• Toxins/Infection phases

• Secretion Systems

ADP-ribosyltrans-

ferases(G-protein constantly

active, increased

cAMP/Choleratoxin)

Proteases(inhibition of presyn-

aptic vesicle fusion/

BotulinumtoxinA)

Endotoxin: LPS, LTA

N-Glykosidases(inhibition of protein

synthesis, rRNA-depu-

rination/Shiga-toxin)

Adenylate cyclases(increased cAMP-level/

edema factor

of Anthraxtoxin)

External function

Membrane damaging toxins

Internalized toxins

Exotoxinsspecific secreted proteins

Short Repetition

Genotoxins(arrest of cell cycle

cytolethal distending

toxins)

General mechanism of neuronal Signaling

Short repetition: Botulinum-Toxin: Zn2+ /Metalloproteases

Mechanism of botulinum toxin activity:

Toxin type: zinc metalloprotease

- hydrolysis of integral proteins

of synaptic vesicles

(membrane-docking-complex)

- cleaves synaptobrevin

(=VAMPs), NAP25, syntaxin

- blocks fusion of acetylcholine-

containing synaptic vesicle

with synapses at peripheral

nerve endings

- blocks transmission of

neuronal stimulus at

neuromuscular nerve endings

Result: flaccid paralyses of

muscles (respiratory paralysis)

Short repetition: Botulinum-Toxin: Zn2+ /Metalloproteases

Botulinumtoxin A

• acts at presynaptical

membran activating neurons

• inhibition of acetylcholine

release

Paralysis of muscles

Brock Mikrobiology

Tetanustoxin

• transport to inhibitory

interneurones

• inhibition of Glycin/GABA

release

Non-controled release of

acetylcholin into synaptic

groove leads to spastic

paralysis

Short repetition: Zn2+ /Metalloproteases

Mechanism of toxin activity

• toxin is endocytosed

• released from ER into cytoplasm

• ADP-ribosylation of trimeric

GTP- binding protein Gs

- modified Gs cannot

dephosphorylate GTP

(Inhibition of GTPase activity,

G-protein remains active)

- Gs massifely stimulates host

adenylate cyclase, leading to

constitutive synthesis of cAMP

- unphysiological activation of

cAMP- dependent protein kinase

A induces disease symptoms

protein kinase A

receptor

αs

ß γ

GTP GDP

Gs

Cholera toxin

A-Subunit

Adenylate

cyclase+ATP cAMP

ADP-ribose

NAD+

Nicotinamid

Cholera Toxin

GM1

Activation of chloride channels

block of chloride- and sodium

absorption

Outpouring of intracellular water,

extreme loss of fluids

membrane

Short repetition: ADP-Ribosyltransferases

Expl:

Cholera-Toxin

Corda, Girolamo, 2003, EMBO J.

Consequences of toxin activity

• dysregulation of ion channels

leading to massif loss of chloride and

hydrogene carbonate ions, and water

• In addition: inhibition of Na+/H+-transport

channel leads to loss of sodium ions

• symptoms: dehydration

> loss of up to 25 L water/day

> loss of electrolytes

Normal

Blo

od

stre

am

Lu

me

no

fin

tes

tin

e

Na+

H2O

Na+

+ Cholera Toxin

Na+

Cl-

Na+

Cl-

H2O

Na+

Cl-

K+

K+Cl-Na+ ↑cAMP

Increased activity of

adenylate cyclase

Dia

rrh

ea

Compare to: Pertussistoxin (Bordetella pertussis,

Keuchhusten)

• ribosylation of inhibiting small GTPase (Gi),

inhibition of adenylate cyclase-inhibiting G-protein,

uncontroled muscle contraction, whooping cough

Compare to: Diphteriatoxin fromCorynebacterium

diphteriae, causes severe throat infections,

• ribosylation of EF2, inhibition of protein synthesis

Short repetition: ADP-Ribosyltransferases

Expl:

Cholera-Toxin

Protective Antigen (PA) = B-subunit:

• secreted as single peptide, mediates inter-

nalization of anthrax-toxin, highly immunogenic

• binds to cell surface anthrax-toxin receptor (ATR),

host cell protease cleaves 20 kDa peptide from toxin

• formation of heptameric prepore

• binding of the edema factor and lethal factor to

receptor-PA-complex induces endocytosis

Mourez, 2001, Trends Microbiol.

Anthrax Toxin – a toxin with three players:

•Anthrax toxin: A2B- toxin

- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)

- 1 B: subunit: Protective Antigen (PA)

• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)

PA and EF = Edema Toxin (ETx)

Short repetition: Adenylatecyclases (Anthraxtoxin)

Anthrax Toxin – a toxin with three players

Edema factor (EF)• bacterial adenylatecyclase activity increases level of cAMP

• increase of cAMP activates protein kinase A, induces water influx

•Anthrax toxin: A2B- toxin

- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)

- 1 B: subunit: Protective Antigen (PA)

• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)

PA and EF = Edema Toxin (ETx)

Short repetition: Adenylatecyclases (Anthraxtoxin)

Lethal factor (LF)

• complex of LF with PA generates lethal toxin

• Zn-dependent metallo protease activity cleaves

MAPKK and reduces kinase activity

• Consequence is necrotic cell damage (black)

Short repetition: Adenylatecyclases (Anthraxtoxin)

Anthrax Toxin – a toxin with three players

•Anthrax toxin: A2B- toxin

- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)

- 1 B: subunit: Protective Antigen (PA)

• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)

PA and EF = Edema Toxin (ETx)

ADP ribosyltransferases(transfer of ribosylgroup)

target expl.: Rho,Rac,CDC42,EF2

Bacterial Adenylatcyclases(generation of second messenger cAMP)

Bac.adenylate-cyclase

Proteases(cleavage of proteins)

target expl.: synaptobrevin, syntaxin, SNAP-25, E-Cadherin, desmoglein

Short Repetition

• subset and activity of virulence factors determines virulence and host adaptation

Phases of Infection

Initial phase Establishment Chronification

Host entry

• Vectors

• Wounds

Adapataion

to host,

proliferation

• Metabolism

• Siderophores

• …

Persistence

• spores, antibiotic-

• resistences

Invasive

Dissemination,

Host damage

• Invasins

• Secretion systems

• Toxins

(i.e. proteases)

S. pyogenes invasion in

epithelial cells (M. Rohde, HZI)

Immune defense

• protection against

phagocytosis via capsules

• IgG-cleaving enzymes

• Inhibition of Complement

Phagocytosis of N. meningitidis by human granulocytes

(sciencedirect.com)

Tissue-

adhesion

• Adhesins

• cellprotrusions

(Pili, Fimbria)

Pneumococcus adhesion to

epithelial cells (M. Rohde, HZI)

Phases of Infection

Initial phase Establishment Chronification

• subset and activity of virulence factors determines virulence and host adaptation

Wound-

infections

hivwebstudy.org

tissue-

infections

microbiologyspring2010.wikispaces.com

Organ-

infections

peir.path.uab.edu

Systemic

infections/

Sepsis

beforeitsnews.com

Chronic

infections

emedmd.com

Symptomless

infected

mevis-research.de

Host entry

• Vectors

• Wounds

Adapataion

to host,

proliferation

• Metabolism

• Siderophores

• …

Persistence

• spores, antibiotic-

• resistences

Invasive

Dissemination,

Host damage

• Invasins

• Secretion systems

• Toxins

(i.e. proteases)

S. pyogenes invasion in

epithelial cells (M. Rohde, HZI)

Immune defense

• protection against

phagocytosis via capsules

• IgG-cleaving enzymes

• Inhibition of Complement

Phagocytosis of N. meningitidis by human granulocytes

(sciencedirect.com)

Tissue-

adhesion

• Adhesins

• cellprotrusions

(Pili, Fimbria)

Pneumococcus adhesion to

epithelial cells (M. Rohde, HZI)

On schedule for 15th of july:

• Zipper- and trigger mode of bacterial uptake

• Overview secretion systems

A) Zippering

1. step: tight receptor binding by bacterial adhesins

2. step: cytoplasmic receptor domain induces signaling cascades

3. step: signaling results in cytoskeleton-mediated zippering of host

plasma membrane via filopodia

4. step: Engulfment and uptake of the bacterium

B) Triggering

1. step: bacterial colonization of the cells

2. step: Injection of effectors via type III or type IV secretion systems

(e.g. Salmonella, Shigella) into host cell

3. step: effectors trigger host signalling, i.e. activation of Rho-

GTPases, which leads to reorganization of cytoskeleton,

induction of membrane ruffling via lamellipodia

4. step: Internalization of this bacteria into a vacuole

Trigger- or Zipper ???: EM of invading C. jejuni (yellow arrows), membrane ruffles (red arrow), and filopodia (blue arrow).

From: T. Ó Cróinín & S. Backert, Frontier in Infection and Cellular microbiology

(Filopodien)

• Induced by bacterial adhesion to cell receptors

• Used by Gram-Positives and Gram-Negatives!

α5β1-integrin receptors

ECM

Actin

Bacterium

ECM

Actin

Integrin receptors

Example for Yersinia, adapted from Prof. Dr. Dersch, HZI

Colorized EM of Yersinia invasion

(M. Rohde, HZI)

Expl. for direct mode of integrin-mediated uptake via invasin (Yersinia)

= invasin

Induction of own bacterial uptake

by a receptor-independent mechanism

Membrane ruffling

• Requires an secretion system with injectisome/needle!

• So far exclusively described for Gram-Negatives!

• Gram-Positives use sec, tat, holins, type

IV pili, type VII SS, and outer membrane

vesicles (OMV = type 0)

• but no injection-needle-formation, no

trigger-like uptake mechanisms reported!

• Gram-Negatives might use all currently described

secretion systems (type 0 - type IX)

• either membranes-spanning one-step secretion across

both membranes to outer environment or into host cells

• or two step translocation, first from cytoplasma to

periplasma and second from periplasm to

outer environment

• generate injection needels and

induce trigger-like uptake processes,

membrane pedestal formation and

other cytoskeletal rearrangements

Function in Pathogenesis of Bacterial Infection:

• Export of virulence factors (proteases, adhesins, toxins,…)

• Export of bactericidins for species competition in host environment

• Export of transporter proteins for iron and nutrient aquisition

• Translocation of effector proteins into host cytosplasm for host cytoskeleton rearrangements

• Assembyl of pilus and curli for bacterial adherence

• Assembly of flagella and type IV pili for bacterial motility

• Generation of conjugation pili for interbacterial DNA-transfer

• Export of proteins, carbohydrates for biofilm formation

• Export of antibiotics to enhance

antibiotic tolerance / resistance (OMV)

From: Galan and Collmer, „TypeIII secretion“, Science, 1999

Plus T9SSPlus OMV

Transport of factors only into periplasma

Plus T9SSPlus OMV

Transport of factors into exterieur or host cells, crossing IM and OM

Plus T9SSPlus OMV

Formation of pilus filamtens or curli

Plus T9SSPlus OMV

Transport from periplasam to exterieur, +/- pilus

Plus T9SSPlus OMV

Secretion systems & pili types also present in Gram-Positives!

Plus T9SSPlus OMV

Transport of factors only into periplasma

Plus T IX SSPlus OMV

Transport from periplasam to exterieur, +/- pilus

• Importance in virulence and in cell physiology

•Transport of proteases, lipases, toxins (e.g. ETEC heat labile toxin, Cholera toxin)

• Role in pilus biogenesis

• 2 step process:

- Sec or TAT across IM

- TypeII SS to cross OM

- associated with typIV pilus formationArchitecture of TIISSs

• OM complex

– secretin

– pilotin

• IM complex

– platform proteins

– cytoplasmic ATPase

• SDA protein (secretin dynamic

associated)

– interacts with secretin and IM

components

– may act to transduce energy

• Three subtypes: Va,Vb,Vc

• common features: N-terminal leader sequences for IM transport,

formation of periplasmic intermediates, formation of beta-barrel pore in OM

Type Va: Autotransporters (AT) similar to Vc

(Oca-system)• secretion starts wit Sec-translocation through IM

• secreted proteins induces its own translocation:

- N-terminal signal sequence (passenger domain)

for IM-transport

- C-terminal ß-barrel for pore-formation in OM

• Type 5c requires formation of a coiled Oca-

protein heterotrimer in periplasma for transport

Sectreted proteins: adhesins, degradative enzymes, cytotoxins

- Neisseria gonorrhoeae IgA protease

- Yersinia pseudotuberculosis InvA

Type Vb: Two partner secretion

Type Va: Autotransporters (AT) similar to Vc

(Oca-system)• secretion starts wit Sec-translocation through IM

• secreted proteins induces its own translocation:

- N-terminal signal sequence (passenger domain)

for IM-transport

- C-terminal ß-barrel for pore-formation in OM

• Type 5c requires formation of a coiled Oca-

protein heterotrimer in periplasma for transport

Sectreted proteins: adhesins, degradative enzymes, cytotoxins

- Neisseria gonorrhoeae IgA protease

- Yersinia pseudotuberculosis InvA

Type Vb: Two partner secretion

• Similar to autotransporter but 2 proteins

• secreted protein (TpsA) with TPS-domain at N- terminus

• channel forming β-barrel (TpsB protein) in OM,

• TPS proteins in pathogens:

- Haemophilus influenzae adhesins (HMW1)

- Bordetella pertussis hemagglutinin (FHA)

- Serratia marcescens hemolysin ShlA, ShlB

Plus T9SSPlus OMV

Transport of factors into exterieur or host cells, crossing IM and OM

• Inner membrane complex

– ABC-transporter (with ATP-binding cassette)

and trimeric MFP

• Substrate recognition

– C-terminal signal (not cleaved)

– Proteins remain unfolded during transport

• MFP contacts trimeric outer membrane

proteins (OMP)

– conformational change in MFP

– transient complex

• Energy provided by ATP and PMF

• Proteins secreted by T1SS:

- Pore-forming leukotoxins (E. coli hemolysin)

- proteases, lipases

- Pseudomonas aeruginosa HasAp Haemophore

The most complex secretion system so far

- encoded on pathogenicity islands

(SPI-1 and SPI-2 in S. typhimurium)

- Two different major functions:

A) transport and injections of effectors into host cells

B) exportsystem for assembly of flagellae

• Needle-like channel

- needle length controlled by molecular ruler:

length of protein determines length of needle

(EscP in EPEC/EHEC, YscP in Yersinia)

• Effector translocation

- blocked by gate keeper substrate (SepL in EPEC/EHEC)

- relieved upon low calcium signal (host cell contact)

• Architecture:

- Basal complex with ATPase, Needle &

translocation pore

Plus T9SSPlus OMV

Transport from periplasam to exterieur, +/- pilus

?

Found in Gram-Positives and -Negative

but with different functions:

a) Conjugative TIVSS delivers plasmids &

mobile elements (transposons)

b) DNA uptake and release

c) Effector translocation (only Gram-Negatives);

delivers effector proteins into eukaryotic cell:

Proteins, DNA, Protein-DNA complexes

• ATP hydrolysis drives secretion

• Homologous to Conjugation Machinery of

bacteria and to flagellar system of archaea

• Virulence proteins secreted into eukaryotic cell:

- Bordetella pertussis, pertussis toxin;

- Agrobacterium tumefaciens, T-DNA portion of

T1 plasmid

- Legionella pneumophila, Coxiella burnetii, Dot/Icm system

• Phage-tail-spike-like injectisome

•Evolutionary similarity to components of bacteriophage tails

•Highly analogous to TypeIII and TypeIV SS

•Encoded on pathogenicity islands

(Exampl.: Pseudomonas aeruginosa, Francisella

tularensis, Burkholderia mallei, Vibrio

cholera, Campylobacter,

also Agrobacterium tumefaciens)

•Used by bacteria to attack competing bacteria

within the same niche

•Architecture

- protein complex connects IM & OM

- enery sorce is ATP hydrolysis (ClpV),which also acts

as disassembly protease

- Tail is formed by Hcp tube polymerization, covered by VipA/B dimers

- VgrG formes tip of Hcp tube,

Contact with host cell

triggers contraction of

VipA/B sheath

and Hcp-tube is fired

Ates et al., 2016

• present in Actinobacteria & Firmicutes including

pathogenic M. tuberculosis, M. leprae, C. diphteriae,

Staphylococcus aureus

- specialized system to transport substrates through

cell wall with high lipid content (i.e. mycolic acids layer)

- In Gram- negatives: conserved IM protein complex identified

- energy derives from ATP hydrolysis by EccC

- Transport of substrates with conserved C-terminal signal

sequence

- putative membrane channel consists of EccB-E

- OM pore complex unknown, secretion process barely

understood

- It is unknown whether it is a one- or two-step process

Model for Gram-Negatives

Model for Gram-Positives (Mycobacteria)

Functional aspects of Type 7SS in Mycobacteria

• ESX-1 is used by M. tuberculosis, M. marinum, and M.

leprae in the macrophage infection cycle

• Only ESX-1 expressing mycobacteria translocate from the

phagosome to the cytosol

• bacteria start to replicate and ultimately induce a necrosis-

like cell death

• escape from phagosomes into the cytosol is essential for

full virulence

• phagosomal escape is not observed for M. bovis BCG or

esx-1 mutants of M. tuberculosis and M. marinum

• ESX-1 system is used to lyse erythrocytes and are

supposed to transport proteins with membrane-disrupting

potential

• Gram-negative specific pathway for

secretion and assembly of prepilins for

fimbriae biogenesis, the prototypical curli

• Also called: extracellular nucleation-

precipitation (ENP) pathway

• TVIIISS differs in that fibre-growth occurs

extracellularly

• thin aggregative fimbriae (Tafi) are the

only fimbriae dependent on the TVIIISS

• Tafi were first identified in Salmonella spp

• Associated with adherence and biofilm

formation

Chapman et al., 2002

PseudomonasSalmonella/E.coli

Lasica et al., 2017

• Also known as Por-secretion system (PorSS) or perioGate

• Describes first in 2017 in Porphyromonas gingivalis (cause of aggressive Parodontitis),

also present in Flavobacterium spec. commensals

• Several lipoproteins identified, but ß-barrel-proteins forming a translocation pore in OM still unknown

• Cargo protein with two sorting signals: N-terminal signal peptide (SP) for sec-translocation and

conserved C-terminal somin recognized by type IX SS

• After translocation of periplasmic intermediate by type IX translocon, C-terminal signal sequence is

cleaved off by PorU-sortase

• OMV/MV pinched off from cell surface as membranous nanoparticles

• OMV/MV now considered as true secretion system of Gram-negative & -positive bacteria

• OMV/MV filled with membrane-associated and soluble proteins, nucleotides, etc.

• involved in a series of biological functions, including nutrient acquisition, iron

scavenging, antibiotic resistance and biofilm formation

• Contribute to pathogenesis by delivering virulence factors

- P. aeruginosa MVs enable long-distance delivery of multiple virulence factors including

alkaline phosphatase, hemolytic phospholipase C and Cif, a toxin that inhibits CFTR-mediated

chloride secretion in the airways

• Importance in antibiotic resistance, response to ß-lactam by secretion of OMV filled with ß-lactamases,

increase ß-lactam tolerance of P. aeruginosa, H. influenzae

• bacterial infection is a multi-step event

• non-phagocytic uptake occurs via receptor-binding mediated

„zipper“mechanism or effector-mediated „trigger“-mechanism

integrin receptors often involved

in receptor-mediated „zipper“-

uptake

type III, IV, and VI-secretion

systems involved in „trigger“- type

uptake (only Gram-Negatives)

• bacteria use up to 9 (10) different secretion systems for export of virulence factors

• Sec, tat, holins and type VII-secretion are ubiquitously used for proteins export

• Gram-negative bacteria express injectisomes spanning both membranes

• exocytosis of outer membrane proteins by both Gram positives and Gram –

negatives is defined as type 0 secretion system

• bacteria secrete exotoxins with specific enzymatic activity and host cell tropism

Questions for Repetition

- Describe the principal toxin mechanism of botulinum and tetanus toxin.

- Name the bacteria specis expressing Cholera toxin and describe the toxin mechanism

- What kind of toxic activity is mediated via the two A domains of the anthrax toxin?

- What properties are required if you would like to synthesize a powerful bacterial toxin? Name 5 properties.

- Explain the trigger and the zipper-like bacterial invasion mechanisms.

- Describe 6 biological functions of bacterial secretion systems.

- Which bacterial secretion systems are involved in trigger-like uptake processes?

- Which secretion system has been described for Gram-positive bacteria?

- Describe the principal architecture of a type VI secretion system, where do we find structural analogies?

- Describe the biosynthesis and the nature of type zero secretion system

- Which secretion system is also used for bacterial conjugation and DNA-transfer?

- Which secretion system is used for twichting motility? What is twichting motility?

- Describe the two energy sources which can be used for energizing secretion processes.

- What are key features/differences between sec, tat, and holin-pathway?

Video

https://www.youtube.com/watch?v=nP4

Ou2eoq4c

Tit-for-Tat (Type 6 Secretion fighting):

Video https://youtu.be/OBf64TEo7gA

Salmonella type III secretion system

3:32