9

3 0.

0001

7

STAA

AVSQ

IGD

GQ

VQAT

TTTL

APK

3 93

4.78

97

3 24

.79

0.01

5

STAA

AVSQ

IGD

GQ

VQAT

TTTL

APK

4 98

8.80

86

3 21

.4

0.03

5

STAA

AVSQ

IGD

GQ

VQAT

TTTL

APK

5 10

42.8

258

3 31

.81

0.00

29

TS

GTL

EMN

LK

1M

636.

3047

2

31.5

2 0.

0007

TTAA

AVSQ

IGD

GQ

IQAT

TKTT

SAK

4 99

9.81

57

3 22

.34

0.03

1

TTAA

AVSQ

IGD

GQ

IQAT

TKTT

SAK

5 10

53.8

333

3 29

.79

0.00

54

TT

AAAV

SQIG

DG

QVQ

ATTK

1

1005

.004

2 2

24.4

7 0.

0091

TTAA

AVSQ

IGD

GQ

VQAT

TK

2 10

86.0

314

2 37

.19

0.00

066

Chapter 5

124

TT

AAAV

SQIG

DG

QVQ

ATTK

3

778.

3724

3

47.0

5 6.

8E-0

5

TTAA

AVSQ

ISD

GQ

IQAT

TTTL

APK

5 10

62.1

732

3 38

.88

0.00

08

TT

AAAV

SQIS

DG

QIQ

ATTT

TLAP

K 6

1116

.188

8 3

27.2

5 0.

012

LysN

KS

TAAA

VSQ

IGD

GQ

VQAT

TTTL

AP

3 93

4.79

07

3 33

.97

0.00

19

KS

TAAA

VSQ

IGD

GQ

VQAT

TTTL

AP

6 10

96.8

432

3 23

.92

0.01

6

KTSG

TLEM

NL

1 62

8.30

78

2 46

.22

0.00

005

628.

3077

2

44.7

9 0.

0000

7

Q

0318

0 Pi

r3

AspN

D

GQ

VQAT

AEVK

1

654.

3193

2

14.3

4 0.

037

LysC

AA

ASQ

ITD

GQ

IQAS

KTTS

GAS

QVS

DG

QVQ

ATAE

VK

3 13

07.2

903

3 44

.9

0.00

017

P470

01

Cis

3 As

pN

DAK

GR

IGSI

VAN

RQ

FQF

1 69

0.36

25

3 14

.64

0.03

4

Ly

sC

ISSS

ASKT

STN

ATSS

SCAT

PSLK

1

817.

0563

3

50.7

2 3.

5E-0

5

ISSS

ASKT

STN

ATSS

SCAT

PSLK

2

871.

0743

3

28.4

5 0.

0066

ISSS

ASKT

STN

ATSS

SCAT

PSLK

3

925.

0918

3

32.8

8 0.

0026

ISSS

ASKT

STN

ATSS

SCAT

PSLK

4

979.

1102

3

29.1

6 0.

0053

97

9.10

95

3 64

.96

1.2E

-06

IS

SSAS

KTST

NAT

SSSC

ATPS

LK

5 10

33.1

272

3 20

.55

0.02

9

NSG

TLEL

TLK

1 61

9.32

96

2 19

.25

0.01

9

TSTN

ATSS

SCAT

PSLK

1

894.

911

2 60

.33

1.5E

-06

TS

TNAT

SSSC

ATPS

LK

2 97

5.93

76

2 31

.49

0.00

12

TS

TNAT

SSSC

ATPS

LK

3 10

56.9

64

2 23

.9

0.00

43

TS

TNAT

SSSC

ATPS

LK

4 11

37.9

908

2 24

.41

0.00

36

No

t assig

ne

d P

IR

seq

ue

nces

AspN

D

GQ

IQAT

TKTT

AAAV

SQIG

2

729.

0282

3

90.2

8 1.

6E-0

9

DG

QIQ

ATTK

TTAA

AVSQ

IG

3 78

3.04

58

3 51

.69

1.7E

-05

D

GQ

IQAT

TKTT

SAKT

TAAA

VSQ

IG

4 99

9.81

57

3 18

.25

0.04

7

DG

QIQ

ATTK

TTSA

KTTA

AAVS

QIG

5

1053

.833

9 3

21.7

9 0.

024

D

GQ

IQAT

TKTT

SAKT

TAAA

VSQ

IG

6 11

07.8

516

3 19

.49

0.04

4

DG

QIQ

ATTK

TTSA

KTTA

AAVS

QIG

7

1161

.867

7 3

21.2

9 0.

033

D

GKG

RIG

SIVA

NR

QFQ

F 1

685.

6916

3

25.8

9 0.

003

D

GQ

IQAT

TKTT

AAAV

SQIG

1

1012

.012

2

50.3

7 1.

5E-0

5

DG

QIQ

ATTK

TTAA

AVSQ

IG

2 72

9.02

82

3 90

.28

1.6E

-09

D

GQ

IQAT

TKTT

AAAV

SQIG

4

837.

0635

3

38.5

2 0.

0004

7

Ly

sC

AAAV

SQIG

DG

QIQ

ATTK

2

661.

6637

3

70.3

4 3.

6E-0

7 99

1.99

04

2 59

.53

0.00

0004

AAAV

SQIG

DG

QIQ

ATTK

TTSA

K 4

932.

4511

3

27.5

7 0.

007

AA

AVSQ

IGD

GQ

IQAT

TKTT

SAK

5 98

6.46

87

3 28

.79

0.00

55

Chapter 5

125

AA

ASQ

ITD

GQ

IQAS

K 1

825.

9122

2

50.0

7 1.

6E-0

5

STAA

AVSQ

ITD

GQ

VQAA

K 1

954.

481

2 54

.26

1.1E

-05

ST

AAAV

SQIT

DG

QVQ

AAK

2 10

35.5

064

2 37

.21

0.00

074

ST

AAAV

SQIT

DG

QVQ

AAK

3 11

16.5

331

2 31

.48

0.00

26

LysN

KS

TAAA

VSQ

ITD

GQ

VQAA

1

954.

4803

2

101.

22

2.5E

-10

KS

TAAA

VSQ

ITD

GQ

VQAA

2

1035

.505

2

99.7

2 4E

-10

No

t assig

ne

d

P3

6135

U

th1

AspN

D

GAV

VIPA

ATTA

TSAA

A 2

905.

9436

2

50.2

2 1.

5E-0

5

Q3E

842

Ym12

2 Ly

sC

ASST

STSA

SASS

SIK

1 76

7.35

95

2 11

5.38

4.

2E-1

2 76

7.35

9 2

80.8

5 1.

2E-0

8

Chapter 5

126

Tab

le S

2.

O-m

an

no

syla

ted

pep

tid

es q

uan

tifi

ed

by S

ILA

C E

TD

- an

d H

CD

-MS

/MS

. The

pep

tides

wer

e pr

oduc

ed b

y a

LysC

dig

est.

The

area

und

er th

e

curv

e (A

UC

) was

det

erm

ined

man

ually

from

a X

IC c

hrom

atog

ram

of t

he c

orre

spon

ding

m/z

. Lig

ht (L

) to

heav

y (H

) rat

ios

wer

e ca

lcul

ated

man

ually

from

the

extra

cted

AU

Cs.

For

eac

h pe

ptid

e, th

e lig

ht a

nd h

eavy

ver

sion

is s

how

n w

ith th

e co

rresp

ondi

ng re

tent

ion

time

(RT)

.

Ac

c.

Pro

tein

S

eq

ue

nce

# M

an

E

TD

H

CD

m/z

z

Exp

ect

RT

[m

in]

AU

C

Rati

o

L/H

m

/z

z

Exp

ect

RT

[m

in]

AU

C

Rati

o

L/H

P323

23

Aga1

SW

VSSM

TTSD

EDFN

K 1

948.

3945

2

2.2E

-06

46.0

4 1E

+07

94

8.39

67

2 N

o M

S2

45.8

1 1E

+07

SWVS

SMTT

SDED

FNK

1 95

2.40

17

2 0.

0002

6 46

.04

1E+0

7 0.9

95

2.40

08

2 0.

024

45.8

1 1E

+07

1.0

O13

547

Ccw

14

APSS

EESS

STYV

SSSK

1

897.

8909

2

1.6E

-05

27.2

1 1E

+07

89

7.89

09

2 4.

7E-1

2 26

.83

2E+0

7

APSS

EESS

STYV

SSSK

1

901.

8982

2

4.6E

-05

27.2

1 1E

+07

1.1

90

1.89

77

2 1.

3E-1

5 26

.83

2E+0

7 1.1

P470

01

Cis

3 IS

SSAS

KTST

NAT

SSSC

ATPS

LK

4 97

9.11

05

3 0.

0073

29

.61

5E+0

6

979.

11

3 3.

7E-0

7 29

.19

7E+0

6

ISSS

ASKT

STN

ATSS

SCAT

PSLK

4

984.

4546

3

No

MS2

29

.53

6E+0

6 0.8

98

4.45

46

3 N

o M

S2

29.1

9 1E

+07

0.7

P533

01

Crh

1 TT

GC

TPD

TALA

TSFS

EDFS

SSSK

1

858.

3748

3

No

MS2

52

.02

5E+0

7

1287

.056

2

1.3E

-08

51.8

2 2E

+07

TTG

CTP

DTA

LATS

FSED

FSSS

SK

1 86

1.04

31

3 3.

2E-0

5 52

.02

5E+0

7 1.0

12

91.0

623

2 5.

1E-0

7 51

.82

2E+0

7 1.0

TTG

CTP

DTA

LATS

FSED

FSSS

SK

2 91

2.39

06

3 2.

2E-0

5 51

.59

2E+0

7

912.

3924

3

No

MS2

51

.36

1E+0

7

TTG

CTP

DTA

LATS

FSED

FSSS

SK

2 91

5.06

16

3 8.

7E-0

7 51

.59

2E+0

7 1.1

91

5.06

2 3

0.01

6 51

.46

1E+0

7 0.9

P428

35

Egt2

LT

EATA

TDK

1

55

6.27

13

2 1.

8E-0

5 23

.54

5E+0

6

LTEA

TATD

K 1

560.

2785

2

4.6E

-05

23.5

4 4E

+06

1.2

LTEA

TATD

K 2

637.

2988

2

No

MS2

20

.69

8E+0

6

637.

2986

2

0.00

016

21.0

6 7E

+06

LTEA

TATD

K 2

641.

3052

2

0.03

1 20

.69

8E+0

6 1.0

64

1.30

47

2 0.

0000

9 21

.06

6E+0

6 1.2

P221

46

Gas

1 IS

PTSA

NTK

1

540.

7746

2

0.00

23

23.1

6 7E

+07

54

0.77

43

2 4E

-06

22.4

9 7E

+07

ISPT

SAN

TK

1 54

4.78

22

2 N

o M

S2

23.1

7 7E

+07

1.1

54

4.78

12

2 8.

8E-0

7 22

.49

7E+0

7 1.1

ISPT

SAN

TK

2

62

1.80

09

2 0.

0000

2 23

.09

1E+0

8

ISPT

SAN

TK

2

62

5.80

77

2 2.

9E-0

5 23

.09

1E+0

8 1.1

ISPT

SAN

TK

3 70

2.82

7 2

0.01

20

.96

1E+0

7

702.

8266

2

3.7E

-05

21.0

6 1E

+07

ISPT

SAN

TK

3 70

6.83

5 2

No

MS2

20

.97

1E+0

7 1.1

70

6.83

37

2 0.

0000

6 21

.06

1E+0

7 1.0

SYSA

TTSD

VAC

PATG

K 1

896.

4004

2

1.2E

-07

32.7

5 3E

+07

89

6.39

99

2 1.

7E-1

0 32

.46

3E+0

7

SYSA

TTSD

VAC

PATG

K 1

900.

4071

2

0.00

097

32.7

5 2E

+07

1.1

90

0.40

67

2 2.

1E-1

1 32

.46

3E+0

7 1.0

SYSA

TTSD

VAC

PATG

K 2

977.

4256

2

0.00

42

31.2

7 2E

+07

97

7.42

66

2 8.

4E-0

8 31

.89

1E+0

7

SYSA

TTSD

VAC

PATG

K 2

981.

4329

2

0.00

043

31.2

7 2E

+07

1.1

98

1.43

35

2 2.

2E-1

1 31

.89

1E+0

7 1.3

SYSA

TTSD

VAC

PATG

K 3

1058

.452

3 2

1.9E

-09

30.4

6 2E

+07

SYSA

TTSD

VAC

PATG

K 3

1062

.459

5 2

2.8E

-07

30.4

6 2E

+07

1.1

TLD

DFN

NYS

SEIN

K 1

911.

4036

2

3.7E

-05

42.3

9 1E

+08

91

1.40

16

2 2.

8E-0

9 42

.08

1E+0

8

Chapter 5

127

TLD

DFN

NYS

SEIN

K 1

915.

4102

2

1.7E

-05

42.3

9 1E

+08

1.1

91

5.40

84

2 3E

-09

42.0

8 1E

+08

1.1

Q03

655

Gas

3 AN

SLN

ELD

VTAT

TVAK

2

985.

9844

2

1.1E

-05

41.8

1 2E

+07

98

5.98

45

2 8.

4E-0

6 41

.5

2E+0

7

ANSL

NEL

DVT

ATTV

AK

2 98

9.99

14

2 1.

7E-0

5 41

.89

2E+0

7 1.0

98

9.99

38

2 N

o M

S2

41.5

2E

+07

1.1

ANSL

NEL

DVT

ATTV

AK

3

10

67.0

111

2 5.

6E-0

6 40

.39

4E+0

7

ANSL

NEL

DVT

ATTV

AK

3

10

71.0

183

2 0.

0000

2 40

.39

3E+0

7 1.1

Q05

164

Hpf

1 AS

SAIS

TYSK

1

588.

7847

2

0.00

24

26.6

3 6E

+06

58

8.78

52

2 0.

025

26.3

8 9E

+06

ASSA

ISTY

SK

1 59

2.79

27

2 N

o M

S2

26.6

3 3E

+06

2.0

59

2.79

27

2 N

o M

S2

26.3

8 5E

+06

2.1

ATSL

TTAI

SK

2 65

8.83

64

2 0.

018

31.1

9 2E

+07

65

8.83

65

2 0.

0004

4 30

.96

3E+0

7

ATSL

TTAI

SK

2 66

2.84

52

2 N

o M

S2

31.2

7 1E

+07

1.5

66

2.84

37

2 0.

0013

30

.96

2E+0

7 1.4

SYTT

VTSE

GSK

4

904.

3823

2

0.03

7 24

3E

+06

SYTT

VTSE

GSK

4

908.

3908

2

No

MS2

24

.09

2E+0

6 1.5

P324

78

Hsp

150

STAA

AVSQ

IGD

GQ

VQAT

TTTL

APK

4 98

8.80

87

3 0.

033

38.5

7 2E

+07

STAA

AVSQ

IGD

GQ

VQAT

TTTL

APK

4 99

1.47

99

3 0.

0085

38

.57

2E+0

7 0.9

PIR

seq

uenc

es

AAAV

SQIG

DG

QIQ

ATTK

TTSA

K 4

932.

4502

3

0.00

84

33.4

7 6E

+06

93

2.45

06

3 0.

0011

33

.28

8E+0

6

AAAV

SQIG

DG

QIQ

ATTK

TTSA

K 4

937.

7954

3

No

MS2

33

.47

8E+0

6 0.8

93

7.79

53

3 N

o M

S2

33.2

8 8E

+06

1.0

AAAV

SQIG

DG

QIQ

ATTK

2

991.

9907

2

0.00

72

35.4

3 2E

+07

99

1.99

24

2 N

o M

S2

35.1

7 2E

+07

AAAV

SQIG

DG

QIQ

ATTK

2

995.

9976

2

0.00

077

35.5

1 2E

+07

0.9

99

5.99

78

2 0.

0003

9 35

.17

2E+0

7 1.0

Q01

589

Sed1

G

TTTK

ETG

VTTK

5

678.

6416

3

0.00

048

20.5

7 4E

+06

GTT

TKET

GVT

TK

5

68

3.98

54

3 N

o M

S2

20.5

7 3E

+06

1.2

GTT

TKET

GVT

TK

6

73

2.66

03

3 N

o M

S2

20.5

7 1E

+07

GTT

TKET

GVT

TK

6

73

8.00

25

3 0.

0048

20

.57

1E+0

7 1.0

SEAP

ESSV

PVTE

SK

1 80

4.87

85

2 N

o M

S2

31.1

1E

+08

80

4.87

67

2 7.

8E-0

8 30

.87

1E+0

8

SEAP

ESSV

PVTE

SK

1 80

8.88

38

2 8.

4E-0

6 31

.1

9E+0

7 1.2

80

8.88

37

2 1.

3E-0

7 30

.87

1E+0

8 1.1

SEAP

ESSV

PVTE

SKG

TTTK

3

807.

7086

3

0.00

27

29.1

9 9E

+06

SEAP

ESSV

PVTE

SKG

TTTK

3

813.

0529

3

No

MS2

29

.19

9E+0

6 1.0

SEAP

ESSV

PVTE

SKG

TTTK

4

861.

7262

3

0.00

25

28.8

7 1E

+07

86

1.72

63

3 0.

0023

28

.52

2E+0

7

SEAP

ESSV

PVTE

SKG

TTTK

4

867.

0703

3

No

MS2

28

.88

2E+0

7 1.0

86

7.06

9 3

0.1

28.5

2 2E

+07

1.0

SEAP

ESSV

PVTE

SK

2 88

5.90

41

2 0.

037

30.3

5 1E

+08

88

5.90

36

2 4E

-07

30.0

5 2E

+08

SEAP

ESSV

PVTE

SK

2 88

9.91

2 2

No

MS2

30

.35

1E+0

8 1.1

88

9.91

05

2 4.

2E-0

7 30

.05

2E+0

8 1.1

SEAP

ESSV

PVTE

SKG

TTTK

5

915.

7435

3

0.04

1 28

.39

1E+0

7

915.

7428

3

0.00

75

28.0

9 2E

+07

SEAP

ESSV

PVTE

SKG

TTTK

5

921.

088

3 N

o M

S2

28.4

7 1E

+07

1.0

92

1.08

71

3 0.

0092

28

.09

2E+0

7 1.1

SEAP

ESSV

PVTE

SKG

TTTK

6

969.

7611

3

0.02

7 27

.31

1E+0

7

SEAP

ESSV

PVTE

SKG

TTTK

6

975.

1057

3

No

MS2

27

.31

8E+0

6 1.2

Chapter 5

128

P276

54

Tip1

LP

WYT

TRLS

SEIA

AALA

SVSP

ASSE

AASS

SEAA

SSSK

1

1278

.288

9 3

2.6E

-08

67.5

4 6E

+06

12

78.2

901

3 6.

6E-0

5 67

.38

8E+0

6

LPW

YTTR

LSSE

IAAA

LASV

SPAS

SEAA

SSSE

AASS

SK

1 12

82.9

677

3 0.

0003

6 67

.47

6E+0

6 1.0

12

82.9

69

3 2.

4E-0

5 67

.38

7E+0

6 1.1

LPW

YTTR

LSSE

IAAA

LASV

SPAS

SEAA

SSSE

AASS

SK

2 99

9.48

29

4 1.

7E-0

6 63

.66

1E+0

8

1332

.307

9 3

0.00

046

63.5

7 8E

+07

LPW

YTTR

LSSE

IAAA

LASV

SPAS

SEAA

SSSE

AASS

SK

2 10

02.9

931

4 N

o M

S2

63.6

6 8E

+07

1.2

13

36.9

881

3 N

o M

S2

63.5

7 8E

+07

1.0

P108

63

Tir1

IT

SAAP

SSTG

AK

1

62

6.81

62

2 1.

2E-0

6 23

.7

1E+0

7

ITSA

APSS

TGAK

1

630.

8235

2

2.2E

-08

23.7

2E

+07

0.7

ITSA

APSS

TGAK

2

707.

8432

2

0.00

094

23.5

8 2E

+07

70

7.84

28

2 1.

9E-0

6 23

.25

2E+0

7

ITSA

APSS

TGAK

2

711.

851

2 N

o M

S2

23.2

5 2E

+07

0.9

71

1.84

97

2 4.

4E-0

7 23

.25

2E+0

7 0.9

ITSA

APSS

TGAK

3

788.

8693

2

0.00

039

20.9

6 2E

+06

ITSA

APSS

TGAK

3

792.

8774

2

No

MS2

20

.97

2E+0

6 1.1

MLT

MVP

WYS

SRLE

PALK

1

728.

7074

3

0.00

68

56.4

8 3E

+07

MLT

MVP

WYS

SRLE

PALK

1

733.

3861

3

No

MS2

56

.48

4E+0

7 0.8

TSAI

SQIT

DG

QIQ

ATK

2 99

3.49

05

2 0.

0007

3 40

.99

8E+0

6

993.

4922

2

No

MS2

40

.7

7E+0

6

TSAI

SQIT

DG

QIQ

ATK

2 99

7.49

73

2 0.

0012

40

.99

8E+0

6 0.9

99

7.49

76

2 2.

3E-0

5 40

.7

9E+0

6 0.7

Q3E

842

Ym12

2 AS

STST

SASA

SSSI

K 1

767.

3604

2

No

MS2

24

.35

6E+0

6

767.

3595

2

7.5E

-08

24.0

2 6E

+06

ASST

STSA

SASS

SIK

1 77

1.36

65

2 2.

2E-0

5 24

.26

5E+0

6 1.1

77

1.36

77

2 N

o M

S2

24.0

2 6E

+06

1.1

ASST

STSA

SASS

SIK

2 84

8.38

51

2 0.

0001

5 23

.75

9E+0

6

848.

3869

2

3E-0

8 23

.54

7E+0

6

ASST

STSA

SASS

SIK

2 85

2.39

27

2 2.

1E-0

6 23

.84

7E+0

6 1.2

85

2.39

28

2 1.

1E-1

0 23

.54

6E+0

6 1.2

ASST

STSA

SASS

SIK

3 92

9.41

21

2 0.

0031

23

.21

1E+0

7

929.

4123

2

3.2E

-09

22.0

2 8E

+06

ASST

STSA

SASS

SIK

3 93

3.42

01

2 0.

0077

23

.22

1E+0

7 1.2

93

3.41

9 2

3.5E

-09

22.0

2 7E

+06

1.1

ASST

STSA

SASS

SIK

4

10

10.4

383

2 2.

5E-0

7 21

.38

8E+0

6

ASST

STSA

SASS

SIK

4

10

14.4

448

2 1.

9E-0

9 21

.38

8E+0

6 1.1

ASST

STSA

SASS

SIK

5

10

91.4

646

2 1.

7E-0

7 21

.06

3E+0

6

ASST

STSA

SASS

SIK

5

10

95.4

733

2 N

o M

S2

21.0

6 3E

+06

1.1

Chapter 5

129

130

Chapter 6

Discussion and

future perspectives

131

The aim of this PhD thesis was to characterize defense strategies of fungi against other

microbes, in particular bacteria, at the peptide and protein level. We focused on cell wall and

secreted antibacterial proteins.

O-mannosylated cell wall proteins of S. cerevisiae

Besides a number of other biological functions, the cell wall of fungi acts as a protective line for

competing microbes. O-mannose (O-Man) glycans are one type of glycosylation found on cell

wall proteins (CWPs) crucial for its integrity. We identified 24 unique O-mannosylated CWPs,

combining a protein specific cell wall extraction workflow with electron transfer dissociation

(ETD) and higher-energy collisional dissociation (HCD) mass spectrometry (MS).

The analysis revealed an enormous heterogeneity of this type of glycosylation, which was so far

not shown for another protein modification. An in-depth description of the complex pattern and

stoichiometry of O-mannosylations is not possible with the currently available technologies.

When restricted to a certain level of complexity, a relative quantitative analysis by implementing

SILAC (stable isotope labeling by amino acids in cell culture) is feasible, as we demonstrated in

our studies. However, different steps of the cell wall extraction workflow should be optimized

further to achieve a higher reproducibility. For example, a pre-treatment of the cell wall with

hydrolytic enzymes (glucanases, chitinases) could release CWPs and preserves O-Man glycans.

ETD has still a very low efficiency in generation of fragment ions in contrast to HCD. Therefore, if

the localization of the mannoses is not of interest, an HCD based approach combined with a

neutral loss scan is as efficient as an ETD fragmentation.

The SILAC-MS workflow is a valuable tool for an analysis of, for example, pmt (protein

mannosyltransferase) mutant strains. A relative quantification of specific O-Man sites can

answer questions about the functionality and regulation of this modification upon intra- and extra-

cellular stimuli. One of the most interesting open questions is the interaction of O-

mannosylations and other glycan modifications. Recently, it could be shown that the mucin

domain of α-dystroglycan is heavily occupied by O-GalNAc and O-Man moieties, both attached

to serine and threonine residues 1. ETD and HCD measurements revealed that specific sites

serve as acceptors for both types of O-glycoconjugates. These findings indicated a certain

competition in site occupancy of O-GalNAc and O-Man glycans in higher organisms. An

interaction between N-glycans and O-mannosylations was previously found on the yeast cell wall

protein 5 (Ccw5/Cis3) 2. In this case, Pmt4 transfers a mannose to a particular threonine side

chain, which prevents the attachment of the N-glycan in a NAT motif. In our studies, we could

confirm that this threonine position and adjacent regions are heavily modified by O-Man glycans.

On Ccw12, we identified O-mannosylations in close proximity to an N-glycan motif (N81), which

was shown to be modified by an N-glycan structure 3. In a targeted MS approach, it would be

Chapter 6

132

feasible to combine the analysis of N-GlcNAc and O-Man residues, as we cleave the N-glycans

in our workflow to a single GlcNAc moiety.

Bacterial-fungal interactions

An appropriate model system is crucial for studying the interactions of bacteria and fungi. We

tested several substrates, such as glass wool, sand, or vermiculite, to grow C. cinerea on and to

extract enough material for the analysis of secreted proteins. Finally, we selected inert

borosilicate glass beads to cultivate the fungus on a solid surface and to extract medium

repetitively. This setup allowed for a co-cultivation with bacteria that were added to the medium

underneath the fungus. We found a strong interaction between C. cinerea and different bacteria,

such as B. subtilis and P. aeruginosa. The bacterial species tested are not necessarily relevant

for the fungus on herbivorous dung. Nevertheless, they represent model systems to study

bacterial-fungal interactions at the molecular level. It would be interesting to extract the

secretome of C. cinerea grown on a substrate resembling better is natural environment. Its

physiology and certainly also its protein expression profile vary strongly when grown on horse

dung instead of artificial medium. For example, the dung could be placed on top of a glass

beads layer, separated by a membrane selectively permeable for low molecular mass

substances. The medium that connects the beads layer and the dung would allow for an easy

extraction of secreted molecules underneath the dung.

Antimicrobial peptides and proteins

The analysis of the secretome and a newly developed purification workflow revealed a set of

antibacterial peptides and proteins secreted by C. cinerea. Two AMPs (CC92 and copsin) and

the lysozyme CC49 were expressed in Pichia pastoris and the antibacterial activity was shown in

a standard disk diffusion assay on B. subtilis. For each of these three proteins, several

homologous proteins were identified in the genome of C. cinerea and one of them, CC82, was

heterologously expressed in P. pastoris.

Discovery strategies for new lead molecules are of great importance and are a key step in

development of medical drugs. Nowadays, high-throughput genome mining is one of the most

prominent ways for the identification of genes or gene clusters that code directly for an antibiotic

or for enzymes catalyzing the synthesis of a secondary metabolite. Our strategy was a

combination of a ‘classical approach’ of fractionation with a quantitative mass spectrometry

measurement, which led to the identification of copsin. It would be certainly possible to perform a

genome wide search for specific cysteine patterns to identify these AMPs. However, a

fractionation and identification at the protein level allowed us to select for criteria such as

temperature stability, solubility, protease sensitivity, and a maximum of activity in vitro.

Chapter 6

133

The wide range of antimicrobial proteins encoded by C. cinerea is not surprising. As fungi lack

an adaptive immune system, they depend strongly on a diversified and highly effective innate

immune system. Especially as a late fruiter on herbivorous dung, C. cinerea interacts and

competes with a heterogeneous group of fungi and bacteria for nutrients and space. C. cinerea

and most other fungi are referred to as saprotrophs. Per definition, saprophytic nutrition is a

process of extracellular digestion and uptake of dead organic matter. This lifestyle requires the

secretion of numerous digestive enzymes, such as proteases, lipidases, and glycosidases.

Indeed, the analysis of the C. cinerea secretome revealed a diversity of these hydrolytic

enzymes. For copsin and very likely also for CC82 and the lysozyme CC49, we found a lytic

effect on bacteria. In the context of its saprophytic lifestyle, an induced lysis of bacterial cells

could provide the fungus with essential metabolites from bacteria. To study the potentially

synergistic effects of digestive enzymes, secondary metabolites, and AMPs, an appropriate

model system is needed that allows for a quantitative measurement of secreted substances. The

beads system together with a quantitative MS-SILAC approach can be a powerful tool to perform

such an analysis. Furthermore, genetic approaches for targeted mutations of genes are

necessary to study the impact of relevant proteins and AMPs on this defense network. The effect

of changed expression levels of AMPs were studied for species of the cnidarian Hydra. It was

shown that AMPs are involved in selection and regulation of a suitable bacterial community 4.

Time and space are two parameters that were not addressed, so far. From an ecological and

energetic point of view, in makes little sense that the fungus secretes all of these antibacterial

substances constitutively over its entire mycelium. It can be expected that particular bacteria and

other microbes trigger the secretion of AMPs and enzymes locally. One example is the bubble

protein, a defensin from the ascomycete Penicillium brevicompactum 5. It is specifically secreted

into exudate bubbles on the fungal surface, where it reaches high local concentrations and acts

antifungal. Even though technically very challenging, microscopy is one strategy to monitor the

secretion of antibacterial proteins in space and time. It was possible to attach a polyhistidine-tag

at the C-terminal end of copsin, which did not dramatically affect the antibacterial activity.

Whether eventually also a fluorescent tag can be added has to be tested for copsin and the

other proteins identified.

A feature common to most defensins is the high density of charged amino acids. The

amphipathic structure of many AMPs is often related to cell membrane perturbation

mechanisms. Even though fungal defensins characterized contain a high number of charged

side chains, it could not be shown that they are essential for antibacterial action by membrane

pore formation. Lipid II was determined as the molecular target of copsin located at the outer

surface of the bacterial membrane. The third position in the pentapeptide side chain of lipid I was

shown to be crucial for binding, independently of whether it is a lysine, DAP (diaminopimelic

acid), or elongated by a pentaglycine interpeptide bridge. According to the acquired data, we

Chapter 6

134

assume that the pyrophosphate moiety and the lipid tail are also involved in binding. As for

plectasin, NMR based binding studies of lipid II and copsin could provide more insights in the

binding pattern. Mechanistically, copsin can block further steps in peptidoglycan synthesis, such

as transglycosylation and transpeptidation and most likely interferes with the membrane integrity.

Furthermore, an accumulation of a polypeptide, such as copsin or plectasin, close to the

bacterial membrane could also induce a fatal stress response, as it was demonstrated for

mammalian peptidoglycan recognition proteins (PGRPs) 6. PGRPs bind to peptidoglycan and

can activate the CssR-CssS two component system in Gram positive bacteria, which triggers

removal of misfolded or aggregated proteins at the extracellular side of the cytoplasmic

membrane. A constitutive induction of this system that also exists in Gram negative species

induces a membrane depolarization and production of toxic radicals, consequently leading to cell

death.

In contrast to target structures, often little is known about the path of reaching these molecules.

The outer membrane of Gram negative bacteria or teichoic acid in the cell wall of Gram positive

species are known to be involved in attracting or repelling AMPs. A more extensive description

of such off-target interactions would be important to explain the often very distinct antibacterial

profiles of defensins, also found for copsin.

Heterologous expression of AMPs and pharmaceutical applications

A profound understanding of the antibacterial mechanism is important for further optimization of

the activity of an AMP. However, other topics dominate in literature of antibiotic drug

development, such as a low solubility, toxicity, or a short half-life intravenously. The biggest issue

in the field of antibiotics is the low profit made on the market. Nowadays, an intravenous

treatment with a commonly applied daily dose of 2 g of vancomycin costs in the range of CHF 90

(http://kompendium.ch). The generally high amounts of antibiotics needed for systemic

treatments require an optimized and high yield manufacturing procedure. This task should be

addressed as early as possible in research and development procedures, to reduce the overall

cost of a project. In a first attempt, copsin was produced in the Pichia pastoris system. A small

scale bioreactor expression yielded 20 mg/l purified and active peptide, a rather low value in

comparison to other fungal proteins expressed in Pichia. Further optimization of the expression

system is necessary and can be achieved at different steps. A multicopy strain optimized for

secreted proteins would be desirable, but is often laborious to construct. In bioreactor cultivation,

different parameters can be adjusted, such as pH, feeding rate, or time of cultivation. For

example, a reduction of temperature and aeration showed unexpectedly a strong increase in

yield for different proteins. Unfortunately, we found hyperglycosylation of copsin, a problem of the

Pichia system that was already discussed for the lysozyme CC49. In one stock, we identified 10-

15% of copsin modified by O-hexoses mainly at the C-terminal end. Such glycan structures can

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lead to a strong antigenic reaction when introduced intravenously and are commonly rapidly

cleared from the blood stream in the liver 7. As we identified these, most likely, mannose

structures only in one sample, it is possible that the hyperglycosylation depends on the culturing

conditions. More straight forward improvements could be achieved in downstream processing.

We estimated that up to 90% of the active peptide is lost during purification. One of the biggest

obstacles is an efficient separation of the massive amount of cells from culture supernatant and

peptide, which was done by a centrifugation step. A cross flow filtration system is an option for a

rapid separation of cells. An integrated downstream workflow could include a sedimentation step

in the bioreactor, followed by cross flow separation of remaining cells and a direct capturing and

purification on a cation exchange column. A longer sedimentation time is acceptable, due to the

high stability of copsin and would allow for an additional wash of the cells. However, other

expression hosts should be tested, such as Aspergillus oryzae, which was successfully used for

the heterologous production of plectasin. Further possibilities are mammalian systems, in

particular, immortalized Chinese Hamster Ovary (CHO) cells. CHO cells are well established for

the expression of protein therapeutics with final product titers of 1-5 g/l, routinely obtained in

fedbatch cultures 8. A critical factor in these higher organisms is the correct processing of the

pro-peptide, since Kex protease recognition sites can vary strongly. All expression systems have

in common that many parameters have to be determined in a matter of trial and error, which is

often very time consuming.

Today, antibiotics in clinical pipeline originate predominantly from secondary metabolites and

synthetic molecules 9. Based on the available knowledge and experience, it is not easy for AMPs

to compete with these conventional antibiotics. Nevertheless, AMPs could provide a new

generation of antibacterial drugs with additional effects on the immune system.

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References

1. Vester-Christensen, M. B. et al. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins. Proc. Natl. Acad. Sci. U. S. A. 1–6 (2013). doi:10.1073/pnas.1313446110

2. Ecker, M. et al. O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein. EMBO Rep. 4, 628–32 (2003).

3. Ragni, E., Sipiczki, M. & Strahl, S. Characterization of Ccw12p , a major key player in cell wall stability of Saccharomyces cerevisiae. Yeast 24, 309–319 (2007).

4. Franzenburg, S. et al. Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc. Natl. Acad. Sci. U. S. A. 110, E3730–8 (2013).

5. Seibold, M., Wolschann, P., Bodevin, S. & Olsen, O. Properties of the bubble protein, a defensin and an abundant component of a fungal exudate. Peptides 32, 1989–95 (2011).

6. Kashyap, D. R. et al. Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems. Nat. Med. 17, 676–83 (2011).

7. Cregg, J. M., Cereghino, J. L., Shi, J. & Higgins, D. R. Recombinant Protein Expression in Pichia pastoris. Mol. Biotechnol. 16, 23–25 (2000).

8. Jayapal, K. P., Wlaschin, K. F. & Hu, W.-S. Recombinant Protein Therapeutics from CHO Cells — 20 Years and Counting. Chem. Eng. Prog. 103, 40–47 (2007).

9. Butler, M. S., Blaskovich, M. A. & Cooper, M. A. Antibiotics in the clinical pipeline in 2013. J.

Antibiot. (Tokyo). 66, 571–91 (2013).

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Curriculum vitae

Andreas Essig

Address: Ausserdorfstrasse 147, 5276 Mettauertal

Nationality: Swiss Date of birth: 10.06.1982

Education

12/2009 - present PhD Thesis, Group Markus Aebi Institute of Microbiology, ETH Zürich 2007 - 2009 Master of Science in Biology, Major in Systems Biology ETH Zürich

2004 - 2007 Bachelor of Science in Biology, Major in Chemistry ETH Zürich 2002 - 2004 Matura, Major in Natural Sciences and Mathematics AKAD College Zürich 1998 - 2002 Graduate Electronics Engineer ABB Switzerland

Work Experience

08/2002–10/2003 ABB Switzerland Electronics engineer, Research and Development Communication Products

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