SspB CYSTEINE PROTEASE OF Staphylococcw aurem PROMOTES DETACHMENT OF HUMAN KERATINOCYTES AND DEGRADES

FIBRONECTIN AND VITRONECTIN

Isabella Massimi

A thesis submitted in confonnity with the requirements for the degree of Master of Science

Graduate Department of Laboratory Medicine and Pathobiology University of Toronto

O Copyright by Isabella Massimi 2001

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ABSTRACT

SspB cysteine protease of S t a p h y l ~ ~ ~ ~ ~ u s aureuc promotcs detachment of human keratinocytcs and degrades fibronectin and vitronectin

babella Massimi, M.Sc., 2001 Deparmient of Laboratory Medicine and Pathobiology

University of Toronto

SspB cysteine protease of S. aureus is coded for by the sspB gene, the second open

nading h e of the ssp operon in which it is preceded by the sspA gene (V8 protease)

and followed by the sspC gene. The 40 kDa precursor form of SspB (p-SspB) was

expressed alone and in tandem with the sspC gene. In the presence of SspC, p-SspB

showed greater activity by gelatin zymography. This may indicated a chaperone function

for SspC. Purified p-SspB was processed by V8 protease to the mature, 22 kDa m-SspB.

Purified m-SspB was shown to promote keratinocyte detachment. Also, m-SspB was

shown to cleave fibronectin and vitronectin but not BSA, IgG or fibrinogen. These

results may demonstrate that SspB is expressed as a zymogen, that is converted to an

active protease, m-SspB, and that m-SspB may have an important virulence hinction in

S. aureus pathogenesis through proteolysis of host proteins.

- -a

ACKNOWLEDGErnNTS -- - - - - - - -

I would like to extend my gratitude to Dr. Martin J. McGavin for allowing me the

opportunity to pursue graduate studies in his lab and under his guidance. It is only with his expert

leadership, support, patience and encouragement that this work has been successfully

accomplished.

1 would also like to thank the members o f my advisory cornmittee, Dr. Joyce De

Azavedo, Dr. Miles Johnston and Dr. Daniel Sauder, for their direction and encouragement.

l am gratefu t to Dr. Daniel Sauder and the dermatology lab for their extensive

collaboration in this work. Special thanks are extended to Invin Freed for his invaluable

assistance and steady willingness.

To the good friends that I made in S-wing at SWCHSC, 1 thank you for offerinp a Iielping

hand, lending a tistening ear, providing constant support but most o f dl for laughing with me and

making every day a lot more fun. To Kelly, Robert, Greg, Dareyl, Mario, Sue, Invin, Brandon,

Elena, Maria, El len and Shirley, it wouldn't have been the same without you guys, thanks for the

memories. To my friend Rosa Caldarelli, whose optimism and positive encouragement help me

to stay focused. Grazie per la tua amicizia, ti voglio un mondo di bene.

To my loving parents and my brother Gianfranco, thanks for allowing me to discover

who 1 am and k i n g there while 1 continue to do so. Your love and support sustain me.

Gmie per la vita che mi avete creatoe l'amore che mi avete data Senza di voi non sono niente.

Ti amo.

DEDICATION

This thesis is dedicated to my Lord and Savior Jesus Christ, who grants me strength,

wisdom and peace fiom day to day, to my parents Rosa and Emesto Massimi, whose love

and personal sacrifice have made my success possible, and to my cherised brother

Gianfranco, who I leam from consistently and whose faith in me inspires me to cary on.

TABLE OF CONTENTS

II*

S ~ ~ ~ ~ Y I O C O C C U S U U ~ U S . . ~ ~ ~ ~ ~ ~ * ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 1 (i) Identification (ii) History of epidemiology (iii) Emcrgence and developrnent of antibiotic resistance (iv) Virulence strategy of S. aureus

III. Invasion Factors ........... b b b ~ b b b b b ~ . b ~ b b b b m b b b ~ b ~ ~ b . ~ b ~ . . ~ b b b ~ ~ b ~ b b b b b b b ~ ~ b b ~ ~ b ~ b b b b b b b 14 (0 Invasion and dissemination (ii) S. aureus exfolistive toxin (iii) S. aureus a-toxin (Hla) (iv) S. aureus serine (V8) protease (Ssp) (v) Cysteine proteiises of S. aureus (vi) Cysteine protease, SpeB, of Streptococcus pyogenes (vii) Cysteine proteases of Porphorymonas ginigivalis

IV. Regdation of pne expression inS. aunus . ~ . a b ~ a b b b ~ ~ b e b b b ~ ~ b ~ b ~ b b ~ b b b b b ~ b b b ~ 34 (i) Growth phase dependent virulence factor expression (ii) The accessory gene regulatory (agr) system

PART at MATERIALS AND METHODS ........~... .a....a.a...aa..a.....m.o.aao.m 40

1.

II.

m.

W.

v.

VI,

m.

vm.

Ixb

x.

Bacterial Strainsand Growtb C o n d i t i ~ ~ ~ ~ ~ . ~ ~ ~ . . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 42 (i) Bacterial Str;Uns (ii) Standard Growth Media (iii) Stanàanl Growth Conditions

Molecular Biology T e c b d q ~ ~ a ~ ~ ~ ~ ~ a ~ a ~ a a a ~ ~ a ~ ~ a ~ ~ a ~ ~ ~ a ~ ~ a ~ ~ ~ ~ ~ ~ ~ ~ ~ a ~ ~ 45 (i) Isolation of Plasmid DNA (ii) Polymerase Chain Reaction

(ii) a. Construction of Staphylococcal Expression Vector (ii) b. Amplification of sspB and sspB + C

(iii) DNA Manipulations (iii) a. Construction of StaphyIococcal Expression Vector pIML 1 (iii) b. CloningofsspBandsspB + CintopIMLl

(iv) Transformation procedures and Electroporation Conditions (iv) a. Transformation of E. coli (iv)b. TransformationofS. aureus

Protease n i F L n ~ â t i o û ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ 54 (i) Ammonium Sulfate Precipitation of Starionary-phase

Culture Supernatant (ii) Purification of prccursor SspB (iii) Purification of mature SspB

Trntment of Keraümcytes witb ~ B a b a b b b b a b b b m b ~ m b m a b b b a b b a b a ~ b b o a o a a b b 59 (i) Harvcsting of Keratinocytes (ii) Passaging of Keratinocytes (iii) Trcatmcnt of Keratinocytcs with SspB (iv) Cell Viability

p-... ........ Xt; ~ t c a ~ o t P n i ~ S o b s t r r i a s b p S s p B ..............e.........a....... 61 (i) Purifieci Protein substrates (ii) Resorufin-labeled casein

PART III: RESULTS .................................................................... 64

1 . Construction of Stapbyloamal Expression Vector pIML1 ................ 64

tt. Expression of SspB and SspBc ..............................m.......m...... 65

.......................... ïIï . h t e a s e Purification and N-terminai Sequencing 68

V . Activity of SspB on Beaaayl-Ro-Phe-Arg p n i t r o a a i o d c ~ 7 2

........ VI . Mature SspB promotes detachment of primuy huann keratinocytes 74

.................................................................. . VU CeU Viabiiity 78

................................. Vm. Proteolysis of Extracellular Matrix Proteins 80

IX. m-&PB Substrate Sclectivity ............................................... 85

PART IV: DISCUSSION... ............................................................... 88

Im Exprossion of SopB pprreusor.... ............................................. 88

........................ a SspB ~ctivity on Be~z~yl-Ro-Pbe-A~'g pnitroaniiide 89

.................................... iII . Detacbment of Kentlweytes and m&pB 90

I V . Degradation of Fibnnectin a d Vitronecâin by m.SspB ..................... 92

............................................... V . Substrate Sdcetivity of mSspB 97

.............................................. V I . Sumaury and Future Pnispects 99

vii

LIST OF ABBREVIATIONS RP;--Rlkatme Phosphatase

B. burgdofeeri---Bordela burgdoferi

BHI- Brain Heart Infusion

BSA---Bovine Serum Albumin

ddH20--- double-distilled water

dN'iFs---mixture containin al1 four deoxynucleotides (dATP, dGTP, dTïP, dCTP)

E. coli--- Escherichia coli

Fbg-Fibrinogen

Fn---Fibronectin

FnBP---Fibronectin-Binding Protein

H. influenzae---Haemophilus influenza

Ig G--Immunoglobulin G

K-SFM---Keratinoc yte Serum-Free Medium

LB-Luria Bertram

MEM---Eagle's Minimal Essential Medium

m-SspB--the mature 22 kDa form of SspB

MSCRAMMS-Microbial Surface Components Recognizing Adhesive Matrix Molecules

Na2HP&-- Sodium

Na3---Sodium Azide

NaCl---Sodium Chloride

OD-Optical Density

P. @ngivalis---Porphyronronas gingivalis

PBS--Phosphate Buffer Saline

PCR---Polymerase Chain Reaction

RB---reducing buffer

rpm---revolutions per minute

SDS--Sodium Dodecyl Sulfate

S. pyogenes-Srreptococc~rs pyogenes

SSSS-Staphylococcal Scalded Skin Syndrome

TCA---Trichloroacetic acid

Tris---tris@ ydroxy me th y l)aminome thane

UNITS: Da---Dalton O Ca--degree Celsius g---gram g-gravity h - - - h o ~ kV-- ki lovolt L-litre pF-microf faraday

pg--microgram SZ--Ohm pl---microlitre %---percent m1-millilitre S--0second min-minute U-enzyme unit activity M---Molar V---volt mM---rnillirnolar ng-nanogram nm---nanome ter

LIST OF FIGURJ3S . .

.............................. Depiction of Various Strains Used in This Study 44

.................. Construction of Staphylococcal Expression Vector plML1 51

...................... 7.5% SDS-PAGE of 8 h Culture Supernatant Roteins .66

Geiatin Zymography of 8 h Culture Supernatant Proteins ................... 67

SDS-PAGE and Gelatin Zymography of Puri fied SspB ..................... 70

................................. Rocessing of SspB Precursor by V8 Protease 71

Treatment of Primary Human Keratinocyte Ce11 Culture with SspB ...... 75

Gelatin Zymography of Keratinocyte Ce11 Culture Supernatant ............ 77

...................................................... Degradation of Fn by SspB 82

...................................................... Degradation of Vn by SspB 83

Western Blot of Fn fkom Keratinocyte Cell Culture Supernatant ............ 84

................................................. Substrate Selectivity of m-SspB 86

-= -u

LIST OF TABLES . . ...

...................................... 1 . Virulence Factors of S taphylococcus aureus 7

....................................... 2 . Description of Rimers Uses in this Study 48

..................... 3 . Activity of SspB on Benzoyl-Pro-Phe-Arg pnitroanilide 73

............................................................... . 4 Ce11 Viability Results 79

.............................................. 5 . Statistical Analysis of Ce11 Viability 79

.......................... 6 . Lack of Activity of SspB on Resorufin-labeled casein 87

PART 1: INTRODUCTION =- 5-= - . 4 -.

1. Staphylococcus aureus

i ) Identification

S. aureus is a Gram-positive opportunistic pathogen which was first described by

Sir Alexander Ogston in 1880, who by naming the organism, adequately depicted its

appearance (137). The narne "Stayhylococcus aureus" is well suited, inasmuch as it

describes the appearance of the organism with respect to its shape, colour and the

arrangement in which it exists. Staphylococcus comes from staphyle meaning bunch of

grapes and coccus, meaning berry, both terms coming directly from Greek. Thus, the

species designation describes both the spherical shape of the individual cells which are

approximately lpm in diameter, by likening them to berries, as well as the physical

arrangement of these cells into "grape-like" clusters as seen under the light microscope

(122). The term aureus comes directly from Latin and is used to discem the genus of this

organism within the Staphylococcus species by describing its golden colour when grown

on solid media (122). Biochemically, S. aureus is described as a catalnse positive,

manni tol fermenting facultative anaerobe ( 122).

Since its original identification S. aureus has proven to be an important pathogen

and thus its proper identification in the clinical laboratory has become imperative.

Although there are several ways in which S. aureus is isolated arnong other

staphylococcal species in the clinical laboratory, the most commonly used identification

factor in this respect is its ability to produce coagulase. Coagulase is an enzyme that

possesses an important role in evasion of the immune response by inducing fibrin dot

formation at the focal infection site within the host (6). Since al1 strains of S. aureus

appear to harbour a coajplase gene (coa), other memben of the staphylococccd species -=A2 .-- L - 2- - -- - - - -

are referred to as coagulase-negative S. aureus (CONS) (187).

(ii) History of epidemiology

From a histoncal perspective, Staphylococcus aureus has proven to be one of the

most persistent and pemicious of pathogens. It is an important opportunistic human

pathogen and its ability to cause disease, through abscess formation and sepsis, was first

described in 1880 by Sir Alexander Ogston, who dso gave the organism its name (136).

The severity of S. aureus pathogenesis was also documented in 1941 when the mortality

rate of 122 patients with S. aureus bacteremia at the Boston City Hospital was reported to

be 82% (174). S. aureus has endured as a pathogen and today is a leading cause of both

nosocornial and community acquired infections, which have ken on the steady increase

(108). The clinical importance of S. aureus is exemplified by its prevalence in the

hospital setting. S. aureus was narned the leading overall cause of hospital-acquired

infections by the National Nosocorniai Infections Surveillance system for the years 1990-

1992 (4). More specificaily, it has been characterized as the leading cause of hospital-

acquired pneumonia, empyema (infection of the pleural space) and surgical wound

infections, and the second leading cause of hospital-acquired bacteremia (47; 108; 182).

In the comrnuni ty setting, S. aureus is a major cause of skin infections and it is

estimated that it is responsible for approximately 50% of al1 skin infections, including

cellulitis, folliculitis and carbuncles (182). These types of primary infections can lead to

more serious secondary infections such as osteomyelitis, endocarditis, bacteremia or

toxernias, when the mucosal or skin barrier is breached or overcome by the bacteria

(108). This fact is substantiated by the report that 10 to 40 % of d l community-acquùed -FA-- . A- ..* ---. - ..- - - - - - - -

S. aureus bacteremia, which makes up 11 to 38% of comrnunity acquired bacteremia

overaii, progresses to endocarditis (182).

The capacity of S. aureus to cause such a wide spectrum of infections can be

attributed to several factors. Perhaps the most significant is the ability of this organism to

colonize a number of diverse sites within the human host. These include the nares,

perineum, axilla and vagina (1 22; 182). Risk factors for extended S. aureus colonization

include being a hospitalized patient or health care worker (182). Among the population of

healthy adults, approximately 20 % are permanently colonized whereas transient

colonization occurs in about 30 to 50 % of the same population (108; 182). It follows

that an individual who is already colonized with S. aureus is at increased risk for

infection if the individual is immuno-compromised and/or if the skin or mucosal banier

is abrogated due to tissue trauma or surgicai procedures. In general tenns S. aureus is

mainly transmitted by direct person to person contact, which in the hospital setting

mainly occurs through colonized hands of health-care workers (108).

(iii) Emergence and development of antibiotic resistance.

The continuai cycle between antibiotic development and the emergence of S.

aureus strains resistant to these antibiotics has been a major factor contributing to its

persistence and success as an infectious organism. This cycle begm with the

development of penicillin in the 1940s (93). Penicillin is a member of the p-lactam

family of antibiotics and thus has a four-membered p-lactarn ring structure whose target

is a class of bacterial ce11 wall enzymes known as penicillin-binding proteins (PBPs)

- - -

(160). PBPs catalyze the crosslinking transpeptidation reaction that occurs between P A L - - L - - L - - . - >-

peptidoglycan subuni ts in ce11 w al1 synthesis (1 60). S ynthesis of peptidogl ycan,

therefore, is blocked by the binding of -1actams to PBR and this ultimately results in

death of the bactena due to ce11 lysis (160). With the advent of penicillin came the

emergence of hospital-acquired strains harbouring resistance to penicillin. The

mechanism of penicillin resistance involves the inactivation of the antibiotic due to the

production of a serine protease enzyme called p-lactamase, which is capable of

hydrolyzing the p-iactam ring of penicillin (160). The appearance of penicillin resistance

was first observed in the 1950s, and today greater than 90 8 of S. uureus isolates produce

fblactarnase (108;160). To confront the problem of penicillin resistance, methicillin was

implemented as a clinical solution in 1959 for treatment of infections resulting from

penicillin-resistant strains(93). Methicillin is a semi-synthetic p-lactam antibiotic that is

less sensitive to hydrolysis by fl -1actamases (160). Although methicillin appeared to

effectively combat penicillin-resistant strains, methicillin-resistant S. aureus (MRSA)

was first isolated in 1961 and for the next several decades the incidence of MRSA

infection steadily increased (93). The mechanism of methicillin resistance involves the

acquisition of the mecA gene. The mecA gene codes for a penicillin-binding protein,

PBPZa, that has a low affinity for p-lactam antibiotics(l4û). PBP2a replaces the cell

wall synthesis hinction of normal PBPs that have been inactivated by methicillin, and

thus confers resistance to the h g . The mecA gene is found on a segment of the

S. aureus chromosome called "mec", which is approximately 50-60 kb in length (66).

Upstream of the mecA gene are genes important for mec replation, namely mecRI and

mecl (66). There is evidence that the acquisition of methicillin resistance has occurred

through horizontal transmission between strains because although many MRSA strains - - -

appear to have descended from a small number of clones, some appear to have

multiclonal origin (5;95).

The tnatment of MRSA has become a grave problem in the clinical setting due to

the fact that many MRSA isolates have also acquired resistance to a range of antibiotics

such as chloramphenicol, macrolides and tetracycline (145;160). The last resort for

matment of multi-dmg resistant MRSA infections is treatment with vancomycin, a

glycopeptide that blocks ce11 wall synthesis through binding of the terminal d-Ala-d-Ala

of the Mur-Nac-pentapeptide (1 60). It is becoming apparent that the history of S. aureus

antibiotic resistance will likely repeat itself with respect to vancomycin. Many strains of

Enterucoccus faecium, a gram-psi tivc pathogen which is also a nosocornial concem,

have acquired nsistance to vancomycin, (59) and it has also k e n reported that MRSA

isolated from patients that have undergone long-tenn vancomycin treatment show

reduced glycopeptide susceptibility (160). This suggests that the only barrier to MRSA

becoming resistant to al1 antibiotics currently available for clinical use is time, and that

alternative therapies in this regard are required.

(iv) Virulence strategy of S. aureus

S. aureus is unique arnong pathogens in the vast number and variety of infections

which it is capable of causing. Moreover, S. aureus is not equated with other pathogens in

that its ability to cause disease is generally not associated with the elaboration of a

particular toxin (48). Rather, S. aureus disease is characterized by its rapid multiplication

and induction of the infîamrnatory response at the site of infection followed by

dissemination and initiation of metastatic infection (172). Typically, the infection process -- L & - . -- - - - - - - - - - -. -

praceeds in 6 distinct stages including l), colonization of traumatized host tissue; 2).

growth/multiplication resulting in the establishment of a local infection; 3). invasion of

deeper tissues; 4), systemic dissemination andor sepsis; 5), metastatic infection; and 6).

toxinosis (4). The different stages of infection place different phenotypic requirements

on the bacteria and these changing requirements are met by the coordinated expression of

a broad range of virulence factors, which include cell-surface associated factors as well as

secreted factors (108) (Table 1).

Like most microorganisms S. aureus possesses a -phase strategy of infection

which includes colonization and invasion. The expression of virulence factors that are

required for colonization may not be conducive to invasion and vice-versa. Thetefore,

virulence factors are expressed in a growth-phase dependent fashion. To facilitate

specific adhesion and colonization to host tissue and plasma components, S. aureus

possesses a number of cell-associated proteins known as adhesins or colonization factors

which, in vitro, are expressed during the colonization/exponentid phase of growth.

These factors specifically interact with host proteins to promote adhesion and evasion of

the immune response such that the formation of a microcolony results. Once colonization

has occurred, the microcolony increases in cell-density and growth slows as the bacteria

reach the stationary phase of growth. The transition between the exponentiai phase and

the stationary phase is marked by the upregulated expression of secreted virulence factors

Table 1: Virulence factors of Staphylococcus aureus -S. 2 - ----a - -- -

1 Virulence Factor

Factors involved in attachment

Clumping factor

Fibrinogen-binding protein

Fibronectin-binding protein A

Fibronectin-binding protein B

Collagen-binding protein

Coagulase

Polysaccharide/adhesin (PSA)

Polysaccharide intracellular adhesin

Factors involved in evasion of host defenkes

Enterotoxins A B Cl-3 D E H

Toxic shock syndrome t a i n

Protein A

Lipase

V8 protease

Fatty acid modifying enzyme (FAME)

Panton-Valentine leukocidin

-

gene

sea-h

tst

sspA

Leukocidin - - - - R - -

Capsular polysaccharide type 1

Capsular polysaccharide type 5

Capsular polysaccharide type 8

Staphylokinase

Factors involved in invasion/tissue ene et ration

Alpha-toxin

Be ta- toxin

Exfoliative toxins A, B

Garnma- toxin

Delta-toxin

Phospholipase C

Metalloprotease (elastase)

Hyaluronkiase, hyaluronate lyase

IukF-R, hkS-R

cap l locus

cap8locus

sak

Hla

Hl&

eta, etb

U g A , hl@, hlgC

Hld

Plc

SepA

h ysA

such as toxins and enzymes that degrade host tissue which facilitate invasion and

dissemination. Concomitant with the upregulation of expression of secreted factors is the

downregulation of expression of cell-associated factors. This type of regulation is crucial

to the infectious process because if adhesive factors were expressed throughout the

course of infection it would theoretically diminish the ability of the bactena to

invade and disseminate to deeper tissues. Similarly, if toxins and tissue-degrading

enzymes were expressed dunng the initial phases of infection it could promote clearance

of the bactena through activation of host-defence mechanisms. This scenario is

prevented by strict control of virulence factor expression through the action of several

global regulatory pathways (see Part IV). Though much is known about the clinical

significance of S. aureus as a pathogen, with the exception of a few, the specific

functions of virulence factors and their contribution to S. aureus infection and disease

remains poorly understood (108).

II . Colonization Factors

(i)Colonization of host tissue and the extracellular matrk

in order for a pathogenic organism to successfully estabîish an infection within

the human host it must first overcome or penetrate its first line of defense (122). That is,

the skin and mucosa of the body (122). As an opportunistic pathogen, S. aureus takes

advantage of traumatized tissue as an entry portai. Host tissue trauma can be iatrogenic

in origin as is the case in surgical wounds or implanted foreign materials, or it can be the

result of injury. Traumatized tissue is a rich environment of potential colonitation sites

because the extracellular matrix (ECM) becomes exposed (143; 19 1). The ECM is a = - - -

fibrillar network of xnacromolecules within the extracellular space that surrounds

connective tissues and serves as a scaffold or support system for cells (143;191). The

ECM also possesses roles in signalling, cell adhesion, migration proliferation and

differentiation (191). The components of the ECM interact with one another fonning a

complex biologicaily active network of proteins which also interacts with components on

the surface of endothelial or epithelial cells (191). The ECM is composed of collagen,

fibronectin, vitronectin, fibrinogen, laminin and tenascin (191). Cornponents of the ECM

possess several important roles that go far beyond scaffolding, support, and filtration to

include roles that are vital to the host immune response, ce11 signalling and prevention of

abnormal cell proliferation. The integrity and overall structure of the ECM is partly

regulated and maintained by a family of enzymes called the matrix metalloproteases

(MMPs). MMPs are a family of zinc-dependent enzymes that participate in many normal

biological processes (e.g. embryonic development, blastocyst implantation, organ

morphogenesis, nerve growth, ovulation, cenical dilatation, postpartum utenne

involution, endometrial cycling, hair follicle cycling, bone remodeling, wound healing,

angiogenesis, apoptosis, etc.) and pathologicd processes (e.g. arthritis, cancer,

cardiovascular disease, nephritis, neurologicai disease, breakdown of blood brain banier,

periodontal disease, skin ulceration, gastric ulcer, corneal ulceration, liver fi brosis,

emphysema, fibrotic lung disease, etc.) (128). The main function of MMPs is removal of

ECM during tissue resorption and progression of many diseases, but it is notable that

MMPs also alter biological functions of ECM macromolecules by specific proteolytic

activity (SS;69; 146) . Other than a few members that are activated by furin, most are

secreted fmm the ce11 as inactive zymogens (128). Secreted pro-MMPs are activated in --

vitro by proteases and by non-proteolytic factors such as SH-reactive agents, mercdal

compounds, reactive oxygen, and denaturants (128). Activation always requires the

disruption of the cYs4n2+ (cysteine switch) interaction, and the removal of the

propeptide proceeds often in a stepwise manner (127). In vivo, most pro-MMPs are likely

to be activated by tissue or plasma proteinases or opportunistic bacterial proteinases (20).

S. aureus expresses several cell-associated proteins that interact with specific

components of the ECM and in this capacity it is able to take advantage of traumatized

tissue as a site for colonization. Components of the ECM to which S. aureus specifically

adheres include fibronectin, fibnnogen, collagen and elastin and the adhesins that

mediate these interactions usually belong to a class of proteins cailed MSCRAMMS

(microbial surface components recognizing adhesive matrix molecules).

(ii) MSCRAMMS and S. aureus Fibronectin-Binding Protein

There are a few cntena by which cell-surface molecules are classified as

MSCRAMMS. Firstly, the molecule must be located on the ce11 surface (143). Secondly,

it must recognize a macromolecular target within the ECM specifically and bind to that

target with high affinity (143). In the case of S. aureus, MSCRAMMS have an N-

terminal sequence which can range in length from 35 to 40 amino acids and serves to

target the protein, via the Sec-dependent secretion pathway, to the bacterial ce11 surface

(52). Secretion of MSCRAMMS is prevented by a hydrophobie transmembrane domain

and a positively charged tail withh the C-terminal domain which serve to anchor the

protein in the cell-membrane (129). interaction with the bacterial ce11 wall is mediated

by a region which is either made up of serine-aspartate (S-D) dipeptide repeats or nch in - - .-L =-- - - -2- - -

either proline and glycine amino acid residues (52). This ce11 wall-spanning domain is

usually preceded by a conserved Leucine-Proline-X-Threonine-Glycine (LPXTG) motif

whose role is to anchor MSCRAMMS to the ce11 wall(116). The LPXTG motif is

cleaved by an enzyme called sortase, (169) between the threonine and glycine residues

such that the threonine carboxy-terminal is linked, through an amide bond, to the free

amino group within the pentaglycine crossbridge of the ce11 wall dunng the process of

peptidoglycan assembly (129; 168). This process results in transpeptidation of the

adhesion molecule to the peptidoglycan precursor so as to covalently anchor the N-

terminal ligand binding domain to peptidoglycan.

Fibronectin-binding protein (FnBP) is an important colonization factor of S.

aureus that belongs to the MSCRAMM farnily. S. aureus possesses two highly

homologous Fibronectin-binding proteins known as FnBPA and FnBPB encoded by the

fnbA andfnbB genes respectively (50;86). The predicted amino acid sequences of

FnBPA and FnBPB show 45-7596 identity in the N-terminal region and 95% identity in

the C-terminal region (86). The high affinity binding domain of S. aureus to Fn consists

of three tandem motifs named D l , D2, and D3 each of which are approximately 37-38

amino acids in length, and a fourth truncated motif, W (173). Just as classical

MSCRAMMs, both FnBPs of S. aureus 8325-4 contain a N-terminal signal sequence, a

proline-rich cell-wdl spanning domain, a LPXTG motif, a membrane-spanning domain

and a positively charged cytoplasmic tail. Like most colonization factors, both FnBPA

and FnBPB are optimally expressed during the exponential phase of growth and

disappear rapidly from the ce11 surface as growth appmaches the stationary phase (1 18).

Regulation at both the transcriptional and pst-translational level controls the growth- - - 3 - L - - - - - -

phase dependent expression of FnBP. At die transcriptional level, the expression of

FnBP is controlled by the global regulatory locus, agr (166). The transition between

exponential and stationary phase expression of FnBP may also be affected post-

aanslationally by a senne protease secreted by S. aureus known as V8 protease (1 18).

(iii) FnBPs and pathogenicity.

There have been conflicting nsults in the literature with respect to FnBP and its

role in the colonization of traumatized host tissues and thus its contribution to

pathogenesis. Infective endocarditis may occur a k r initial damage to the hem valve

endothelium results in the formation of sterile vegetations compnsed of a complex

mixture of platelets, fibrin, inflammatory cells, Rbnnogen and fibronectin which then

serve as sites for S. aureus adhennce facilitating infection (168). The role of S. aureus

FnBPs in this process remains to be elucidated as there are opposing views in the

literanire. A S. aureus mutant which demonstrates low-level fibronectin binding through

transposon mutagenesis of thefnbA gene showed attenuated virulence in a rat mode1 of

endocarditis (98). in a more general sense, it was reported that organisms that are more

frequently isolated fiom endocarditis cases were significantly better at binding

fibronectin in vitro than other organisms (167). Contrary to these findings, a S. aureus

fkbAfnbB double mutant did not display reduced adherence to traumatized rat heart

valves, compared to the strain h m which it was derived (5 1). The conflicting evidence

reported may serve to illustrate that bacterial adherence is a multifactorial phenornenon

w herein the obliteration of one factor is substantiated by compensatory mechanisms.

-. .--.- ----=- - - Whether Fn-binding promotes the invasiveness of S. aureus is an issue that

- -- - . - - - - - - .- - - - -

remains less explored and understood. It has been demonstrated that the invasiveness of

a S. aureus isolate could be positively comlated with its ability to bind Fn, in that

isolates ftom patients with deep tissue infections expressed four times the amount of Fn

receptors than did commensal or inoculated isolates (159). It was also demonstrated that

patients exposed to S. aureus infection produce elevated levels of antibodies directed

against the Fn-binding D-repeat of FnBPA (23).

III. lnvasion factors

( i ) Invasion and dissemination

lnvasion nfers to the second stage of the infectious process where, after an

organism has colonized the host. it is able to penetrate host tissue by breaking down or

evading host defence mechanisms to establish a successful infection. In extreme cases,

primary infections can lead to secondary infections, and eventually bacteremia, which

promotes dissemination and metastatic infection. The invasive process is facilitated by

the production of secreted virulence factors that act to dismantle components of host

defence mechanisms, degrade tissue and darnage host cells. al1 of which contribute to the

growth and spread of the organism within the host. The production of secreted virulence

factors, by S. aureus including tissue degrading enzymes and toxins, is upregulated

following successful colonization, in response to increasing ce11 density. The secreted

factors of S. aureirr can be lwsely classified into two broad categones. Factors that

allow the bacteria to release itself h m a local abscess or site of infection and invade

deeper tissues include exoproteins, lipases? nuclease, hyaluronate lyase, and

staphylokinase (4). Others, which contribute to sepsis and toxinoses, include -a- - - - -

a-, 8-, 6, y- hemolysins (4). As previously mentioned, the production of secreted

factors is upregulated in vitro as cells approach the stationary phase of growth, which is

controlled by a gene regulatory locus to be discussed in Part V. There are several

reasons why it may be beneficial to the bactena to restrict the expression of secreted

factors pcimarily to the stationary phase of growth. Probably foremost of these, early

production of such factors could increase the probability that the host immune response is

activated before a microcolony has been established. This review of secreted virulence

factors will focus on exfoliative toxin, a-toxin, a major lethal toxin produced by S.

aureus, as well as staphylococcal proteases.

(ii) S. aureus exfoutive toxin

Staphylococcal Scaided Skin Syndrome (SSSS) is a blistering skin disease

primarily in neonates and sometimes in adults of which exfoliative toxin is the causative

agent. The presence of a soluble toxin in the pathogenesis of SSSS was the subject of

speculation as far back as the 1950s (99). However, the first clue of its existence came

only after Melish and Glasgow showed that sterile fluid obtained from intact bullae and

from phage group II (120). S. aureus culture medium was able to produce a positive

Nikolsky sign in neonatal mice (120). Even intraperitoned inoculation resulted in

exfoliation, indicating that the toxin had its specific target in the slon (120). Within a

year, the active component in the supernatant was isolated, pwified, characterized, tenned

exfoliatin (ET) (90). Soon thereafter, it was realized that at least two different ET

serotypes existed (99). The second semtype was isolated and characterized as a heat-

labile toxin that has a similar molecular weight to the original toxin (94). This second ET -.- - - - - A - -:-Sc -

serotype was able to elicit a positive Nikolsky sign in the neonatal-mouse bioassay, and

was designated exfoliative toxin B (ETB) (94). These two serologically distinct toxins,

ETA and ETB, are able to cause disease in humans and are produced by a significant

proportion of S. aureur strains (99). Although they differ in some physicochemical

properties, both toxins produce the dermatological effects described above in neonatal

mice (1 1;90). ETA and ETB consist of 242 and 246 arnino acids, respectively, with

molecular masses of 26.9 and 27.3 kDa (10). The genes for ETA and ETB have ken

identified and fully sequenced and possess 40% homology (9; 105; 135). The gene for

ETA is chromosomal, while that for ETB is plasmid located (99). Both genes have been

fully sequenced and expressed in Escherichia coli (78;165). ETA is heat stable and

retains its exfoliative activityeven after being heated at 60' C for 30 min (73;90). Initial

work with EDTA, a metal ion chelator, suggested that ETA but not ETB required a metal

ion for activity (76). Cornparison of the primary amino acid sequences revealed that

amino acid residues 93 to 107 of ETA were significantly homologous to a region of rat

intestinal and placental calcium-binding protein, and supported the evidence that ETA but

not ETB may require calcium for activity (34). On the other hand, calcium-binding motifs

have not k e n identified in the two recently detemined crystallographic structures of

ETA (24; 188). Takiuchi et al. were the first group to provide evidence that ETs may act

as proteases (18 1). Demonstrating that incubation of ET with epidennis from 1 to 3-day

old mice activated a latent caseinolytic activity which could be inhibited by a*-

macroglobulin (18 1). An important step forward foilowed after both ETs were shown to

have significant amino acid sequence homology (25%) to the staphylococcal V8 protease,

particularly in the region containing the protease active site(34). V8 protease is a member - - - -

of the trypsin-like senne protease family and cleaves on the carboxyl-terminal side of

acidic amino acid residues (43). The active site consists of a catalytic triad of senne.

histidine, and aspartic acid which are present in al1 known eukaryotic trypsin-like serine

proteases (43). The high degree of sequence conservation around these regions was

speculated to preserve the active site of possible protease activity of the toxins (34).

The majority of the work done on ETs has been done with ETA and the neonatai-

mouse model. The toxins are made during the stationary phase of bacterial growth, and

absorbed into the systemic circulation (1 1;99). The toxins eventually reach the zona

granulosa of the epidemis by diffising through dermal capillaries(99). Histological

studies have shown that addition of ETs to confluent keratinocyte cultures results in the

disappearance of small vesicles that are normally present between the cells. This is

followed by formation of intercellular fluid-filled spaces in the granulosa-spinosum

interface, which eventually leads to the characteristic midepidermal splitting seen in

SSSS (99; 106). In addition, the biological actions of ETA and ETE3 are not histologically

distinguishable (54). Recently, immunoperoxidase staining on histological specimens by

Gentilhomme et al. revealed that cells that bordered both the upper and lower edges of the

cleavage site caused by the ET were positive for keratohyalin granules (54). Electron

microscopy of femtin-labelled toxin and light microscopy of fluorescein thiocarbamyl

toxin were previously used to show that the toxin bound intensely to keratohyalin

granules in the straturn granulosum of the epidemis and also that ETB had a particularly

high affinity toward profilaggrin (360 kDa) and filaggrin (30 kDa) in epidemai extracts

(175; 176). Profilaggrin is synthesized during the pathway of keratinocpe development

and incorporated into keratohyalin granules, where it is hydrolyzed to intemediate - - - - -

filaggrins and then to amino acids in the seatum comeum (1 1). The authors speculated

that ET binding to these intracellular granular proteins may lead to disruption of

intercellular cohesion, because the keratohyalin granules are linked to the keratin

intermediate filaments, which extend to desmosomes at the ce11 surface (12 1). These

observations fitted well with previous electron micrographic observations suggesting that

desmosomes, which link adjacent cells, were dismpted (121). However, several

histological studies have demonstrated that even though intercellular edema can be

demonstrated, the desmosomes are initially intact and desmosomal disruption is a

secondary event, occurring only as the edema progresses (46;67). However, a very recent

study has indicated that desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-

ce11 adhesion, may be the target of exfoliative toxin A (3). Desmoglein 1 is the target of

autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue

specificity and histology to SSSS and bullous impetigo (3). Amagai et al show that

exfoliative toxin A cleaved mouse and human Dsgl, but not closely related cadhenns

such as Dsg3 (3). It was demonstrated that this specific cleavage occurred in ce11 culture,

in neonatal mouse skin and with recombinant Dsgl. It was therefore concluded that

Dsgl is the target for cleavage by exfoliative toxin A (3). This unique proteolytic attack

on the desmosome causes a blister just below the stratum comeum, which foms the

epidermal banier, presumably allowing the bacteria in bullous impetigo to proliferate and

spread beneath this barrier (3).

(iii) S. aureus ar-toxin (Hla) --- -- -- a --- - - - - - -

S. aureus a-toxin is one of the most potent bacterial toxins known to date (17). It

acts through its ability to lyse host cells by foming pores within the ce11 membrane.

Alpha-toxin is a 33 kDa protein that is secreted as a monomer following cleavage of a 26

amino acid signal sequence. In vivo, a-toxin has been shown to be an important virulence

factor, as mutants defective in a-toxin production exhibit significantly less virulence than

the wild-type strains from which they are derived (53). The mechanism by which a-toxin

acts begins with the binding of the rnonomer to the ce11 membrane. Upon binding,

oligomerization of seven a-toxin monomers results to fonn a cylindrical ring-shaped

pore. which essentially abolishes the selectively permeable property of the ce11

membrane. This in tum promotes the influxlefflux of ions and small molecules resulting

in loss of osmoregulation and ultimately ce11 death (lî;84; 185; 186). The cytotoxic,

neurotoxic and dermonecrotic effects of a-toxin are exerted on monocytes, lymphocytes,

macrophages and epitheliai cells (15;38). In addition to the death of the affected cell,

lysis of activated macrophages and platelets by a-toxin results in the release of their pro-

inflammatory cytokines and procoagulatoy compounds, respectively (17). These events

are thought tu conhibute to the systemic effects of a-toxin on the cPrdiovascular system

and lungs (17).

(iii) S. aureus serine (V8) protease (Ssp)

Serine (V8) protease was first purifed from S. aureus strain V8 and described by

Drapeau et al (44). The activity of V8 protease was inhibited by diisopropyl

fluorophosphate (DFP) which is a specific senne protease inhibitor. However, protease

20

activity was not sensitive to chelating agents such as EDTA. V8 protease was therefore --- .--- . - - A- - - a - - - -

classified it as a member of the senne protease family (39;44). Continuing work

demonstnited that the substrate specificity of V8 protease was quite limited, in that

cleavage occurred preferentially at the carboxy-terminal side of glutamic acid (40;44).

Sequence analysis of V8 protease revealed the catalytic triad His, Asp, Ser, that is a

conservai sequence among mammalian senne endopeptidases. However, other than the

presence of this motif, V8 protease showed no other regions of high sequence homology

to other serine proteases (42). Therefore, V8 protease has been classified as a member of

the glutamyl endopeptidase family of enzymes (70).

Cloning and sequencing of V8 protease (ssp) h m both S. aureus strain V8 and

ATCC 12600 revealed that V8 protease is translated as a preproenzyme containing a N-

terminai signai peptide of 27-29 amino acids required for secretion, followed by a

propeptide region of 39 amino acids in length (21 ;l94). There is evidence to suggest that

V8 protease is secreted as an inactive precursor that is subsequently pmcessed, to form a

mature protease, by S. aureus metalloprotease. Specifically, the inactive precursor form

of V8 protease was shown to accumulate in a S. aureus mutant strain defective in

metalloprotease (4 1).

The contribution of V8 protease to S. aureus pathogenesis is an area that has

sustained littie progress. However, a recent study using signature tagged mutagenesis

(STM) suggested that V8 protease contributes to in vivo p w t h and survival of S. aureus

(32). Specifically, two V8 protease (ssp) gene mutants showed attenuated growth in a

systemic intravenous model, and in abscess and buni wound models of infection.

Furthemore, a study by McGavin et ai indicated that V8 protease may possess a mle in

the pnmssing of cell-surface FnBP and so act to promote the progression from - - - -

colonization to invasion ( 1 18). In this study, exogenous V8 protease was added to

exponential-phase culture of S. aureus, and caused the rapid elimination of FnBP from

the bacterial ce11 surface ( 1 18). When the same expriment was done with the addition of

a general protease inhibitor found in human plasma, a2-macroglobulin (MM), the

growth-phase dependent loss of FnBP was slowed dramatically (1 18). This result

combined with the fact that V8 protease is maximally expressed during the stationary

phase of growth, lends to the ides that V8 protease activity is important for the transition

h m coionization to invasion.

Rice et al presented the first application of molecular techniques towards

providing a detailed understanding of the hinctions of a secreted proease of S. aureus

(164). Through sequence analysis of the S. aureus strain COL genome

(htt~:llwww.tier.or@, it was found that the ssp structwal gene (sspA) is followed closely

by an open reading frame encoding a cysteine protease, designated sspl. The sspA and

sspB proteases are transcribed as an operon, which also includes a third open reading

frame sspC, of unknown function. Analysis of the nucleotide sequence downstrearn of

ssp revealed two additional open reading frames sspB and sspC. The senne protease gene

designated sspA is followed by sspB, which encodes a hypothetical protein of 393 arnino

acids. A signai peptide cleavage si te was predicted after A1a36, creating a mature protein

of 40.6 kDa. Arnino acids 220 to 393 of SspB possess 47% identity and 64% similarity to

staphopain, a 23-kDa cysteine protease purified h m S. aureus strain V8. A eukaryotic

thiol protease histidine active site consensus pattern (LGHALAWGNA), spanning

amino acids 338 to 348. which is also conserveâ in staphopain. Downstream of sspB is a

thud open reading frame, sspC, which encodes a 109-amino-acid protein with a predicted . %d - - - -- - -

size of 12.9 D a . A cytoplasmic localization was predicted for SspC. SspC did not

possess significant homology to other known proteiris. Following sspC is a hairpin

structure, representing a possible transcription temination signal. Through construction

of a nonpolar allelic replacement mutation, inactivation of sspA was achieved without

affecting transcription of sspB or sspC. This resulted in the creation of S. aureus strain

SP639 1. When analyzed in a tissue abscess mode1 of infection, the sspA mutant, SP639 1 ,

exhibited enhanced virulence relative to the wiltype (164). These results suggest that V8

protease does not contribute to tissue abscess infections in its own right.

The culture supematant of S. aureus SM391 (sspA::ennAB) possessed a new 40

kDa protein that was not present in RN6390. Furthemore, in cornparison to RN6390,

culture supematant of SP6391, exhibited a loss of at least two minor protein bands at 22

to 23 kDa (164). The new 40 kDa protein in SP6391 corresponds to the expected size of

SspB after processing at a predicted signal peptidase cleavage site, and N-terminal

sequencing of this protein yielded the sequence DSHSKQLEINV, which is the exact

sequence of the predicted N terminus of secreted SspB. Therefore, inactivation of sspA

results in accumulation of the 40 kDa SspB protein in the culture supematant of SP6391

(164). Transduction of the inactivated sspA allele (sspA::ennAB) of SP6391 into the agr-

nul1 strain S. aureus RN691 1, created strain SP6912 (agr::tetM sspA::ennAB). A protein

of the same size was aiso present in the culture supematant of SP6912 (agr::tetM

sspA::ennAB) and had the same N-terminal sequence DSHSKQLEïN, confllming its

identity as SspB (164). This protein was not expressed in the agr-nul1 parent strain

RN691 1. Therefore, inactivation of sspA results in accumulation of the 40-kDa SspB

protein in the culture supematant of SP6391, and transduction of the mutant allele into the - - - LA - - -

agr-nul1 stmin S. aureus RN691 1 pennits this protein to be expressed and secreted

independently of agr function by transcription from the ennAB promoter (164).

Zymogram analyses were conducted to examine the effwt of sspA inactivation on

the profile of secreted proteases. Although the 40-kDa SspB protein was present on1 y in

the culhm supematant of SP6391 and SP6912, no new zones of protease activity were

detected by casein zymography. However, in zymograrns containing gelatin. SP6391 and

SP6912 each exhibited a protease activity of a pa te r mass that was not present in either

RN6390 or RN69 1 1. Furthemore, a low-molecular-mass gelatinase of RN6390 was

absent fiom culture supematant of SP6391 (164). When the samples were pretreated with

E-64, a specific inhibitor of cysteine protease activity, the lower-mass gelatinase of

RN6390 and the higher-mass activity in SP6391 and RN6912 were no longer detected

( 164). Therefore, inactivation of sspA resulted in the loss of a low-molecular-mass

cysteine protease and the appearance of a higher-molecular-mass protease. representing

the 40-kDa SspB protein. These observations suggest that the SspA serine protease is

required for proteolytic maturation of the 40-kDa fonn of SspB. Accordingly, when

culture supematant h m SP6912 (agr sspA::ennAB) was treated with purified SspA prior

to zymogram analysis, the precursor form of SspB was processed to fonn lower-

molecular-mass gelatinase activities (164). Furthemore, inactivation of sspA resulted in

loss of two proteins of 22 to 23 kDa, and appearance of the 40 kDa SspB protein in

culture supematant of SP639 1. These observations wen also reflected in the profile of

secreted gelatinase activities. Cumulatively, these data indicate that SspB is expressed as

a 40 kDa precursor protein, which is processecl by SspA to form a mature cysteine -,na- -..--- - - - - - - - - - - - . - - - - - - - - -

protease of 22 to 23 kDa.

( iv) Cysteine proîeases of S. aureus

Before these findings, the 40.6 kDa SspB protein, as part of the ssp operon or

otherwise, had not been previously reported. However, two other cysteine proteases that

are also produced by S. aureus have been previously descnbed. Arvidson et al purified

and characterized a protease from S. aureus strain V8. refemd to as protease II(7).

Rotease II had a molecular mass of approximately 12.5 kDa, was optimally active at pH

8.8 and had a pI of 9.4 (7). The protein was only active in the presence of reducing

agents such as cysteine or 2-rnercaptoethanol(7). The samç enzyme was subsequently

punfied by Potempa et al. from the same strain, who found that the enzyme displayed

restricted substrate specificity but did exhibit elastinolytic activity under physiological

conditions equal to that of neutrophil elastase (150). This may indicate a specific role in

virulence for this pmtease through the damage of connective tissue. However, it remains

to be seen whether this protease is common to most S. aureus strains, as neither the gene

nor the amino acid sequence of this protease has been identified. The second cysteine

protease that has been discussed in the literature is a 23 kDa protein named staphopain

(68). Staphopain was also f î t punfied from S. aureus strain V8 (68). The arnino acid

sequence of staphopain revealed that it contains a eukaryotic thiol protease histidine

active site consensus motif (AGHAMAWGNA), and m e r sequence andysis through

the University of Oklahoma S. aureus genome project revealed that this enzyme is most

likely secreted as a preproenzyme (6;68). The hinction of staphopain with respect to its

d e in virulence and contribution to S. aureus pathogenesis has not been established. -

However, two thiol proteases, both of which had an estimated molecular weight of 23

kDa and an amino acid sequence that was very homologous to staphopain, have been

implicated in contributing to the pathogenesis of avian dermatitis (180).

The role of cysteine proteases in S. aureus disease is an area of interest that remains

poorly understood. Conversely, cysteine proteases in other microorganisms, namely

Streptococcus pyogenes and Porphorymonas g ing ivalis, have ken extensively

characterized and established as important virulence factors.

( v ) Cysteine protease, SpeB, of Streptococcus pyogenes.

Streptococcus pyogenes is a Grampositive bacterium that causes an array of

infections in the human host. These infections range in seriousness ftom less severe

infections like tonsilitis and skin infections, to grave life-threatening bacteremia and

necrotizing fasciitis/myositis. S. pyogenes produces a wide variety of virulence factors

that include cell-surface colonization factors and secreted invasion factors. Among the

plethora of secreted virulence factors, is streptococcal pyrogenic exotoxin B (SpeB).

SpeB is a cysteine protease that is secreted as an inactive 40 kDa zymogen that undergoes

autocatalytic conversion to a proteolyticaily active 28 kDa protease (58). Despite the fact

that almost al1 strains possess the speB gene on the chromosome, interestingly only 59%

of al1 S. pyogenes strains secrete a noticable arnount of SpeB (1 17). There are severai

reports that contribute to the understanding of the regulation of expression of SpeB and

may give insight into the selective expression of SpeB arnong strains of S. pyogenes. A

gene immediately upstream of the speB gene encodes what is known as the Rgg protein

(25). Rgg has been implicated in the positive regulation of SpeB production in that a rgg

mutant displayed a substantiai reduction in the amount of secreted SpeB (25) . in

addition, speB is among the group of virulence genes, which includes the genes that

encode M- and M-like protein, CSa peptidase and serum opacity factor (Sof), al1 of which

are positively regulated by the multiple gene activator (Mga) regulon (148).

Environmental signals may also play a role in the regulation of SpeB production. as speB

gene expression is responsive to the relative amounts of carbohydrates and peptides in the

growth medium (147). in addition, a recent report suggests that the S. pyogenes M-

protein is necessary for the proper folding of the 40 kDa SpeB zymogen. and subsequent

autocatalytic generation of the mature enzyme (31). M-protein of S. pyogenes is a cell-

surface protein, of which there are 80 serotypes, that harbours anti-phagocytic properties

and is expressed maximally in the exponential phase of growth.

There has been much progress made in the understanding of the contribution of

SpeB to S. pyogenes infection. In its active form, SpeB has ken found to cleave several

ECM components including fibronectin and vi tronectin (92) . Also, SpeB was reported

to have a cytopathic effect on human umbilical vein endothelial cells (HUVECs) and this

effect was correlated with the activation of a 66 kDa matrix metalloprotease (MMP-2)

which is responsible for type IV collagenase activity (18). Given that SpeB is expressed

optimaiiy during the stationary phase of growth, (27) and that it possesses the above

proteolytic properties, it cm be infemd that SpeB activity contributes to the tissue

destruction often associated with more serious S. pyogenes infections, and as such cm be

classified as an important vinilence factor. in contrast, it has been proposed that SpeB

acts to facilitate the acquisition of nuaients required during the stationary phase of

27 -

-a*--- - -- growth. Nonetheless, it has been shown that speB mutants of different GAS serotypes

- - - -

displayed no change in growth characteristics in different growth media, which suggests

that SpeB is not directly implicated in the acquisition of nutrients (147). Moreover,

several studies indicate a possible role for SpeB in the host immune response.

Specifically, it has been reponed that SpeB proteolyticaly converts interleukin-1-B (IL-

le) precursor into the mature pro-inflarnmatory IL1 f! cytokine, and also cleaves plasma

H-kininogen to release pro-inflammatory kinins (62;9 1). Furthemore, SpeB may be

implicated in preventing the migration of activated macrophages to the site of infection

by cleaving monocyte urokinase receptor ( 192).

The role of SpeB in the pathogenesis of S. pyogenes disease has ken explored

through the genetic inactivation of the speB gene. In a murine intraperitoneal infection

model, Spe-B deficient mutants have been reported to exhibit increased resistance to

phagocytosis and decreased dissemination to organs wi th respect to the w ildtype strain

(109). The same mutant, when injected subcutaneously in a murine tissue abscess model,

caused significantly smaller abscesses when compared to the wildtype strain that created

larger, necrotic lesions and sometimes disseminated to regions far from the injection site

(1 10). Conversely, another group studying a speB mutant found that speB mutants

showed little or no difference in their ability to cause tissue damage and invasive

infection (8). In addition, it has been shown that SpeB is made in vivo during an invasive

infection, as patients with severe invasive GAS infections have been shown to

seroconvert to SpeB (58). At the cellular level, inactivation of the speB gene in serotypes

M2 and M3 createâ mutants which were intemalized by human endothelial celis and

human fibroblasts to a greater degree than their wildtype counterparts (19). Also, a

SpeB-deficient mutant induced significantly less apoptosis in a human monocyte ce11 line A-=- - - - A - --

when compared to the wildtype from which it was derived (96). This apoptotic effect

was shown to require the activity of SpeB since treatment with the cysteine protease

inhibitor E-64 , or heat inactivation abolished the apoptotic effect (96). in dl, these

studies demonstrate that SpeB contributes to the extracellular growth, survival and

invasiveness of S. pyogenes.

Generally, the contribution of SpeB to the pathogenesis of S. pyogenes is two-fold

in nature, in that it does not only contribute to pathogenesis through its interaction with

host proteins, but also acts to modify its own adhesive phenotype so as to evade the host

immune response and promote invasion. It has k e n reported that purified SpeB, when

incubated witb live M l GAS, is able to release several biologically active fragments from

the surface of S. pyogenes (14). These surface proteins include, the antiphagocytic M1-

protein, IgG-binding protein H and CSa peptidase. C5a peptidase cleaves the host

complement factor C5a and disables its ability to chemotacticdly attract

polymorphonuclear leukocytes (PMNs) (14). CSa and protein H that are released from

the surface of the bacterial ce11 can inhibit granulocyte migration to the site of infection

and form soluble complexes with IgG, respectively. This serves to activate complement

at a site distant from the bacteria. (13). Both of these phenornena can act to help the

bacteria evade host immune responses. M-proteins bind fibnnogen, albumin, and human

keratinocytes mediating attachment to host tissue (14). Cleavage of M-protein from the

surface of the bacterial ce11 could help to release the bacteria fkom r local site of infection

and promote acquisition of fresh nutrients as well as invasion (138). Lastly, SpeB has

also been shown to cleave Ml-protein, such that a 24 amino acid sequence is removed.

This alters its IgG binding ability, which is associated with an increase in invasive --.--A -- - . - -- - - - - -

disease in a murine mode1 of infection (162) (161).

(vi) Cysteine proteases of Pophorymonas ginigivulis

Porphyorymonas gingivalis is a major etiological agent of adult onset periodontal

disease that is characterized by abnormal degradation of connective tissue proteins due to

local, uncontrolled proteolysis (149). The gingipains constitute a group of cysteine

endopeptidases that are responsible for at least 85% of the general proteolytic activity and

1 0 % of the so-called ' 'trypsin-like activity" produced by P.gingivu1is (153; 154) . In

1984 these poorly classitied proteinases were targets for purification, and ai least 24

different variants differing in molecular mass, proteolytic and hemagglutinin activity,

speci ficity and susceptibility to synthetic inhibi tors were described ( 149). Resentl y, al1

of these activities are represented as variants of three gene products encoding cysteine

proteinases which, depending on a specificity for hydrolysis of either Arg-X or Lys-X

peptide bonds, are refemd to as gingipains R and gingipain K (152). Gingipains R are

the products of two related but distinct genes that have been cloned and sequenced from

several strains of P. gingivalis and referred to by many different names (149). Similarly,

the single gingipain iC gene was characterized in a number of strains and given many

different narnes (149). Recently, in an effort to uni@ nomenclature, it was suggested that

the gene encoding gingipain R associated with hemagglutinidadhesion domains should

k referred to as rgpA (arg-gingipain A), and the gene that encodes gingipain R without

this C-terminal hemagglutininladhesion extension be refemd to as rgpB (33). In a similar

manmr the name kgp (lys-gingipain) was suggested as a reference to the gene encoding

gingipain K (33). The translated part of the rgpA gene encodes a polyprotein consisting

of a profragment followed by a catalytic domain and a hernagglutinidadhesion domain. .-----a- -.-- - + .- -- - . -- - - - -- - - - - - -

The initial translation product is apparently subject to posttranslational processing,

leading to formation of ai least three different molecular foms of the enzyme (149). The

most important form of the rgpA gene product, however, is the nontovaient but very

stable complex of the N-terminal catalytic domain with an hemagglutinin/adhesion

domain(s) derived from the C-terminus of the initial translation product. This form of

gingipain R is denoted HRgpA, refemng to the high molecular mass of the cornplex.

Amino acid sequence analysis of its individual components indicate that proteolytic

processing of the precursor protein occurs at four kg-X and one Lys-X peptide bonds,

which precedes cornplex formation (149). In cornparison to the rgpA gene, the rgpB gene

is missing almost the entire section encoding the hemagglutinid adhesion domains, with

the exception of a small C-terminal segment. Apart from this difference, the translated

polypeptides of the rgpA and rgpB genes share 72%,93% and 40% identity wi thin the

profragments, the catalytic domains and the C-terminal extensions, respectively (149).

The rgpB gene is expressed simply as a precursor that requires posttranslational

modification by proteolytic cleavage of the profragment (149).

The structure of the kgp gene is known h m the examination of several strains of

P. gingivalis. It encodes a polyprotein consisting of a typical leader sequence, followed

by a profragment, a catalytic domain, and the C-terminal extension harboring

hemagglutinin/adhesion domains. As in the case of the rgpA initiai translation product,

the nascent Kgp polyprotein is apparently processed at multiple Arg-X peptide bonds and

a single Lys-X bond, leading to the formation of the non-covalent heteromultimeric

complex of the catalytic and hemagglutinidadhesion domains (149). The amino acid

squence of the gingipains has not revealed any significant similarity to known proteins, --A-> - - - - - - - - - - - - - - -

including other proteolytic enzymes (149). This indicates that gingipains are unique

proteinases, and for this reason they have been assigned to a separate family (family C25)

of cysteine proteinases. HRgpA and Kgp assist bacterial cells with an

hemagglutininladhesion ability that is a prerequisite for colonization of host tissues (140)

(101; 102).

The list of host proteins degraded by gingipains in vitro include extracellular

matrix components such as laminin, fibronectin, and collagen types III, IV, and V (149).

Degradation of these ECM components at infected periodontal sites may lead to damage

of basement membranes, extracellular matrix and host cells, but would be rather

insufficient to directly cause the major conneciive tissue damage associated with

periodontitis (149). In spite of several reports,(l2;87), it is clear thai gingipains are

unable to cleave native type 1 collagen. This cleavage, however, can be indirectiy

accomplished by stimulation of the release of MMPs by host cells present in connective

tissue. In fact, it was reported that proteases fiom P. gingivalis culture medium stimulate

collagen degradation by keratinocytes (16). This stimulation is more likely to be caused

by the activation of host pro-MMPs by P. gingivalis proteases (177). It has been inferred

f'rom the cleavage sites of the zymogens that MMP-1 and MMP-9 were processed by

gingipain K, and MMP-3 by gingipain R (36) . Similar mechanisms may also have

contributed to the degradation of ce11 surface and matrix glycoproteins in cultured

fibroblasts, which secrete active foms of collagenase and plasminogen activator when

treated with P. gingivalis proteinases (184). Ruified gingipains can also significantly

enhance the synthesis of latent MMPs (collagenase and stromelysin) in rat rnucosal

epithelial cells and human fibroblasts (35). Collagen-degradation induction in -% -r--- .., P L - - -

proteolytically quiescent epithelial and connective tissue cells can also be mediated by

degradation products of the extraceilular matrix components as was shown in rabbit

synovial fibroblasts (190). Recently, it was demonstrated that gingipains R degrade

fibronectin and its a5p1 integrin receptor on gingival fibroblasts (171). Given that the

aSpl integrin receptor plays an important role in regulating the expression of

collagenase, stromelysin and 92-kDa gelatinase in fibroblasts (77), the degradation of this

receptor may represent a novel mechanism for connective tissue damage in periodontal

disease .

Recently, a cysteine proteinase with the ability to cleave and inactivate al-

proteinase inhibitor, a feature distinguishing it from the gingipains which have no effect

on this serpin, has k e n purified from the culture medium of P. gingivalis HG66 and

designated periodontain (130). Periodontain was separated as a non-covalent

heterodimer with molecular mass of about 75 kDa, and composed of a 55 kDa heavy

chah containing the catalytic active site and a 20 kDa light chah devoid of enzyrnatic

activity, as revealed by gelatin zymography (1 30). The enzyme is a typical cysteine

proteinase whose activity is dependent on the pnsence of reducing agents and is inhibited

E-64 (130). Although a multitude of synthetic chrornogenic peptide-p-nitroanilide

substrates have been screened, none were found to be cleaved by periodontain despite the

fact that the proteinase was able to degrade gelatin, insulin B-chah and reduced,

carboxymethylated lysozyme with very high efficiency (130). In addition, the enzyme

was unable to degrade amcasein, casein, lysozyme, collagen, fibrinogen or plasminogen,

indicating that it could only cleave denatured or easily accessible polypeptide chahs, but

not intact proteins with a defined secondary or tertiary structure (130). The nmarkable - - -

exception from this rule is the proteolytic inactivation of al-proteinase inhibitor by

limited proteolysis within the reactive site lwp (130). This is, however, explainable by

the unique structure of inhibi tors belonging to the serpin superfamily, their reactive site

loop king readily exposed and presenting a susceptible substrate for cleavage by non-

target proteinases (15 1 ; 155). Based on the N-terminal sequence of the heavy and light

chains of periodontain and the gene sequence of r cloned fragment, the entire structure of

the periodontain gene was elucidated from the unfinished microbial genome of P.

ghgivalis W83 (149). The gene encodes an 843 amino acid polypeptide chah with a

calculated molecular mass of 93, 127 Da, which is apparently proteolytically processed

before king released from the ce11 (149). First, the 147-amino-acid propeptide is

removed by cleavage at an Argl47-Thr peptide bond, followed by C-terminal processing

at a Lys629-Asp bond to generate the light chah of the mature heterodimer (130).

Cleavage at basic residues suggests that properiodontain is processed by gingipain.

Penodontain possesses a significant amount of homology to SpeB of S. pyogenes (130).

By analogy to SpeB. which is inactive until the N-terminal profragment is cleaved, it may

be assumed that this enzyme is also produced as ymogen. In addition, SpeB plays a

major role in S. pyogenes virulence while data has yet to be accumulated for the role of

periodontain. It may, however, indirectly participate in connective tissue damage through

inactivation of al-proteinase inhibitor, the major inhibitor of neutrophil elastase.

Neutrophil elastase is not only capable of digesting almost any connective tissue protein

as well as inactivating inhibitors of coagulation (22;85; 158) but, also more significantly,

is an inactivator of tissue inhibitors of MMPs (139). Given that at periodontitis sites

34

MMP regulation is already dishirbed both at the levels of expression and activation, the -- --AL w - -- - local inactivation of tissue inhibitors of matrix MMPs may dismantle the last barrier of

connective tissue defense against proteolytic damage .

IV. Regdation of gene expression in S. aureus

( i ) Growth phase dependent virulence factor expression

As S. aureus progresses from the colonization to the invasive phase of infection

the requirements of the bacteria are changing such that what is conducive to the

colonization phase may not be optimal for invasion. The expression of virulence factors

is thus cwrdinated to meet the changing needs of the bactena, in response to the phase of

infection. The colonization phase represents the time in which the bacteria must establish

itself within the host without king prematurely cleared f'm the host so tbat the

infectious process can continue. As the bacterial ce11 density increases competition for

nutrients increases and conditions within the microenvironment change such that

bacterial growth begins to slow. This marks the transition between the exponential and

stationary phase of growtb whenin the expression of cell-surface adhesins is

downregulated and the expression of secreted virulence factors is upregulated. This

regulation of gene expression is executed in part by a global regulatory genetic element,

die accessory gene regulatory (agr) locus (141).

(ii) The accessory gene regulutory (agr) system

The accessory gene regulatory (agr) locus is a member of the classical two-

component signal transduction systems wherein signals h m environment are transduced

to elicit a response h m the bacteria (141). The environmentaVextracellular signal binds --T-.I..-I-.---- " -..- - - - --__iZ % . =. - % - - - - - - - - - - - - - - -

to the N-termial extracellular domain of transmembrane histidine protein kinase which

acts as an environmental sensor. Once the sensor has bound the signal it becomes

allosterically madified and this modification is tmsmitted to the cytoplasmic, C-terminal

portion of the sensor activating phosphokinase activity, which results in

autophosphorylation of its conserved histidine residue (179). This phosphate group is

then transfened to an aspartic residue on the response regulator protein of the two-

cornponent system, which is in the cytoplasm, activating it (178). The regulator protein,

once activated can either enhance or repress transcription of target genes (178).

Transcription at the agr locus is driven from two divergent promoters, PZ and P3

(133). The P2 operon contains four ORFs, agrB, agrD, agrC, and agrA which code for

the two-component signal transduction system. The cytoplasmic response regulator and

hsuismembrane signal receptor are coded for by the agrA and agrC genes, respectively

(107;124). Whereas, agrD codes for the 46 amino acie extracellular signal peptide which

is processed into a cyclic octapeptide, containing a thiolactone bond between an intemal

cysteine and the C-terminal carboxyl residue, by the agrB gene product (1 15). The

diversity of th AgrD octapeptide sequence serves to divide S. aureus strains into four

interference groups wherein the octapeptide from any one group activates transcription of

RNAm from that group while repressing transcription of RNAn in any other of the t h e

groups (82; 132). This type of bacterial interference may indicate a mechanism by which

some strains of S. aureus inhibit the transcription of virulence genes in other strains

thereby excluding them fiom colonization andor invasion sites (82).

The other promoter of the agr locus, P3, drives transcnption of an untranslated ---- - - - - - -- - --. . * - - --- -- - . . . - - -

RNA molecule, RNAIII, which is the effector molecule of the agr response in that it acts

tum on secreted virulence factor expression dunng the stationary phase (134). Weak

transcription fiom the PZ promoter results in basal levels of the AgrD octapeptide and as

the bacteria progress through exponentiai phase, it accumulates in the sumunding culture

medium. As growth continues to the stationary phase and cell-density increases a criticai

concentration of octapeptide is reached (83). At this critical concentration, the AgrD

octapeptide binds to the transmembrane AgrC protein, inducing autophosphorylation of

the cytoplasmic histidine residue (107). The phosphate group is then transferred to the

AgrA response regulator, which is thought to upregulate the transcription of both the P2

and P3 promoters (107).

Upregulation of the P2 and P3 promoters results in the activation of expression of

the RNAm effector molecule. RNAm acts to downregulate or repress the expression of

colonization factors like FnBP and Protein A (142; 166). while switching on or

upregualting the expression of secreted virulence factors such as V8 protease and a-toxin

(163). Therefore, an ugr mutant displays a pleiotropic loss of stationary-phase secreted

proteins and enhanced or constitutive expression of cell-surface adhesins

(1; 104; 133; 142).

Although the clinical significance of S. aweus as a human pathogen is well

documented, the function of its specific virulence factors remains poorly understood.

The serine pmtease of Staphylococcus aureus strain V8 (Ssp, also known as V8 protease)

was one of the first secreted enzymes of S. aureus to be punfied and characterized in

detail. It is a member of the glutamyl endopeptidase family of enzymes. Rice et al

presented the first application of molecular techniques towards providing a detailed

understanding of the functions of a secreted protease of S. aureus. inactivation of the V8

protease gene, sspA, resulted in the loss of a low-molecular-mass cysteine protease and

the appearance of a higher-molecular-mass protease, representing the 40-kDa SspB

cysteine protease. These observations suggested that the SspA serine protease is required

for proteolytic maturation of the 40-kDa fonn of SspB. The results of this study

indicated that SspB is expressed as a 40 kDa precursor protein, which is processed by

SspA to form a mature cysteine protease of 22 to 23 kDa.

Rior to this work, SspB of S. aureus had not been identified in the literature and

although two additional cysteine proteases have ken reported (reviewed in Part i), little

is lcnown about their specific functions and their contribution to the pathogenicity of S. - -

aureus infections. As reviewed in Part 1, cysteine proteases produced by both P.

gingivalis and S. pyogenes have been extensively characterized as important virulence

factors.

Although S. aureus is a major cause of skin infection, and colonization of human

skin with S. aureus is a common feature in a variety of dermatologie diseases, very little

is known about its interaction with epithelial cells, and virtualiy no work has ken done ---A , - --. - -4 - - .- - - -

toward understanding the contribution of cysteine proteases in this capacity.

P- A - -*- - -

Hypothesis: SspB cysteine protease of S. auteus contributes to the pathogenicity of

S. aureus disease through a specific function in virulence which may include a

detrimental effect on epithelial cells

Objective: To puri@ SspB in its precursor and mature form and evaluate the effect of

SspB in its precursor and mature fom on primary human keratinocytes grown in culture

and assay its proteolytic activity on protein and peptide substrates

P A - ..- PART II: MATERIALS and METHODS

L - - - * - -

1. Bmem and Meàia

Alkaline Phosphate (AP) Developing buffer - 0.5rnM M.gClz, 0.1 M Tris, pH 9.5

Ampicillin stock solution - 50 mglml of the sodium salt of ampicillin in ddHÎO.

Sterilized by filtration and stored at -20' C

Antibody Blocking buffer - 3 96 wlv BSA and 0.02 % NaN3 in PBS

Antibody Dilution buffer - 0.1 % wlv BSA, 0.05 % Tween 20.0.02 % wlv NaN3 in PBS

Brain Heart Infusion (BHI) agar - 15 g of agar per litre of BHI Broth media

BHI Broth media - 37 g of BHI powder per litre of ddH20

Coomassie Destain - 40 % Methanol, 10 % Acetic acid in 5 0 ml ddH20

Coomassie Stain - 2.5 g Coomassie Brilliant Blue, 500 ml Methanol, 100 ml Acetic acii

in 1 L ddH20

Loading Dye for DNA - 0.05 % w/v Bromophenol blue, 40 % wlv sucrose,

0.1 M EDTA, pH 8.0,O.S % wlv SDS

Mcilvaine's Phosphate Buffer -x ml 0.1 M Acetic acid +

( 1 W x mi) 0.2 M Na2HPOJ .2 H20

PBS buffer - 8 g NaCl, 0.2 g KCl, 0.24 g KH2P04, 1.44 g Na2HP04, per litre of ddHzO

Phenol Cholorform Isoamyl alcohol - 25 Phenol: 24 Chlorokrm: 1 Isoamyl alcohol

Plasmid Extraction Solution I - 50 mM glucose, 25 mM Tris.CI (pH 8.0). 10 mM EDTA

pH 8.0

Plasmid Extraction Solution II - 0.2 N NaOH, 1 % SDS

Plasmid Extraction Solution III - 60 ml 5 M Potassium acetate, 1 1.5 ml glacial Acetic

Acid, 28.5 ml dàH20

Rotease assay media - 2 g fbglycerol phosphate, 1 g yeast extract, 0.48 g K2HP&,

0.092 g NaH2Pû4.H20, 2 g Tryptone. 1 ml CaClz in 200 ml ddHzO

Reducing buffer (4X) - 0.64 g SDS, lm12 M Tris (pH 6.8), 2.12 ml H20, 3.2 ml glycerol,

0.08 ml 0.5 % Brornophenol blue. 1.6 ml fbmercaptoethanol

SDS-PAGE Running buffer - 15 g Tris, 72 g Glycine, 5 g SDS, in 5 L of ddH20

SDS-PAGE Blotting Transfer buffer - 3.03 g Tris, 14.4 g glycine, 200 ml methanol

per litre of ddH20

TAE buffer - 4.84 g Tris, 1.14 ml glacial Acetic Acid, 2 ml 0.5 M EDTA, per litre of

ddH20 (pH 8.0)

Wash buffer for western blots and ELISA - 0.05 % Tween-20,0.02 % Na&, in PBS

Zymograrn sarnple buffer (2-buffer) - 50 mM Tris HCl, 2OOmM NaCI, 5mM CaC12,

0.0025 % Tri ton X- 100

II. Bacterial Straim and Growtb Conditions

II, (i) Bacterial Strains

The standard laboratory strain of S. aureus strain RN6390B, wss employed in this

study and has been previously described (144). Strain RN691 1 (agr A::tetM Tc3 is an

agr-nul1 isogenic denvative of RN6390B. wherein the agr locus has ken replaced with a

tetracycline marker. S. uureus strain SP639 1 is an sspA allelic replacement mutant of

strain RN6390B, wherein the sspA gene has been replaced by an erythromycin resistance

cassette (ennAB) (Figure 1) (164). Strain SP6391 shows elevated expression of the

40 kDa form of the cysteine protease, SspB. Strain SP6912 is a derivative of strain

RN691 1 wherein the sspA mutation of SP6391 has been introduced by phage

transduction (Figure 1) (164). E. coli strajn DHS-a was used as a host strain for

construction of recombinant plasmids and transformation of recombinant plasmids into S.

aureus RN691 1 involved initial transformation into the restriction deficient strain

RN4220 (131).

II. (U) Standanl Growth Media

Stodc cultures of the various S. aureus and E. coli strains were maintained in BHI

broth and LB, respectively, at -70°C in 15% glycerol (Sigma; St. Louis, MO). S. aureus

and E. coli strains were propagated by fmt plating ont0 BHI or LB medium (Difco

Laboratories; Detroit, MI) containing 1 3% agar (Becton-Dickinson; Coc keysville, MD)

When required, media were supplemented with 10 pglrnl of erythromycin or

chloramphenicol or 50 pghl of ampicillin when requireci for plasmid maintenance .

- 2 - a - k - - 2

For liquid cultures, either LB, BHI broth or Protease Assay Media (PAM) was used with

or without the appropriate antibiotic.

n. (iii) Staadanl Gmwtb Conditions

Growth of bacteria on solid agar was carriexi out at 37OC in an incubator (ambient

air). Liquid bacterial cultures were grown with shaking at 250 rpm, in a shaker-incubator

also maintained at 37OC. When calibration for ce11 density was required for specific

experiments, the ODm of the overnight culture was determined and sub-cultured into

fresh media such that an initial O&()() of O. 1 W ~ S achieved.

m. Specific Growth Conditions

(i) Profiles of Culture Supernatant Proteins

For visualization of secreted stationary phase supematant proteins bacteria were

grown for 8 h in Rotease Assay Media descnbed by Drapeau. Cell-free supematant

(0.75 ml) collected by centrifugation at 3000 rpm for 20 min at 4OC was mixed with an

equal volume of ice-cold 20 % trichloroacetic acid (TCA), and incubated on ice for 60

min. Rotein pellets were collected by centrifugation at 13,400 g, washed with 1 .O ml of

ice-cold 70 % ethanol, air-dried and then dissolved in 20 pl of L xRB. Pmtein samples

were subjected to SDS - PAGE by loading on a 12 % polyacrylamide resolving gel in

volumes representing equivdent ce11 densities according to the ODm of each culture

from which the supematant was derived.

-, - Fi y r e 1: Diagrammatic representation of ssp o p m n of S. aureus strains

----h ----- - - - -

used in-this s b b y

RN6390 (wildtype)

V8 protease gene

SM912 (ag r-nul 1 derivative)

-%;-%*--- - - IV. Molecular Biolosy Techniques

- -

Ne (i) isolation of Plasmid DNA

Plasmid DNA was isolated fiom E. coli using the mini-preparation alkaline lysis

method. Briefly, single colony transformants were inoculated into LB broth and

incubated ovemight. Cells from 1.5 ml of cultue were pelleted by centrifugation in

eppendorf tubes (SOOOg, 5 min), and subsequently resuspended in 100 11 of ice-cold

Solution 1 and vortexed, A 200 pl volume of freshly prepared Solution Il was added,

and the suspension was mixed by inverting the tube gently 4-6 times, followed by

incubation on ice for 5 min. The reaction was then centrifuged (13,400 g, 5 min), and the

supernatant containhg the plasmid DNA was transferred to a fresh eppendorf tube and

extracted with pheno1:chlorofonn:isoamyl alcohol(25:24: 1) (Sigma), and ethanol

precipitated. The DNA pellets were then nnsed with icecold 70 % ethanol and

resuspended in 20-25 pl of water. For isolation of plasmid DNA fiom S. aureus the same

procedure was used but 20 pglml of lysostaphin was added to Solution 1 and the

suspension was incubated at 37OC for 1 h or until the solution cleared.

--

IV. (ii) Poiyrnerase Chain Reaction ('CR)

IV. (ii) a. Construction of Stapbylococd Expression Vector

For cloning of the sspB gene fragment immediately downstream of the

Staphylococcal Pmtein A promoter, a Staphylococcal expression vector, pIMLl was

constructed. Firstly, inverse PCR was carried out on phagemid vector pG8SAET

(accession # AF130864), so as to delete the E-tag and Gene VIN open reading m e s

- using forward and reverse primers PGI-F and ffi1-R both of which harbor BamHI sites.

This incorporated a B a d I site in frame with the protein A promoter usehil for

subsequent cloning steps. PCR amplification of the resulting vector pSAAG8ET fiom

nucleotides 3 122 to 400 of yielded a 476 bp fragment that includes the Staphylococcal

protein A promoter and leader sequence. Xbal and EcoRl restriction sites were

incorporated into the forward and reverse primers, PS8-F and PS8-R, respectively (Table

1). The reaction was performed in a 25 pl volume, containing 1.0 ng of plasmid DNA

template, 0.2 mM dNTP mix (Roche), 2.0 mM magnesium chloride, 0.625 units of

ArnpliTaq DNA polperase (Roche) and 37.5 pmol of each of the forward and reverse

primers. Thermal cycling was camied out in a PTC-LOOU" machine and consisted of 30

cycles of denaturation (94' C, 1 min), annealing (SOC, 2 min), and extension (72' C, 1

min). The size of the resulting product was confirmed by gel electrophoresis.

IV (fi) b. Amplification of sspB and sspJ3C

Amplification of sspi3 and sspBC (1 kb and 1.5 kb respectively) was performed

using genomic DNA isolated ftom S. aureus strain RN6390B. The forward primer, SspB-

F, includes a 5' BamHI restriction site and was used for PCR amplification of both sspB

and sspC. Reverse primer, SspB-R harbours a 3' EcoR1 restriction site, and SspC-R

anneals to a sequence downstream of an EcoRl restriction site in the ssp operon (Table 2).

The K R reaction and thermal cycling was canied out as described above, but with 1.0

ng of S. aureus RN6390B genornic DNA as template, and the annealing temperature used

was 5Z0 C. Also, the extension period for the sspBC arnplicon was 1 min, 30 sec. PCR

47

products were visualized by gel electmphoresis and gel purified using the Concert Rapid -.--- 2- - 2 -

Gel Extraction System (GIBCO-BRL).

Table 2: Description of primers used in this study

Primer Name

PGI-F

(nts 495-5 12)

PGI-R

(nts 292-75 on minus stranc)

PS8-F

(nts 3 103-3 122)

PSS-R

(nts 400-383)

SspB-F

(nts 1551-1572 of

wp operon)

SS~B-R

:nts 2627-2601 of

rsp opemn)

SspC-R

:nts 3264-3247 of

rsp operon )

Sequence 5' to 3'

cccTCATAGAccc tgattctgtggataacc

cccGGATCCgccgat tcacac tct

Description

Fonvard primer for inverse PCR on pG8SAET

Upper case letters designate BamHI site

Reverse primer for inverse PCR on pG8SAET

Upper case letters designate BamHI site

Forward primer for amplification of protein A

promoter and leader sequence from pSAAG8ET

Upper case letters designate Xbal site

Reverse primer for amplification of protein A

promotcr and leader sequence from

pSMG8ET

Upper case Ictters designate &CORI site

Forward primer for amplification of sspB and

sspB + sspC

Uppercase letters designate BamHI site

- -

Reverse primer for amplification of sspB

Upper case letters designate EcoRI site

Reverse primer for amplification of sspf? +

IV. (M) DNA mialpulations

W. (iii) a. Collstruction of Staphyloeocerl Expression Vector pIMLl

To facilitate the expression of both sspB and sspBC in S. aureus, an expression vector,

pIML1, was constructed (Figure 2). This was accomplished through cloning of the

staphylococcal protein A promoter and leader sequence (arnplified from pSAAG8ET as

descnbed above) into the staphylococcal shuttle vector, previously constructed in the

McGavin laborotory (Figure 2) by insertion of pBluescript II KS + (pBKS+) (accession #

X52327) into the Hind III site of S. aureus plasmid vector pC194 (accession # NC

002013). The resulting shunle vector contains both the E. coli and S. aureus origin of

replication, as well as chloramphenicol and ampiciilin resistance markers. The 476 bp

PCR arnplicon containing the protein A promoter and leader sequence was treated with

proteinase K prior to digestion with restriction endonucleases. Briefly, the PCR

amplicons were exposed to 50 pg/ml of proteinase K in the presence of 5 rnM EDTA,

0.5% SDS, and 10 mM Tris buffer, pH 8.0. The reaction was carried out at 37' C for 30

min, followed by heat inactivation of the enzyme at 75' C for 10 min. The DNA was

then subjected to gel purification using the Concert Gel Rapid Extraction Kit (GIBCO-

BRL) following manufacturer's instructions. The 476 bp PCR amplicon and pMLl

vector DNA were digested with XbaI and EcoRI (New England Biolabs, Beverly, MA) at

37OC for 2 h in an appropriate NEB-supplied buffer. In both cases, for every 2-3 pg of

DNA, 5.0 units of the appropriate restriction enzyme was used, in a total volume of 20 3.

This step was followed by heat inactivation of the restriction enzymes at 75OC for 10-15

min. Dephosphorylation of the XbuEcoRI digested pMLl vector was accomplished

---- *--- - with 2 Units of Calf-intestine Alkaline Phosphatase (Roche) for every 2 pg of vector,

- -

using the appropnate CIAP buffer that was supplied by the manufacturer. The reaction

was carried out at 37' C for 1 h, followed by heat inactivation at 75" C for 10 min. The

476 bp insert was ligated to the vector, overnight at 16' C, in a 3: 1 ratio of insertvector

using 3.0 Units of T4 DNA Ligase (NEB) in the appropnate ligase buffer (NEB) creating

a 6.2 kb vector, pIML1. The resulting recombinant plasmid was transfomed first into E.

coli and then S. aureus RN4220 from which it was prepared for cloning of sspB and

sspBC.

Figure 2: Construction of S taphylococcal expression vector pIML 1

PCR amplification of nucleotides 3 122 to 400 which include protein A promoter and leader sequence.

Rotein A pmmter

Protein A leader sequence

1 Hindlll digest and ligation

m-. Xbal and EcoRl digestion and ligation

IV. (iii) b. Cloning of sspB and sspBC into pIMLl

For expression of sspB and sspBC in S. aureus, PCR amplicons were ligated to

the PCR 2.1 vector (Invitrogen) according to the manufacturer's instructions. Plasmid

DNA was prepared from clones containing each of the inserts and digested with

restriction enzymes B u d I and EcoRl (NEB) at 37OC for 2 h in the appropriate NEB

buffer, as described above. followed by heat inactivation of the enzymes (75OC for 10

min). Inserts were gel purifiecl using the Concert Rapid Gel Extraction System (GIBCO-

BRL) according to the manufacturer's instructions, eluting in 20-25 pl of ddH& These

inserts were then cloned into the Staphylococcal expression vector pML1 (prepared from

S. aureus RN4220)' that was conshucted as described above. pIMLl vector DNA was

digested with restriction enzymes BamHI and EcoRl (NEB) in the appropriate buffer for

2 h at 37OC, and the reaction was set up as described above. This was followed by Calf-

intestine Aikaline Phosphatase dephosphorylation (Roche) and gel purification (GIBCO-

BE). ïnserts were then ligated to pIMLl vector, overnight at 16' C, using T4 ligase

(NEB) as described above, such that sspB/sspB + C were clowd immediately

downstream and in frame with the Staphylococcal protein A promoter and leader

sequence.

N. (IV) TWormation procedures and Electroporation Conditions

N. (i) a. Transformation of E. coli

Electrocompetent E. coli D H S a (GIBCO-BRL) was transformed with - --

recombinant plasmid pIML1. Briefly, the ligation reaction was treated at 70' C for 10

min to heat-inactivate the ligase enzyme and cooled on ice. DNA was then extracted once

with phenol:chlorofom:isoamyl alcohol(25 : 24: 1) (Sigma) followed by ethanol

precipitation. Thawed electrocompetent cells were mixed with 5 pl of the extracted

ligation mixture and allowed to sit on ice for 1 min. Electroporation was c h e d out in

pre-chilled 0.2 cm electroporation cuvettes (Bio-Rad Laboratones; Hercules CA) using

the Gene Pulser0 (Bio-Rad) apparatus at the following settings: voltage of 2.5 kV,

capacitance of 25 pF, and a resistance of 200 R. hmediately following electroporation,

the suspension was supplemented with SOC medium and incubated 37OC for 30 min first

without shaking and for an additional 30 min with shaking (250 rpm) prior to plating on

LB + ampicillin.

IV. (iv) b. Transformation of S. aureus

Following transformation into E. coli, pIML1 plasmid DNA was subsequentiy

prepared and tmsformed into S. aureus strain RN4220. pIML1 DNA prepared from S.

aureus RN4220 was also used for cloning of sspB and sspBC inserts to create pIMB and

pMBC respectively. Plasmids pïMB and pMBC were subsequently transformed into the

S. aureus agr-nul1 strain RN69 1 1, creating strains SP69 13 and SP69 14 respectively. As a

negative control pIMLl was also transformed into RN691 1 to create strain SP6915. in

d l cases, S. aureus RN4220/RN6911 cells and recombinant plasmid DNA used in

transformation procedures were prepared as follows. S. aureus cells were grown to an

ODuo of 0.24 and collected by centrihigation at 3000 rpm for 20 min. Pelleted celis were

54 -

washed with 50,25, and 10 ml of 0.5 M sucrose and finally resuspended in lm1 of 0.5 M -----a- - - - - - -- * - -

sucrose. Plasrnid DNA was prepared using the Qiagen Miniprep Kit (Qiagen) afier

which 10 pg of DNA was ethanol precipitated, washed with 70 % ethanol. air-dried and

resuspended in 160 pl of the prepared S. aureus cells. The mixture was allowed to sit on

ice for 15 min and electroporation was canied out in pre-chilled 0.1 cm elecaoporation

cuvettes (Bio-Rad Laboratories) using the Gene Pulse@ (Bio-Rad) apparatus at the

following settings: voltage of 1.8 kV, capacitance of 25 pF, and a resistance of 400 R.

Immediately following electroporation the suspension was supplemented with SOC

medium and incubated 37'C for 30 min first without shaking and for additional 30 min

with shaking (250 rpm) prior to plating on BHI + chloramphenicol.

V. htease Purification

V. (i) Ammonium Sulfate Recipitation of Stationary-phase Culture Supernatant

For purification of the precursor and mature form of SspB, ovemight cultures of

SP6391 and RN6390B, respectively, were subcultured into 25 ml of PAM and regrown

overnight. The resulting culture was then subcultured into 2 X 200 ml of PAM to

achieve an ODm of 0.1 and subsequently grown for an additional 8 h to stationary phase.

Culture supematants were collected by centrifugation at 3000 rpm for 20 min.

Supernatant proteins were collected by 40 % and 80 % ammonium sulfate precipitation

for the precursor and mature fom of SspB respectively. Briefly, culture supernatant was

kept at 4' C and an amount equivalent to either 40 % or 80 % (wfv) of ammonium sulfate

was added in increments until dissolved over a period of at least 6 h and left ovemight.

Rotein was pelleted by centrihigation of the saturated supernatant at 12000 rpm for 30 - - -

min.

V. (ü) PuFifIcation of precursor SspB

For purification of the 40 kDa precursor form of SspB, the resulting pellet was

dissolved in a minimal volume of dialysis buffer (20 mM Tris-HCL, 0.02 % NaN3, pH

7.4) and dialyzed vs. three changes of the same buffer over a 48 h period. A small volume

of the dialyzed protein preparation was syringe filtered through a 0.45 phif surfactant free

cellulose acetate membrane (Nalgene) and applied to a 5 mi HiTrap Q anion exchange

column (Arnersharn Pharmacia AKTA prime pump system) equilibrated with running

buffer (20 mM Tris-HCl, pH 8.0) and eluted in a linear gradient fiom O - 1.0 M NaCl

with the elution buffer 2OmM Tris-Hcl, 1.0 M NaCl, pH 8.0. Two milliliter fractions

were collected and protein was detected through UV absorbance at 280 nm as well as by

10 % SDS-PAGE. Fractions containing the 40 kDa precursor were pooled and desalted

on the Hi Prep 26/10 Desalting Column (Amersham Phamacia Biotech AKTA prime

pump system) at a flow rate of 10 mumin in 20 rnM Sodium Phosphate, 0.15 M NaCl, pH

7.0. Three rnilliliter fractions were collected and fractions containing the 40 kDa protein

(as visualized by 10 % SDS-PAGE) were concentnited using the Millipore Ultrafiee-15

centrifuga1 filter device (Biomax-SK NMWL membrane 15 ml volume) at 3000 rpm,

4 O C.

V. (ii) Purification of mature SspB

For purification of the 22 kDa form of SspB. the protein pellet resulting nom

-.

56

-- L - - s - 80 % ammonium sulfate saturated culture supernatant of strain RN6390B was used. The

a- -

protein pellet was resuspended in 2 M (NH4)2 S04, 50 mM Na2 HPO4, pH 7.0 and

syringefiltered through a 0.45 pm filter (as described above). Protein was loaded ont0 a

5 ml HiTrap Phenyl Sepharose High Performance column (Amersharn Pharmacia Biotech

AKTA prime pump system) equilibrated in 2M (NH&SO4 and 50 mM Na2 HFQ at a

flow rate of 2.5 mumin and eluted with 50 mM Naz H m , pH 7.0, in a linear gradient

fiom 040 mM Na2 HPû4. Fractions were collected (2 ml) and the W absorbance at 280

nm detected eluted proteins. Those containing a 22 Wla fragment, as visualized by 10 1

SDS-PAGE were pooled and desalted using the Hi Rep 26/10 Desalting Column

(Amersham Pharmacia Biotech AKTA prime pump system) as described above for the 40

kDa protein. Fractions containing the 22 lcDa protein were pooled and diluted with an

equal volume of 40 m M Tris-HCl, pH 8.0 and hirther purified by anion exchange

chromatography using the 5 ml Hi Trap Q Anion Exchange Column (Amersharn

Pharmacia Biotech AKTA prime pump system) as described above. Fractions containing

the 22 kDa protein were pooled and concentrated as described above for the 40 kDa

protein.

VI. (iü) SDS - PAGE and Zymognpby

To detect either purifîed protein or culture supematants were subjected to SDS-

PAGE which was conducted through the buffer system described by Laemmli (100).

Resolving gels were prepared at a concentration of 12% acrylarnide unless otherwise

indicated. Rotein samples to be visualized were boiled for 5 min in 1 X RB and were

subjected to electrophoresis by administering a voltage of 100 V for - 1.5 h. Molecular

weight protein marker from New England Biolabs was used as a size standard. Gels were - A - 7 - -

stained with Coomassie blue and destained with Cwmassie destain solution for protein

detection.

Gelatin Zymography was performed according to Lantz et al (103). Briefly, gels

were made as done for SDS-PAGE, but the separating gel was copolymerized with

1 m g h l of gelatin. Samples were incubated at room temperature for 5 min in zymograrn

sample buffer (Z-buffer) and then run on the gel at lOOV for 1.5 - 2 h. Gels were then

washed two times for 20 min in 2.5% Triton X-100/in dâH2O and subsequently

incubated ovemight at 37OC in development buffer (50 m M Tris HCl, 200mM NaCl,

5mM CaC12, 0.0025% Triton X-100 + 1 mM cysteine or 28 pM E-64) to allow for

maturation of proteins and restoration of any gelatinase activity. Gels were then stained

with Cwmassie Blue and destained with Coomassie destain. Gelatinase activity is

visualized as a zone of clearing against a dark blue background.

W. Processing of SspB precursor by SspA

To directly confirm the hypothesis that SspA processes the precursor form of

SspB into two lower molecular weight fonns of 18 kDa and 22 kDa, the following

experiment was done. 20 pg of purified SspB precursor was treated with 2 pg punfied

SspA in 20 mM Tris, 0.1 M NaCl, pH 7.4 at 37OC for 2 h. The resulting sarnple dong

with 10 ~g of SspB precursor was subjected to 12 % SDS-PAGE.

. A - -

VIIL N-Termlnai Sequencing

To confinn that the 22 kDa band isolated from ammonium sulfate precipitated

culture supernatant of strain RN6390B was in fact the mature f o n of SspB we prepared

samples for N-terminal sequencing. The protein gel was prepared as for 10 % SDS-

PAGE, but the stacking gel and the separating gel were both prepared with 1 M Tris-HCl

pH 8.0 and the gel was allowed to polymerize ovemight. The gel was then pre-run for 30

min at 15 mA with 0.76 g of glutathione in 250 ml of dâH20 in the inner chamber and

SDS - PAGE running buffer in the outer chamber. Buffers were then replaced with fresh

SDS - PAGE running buffer and 5 x 4 pg of mature SspB boiled in reducing buffer was

loaded ont0 the gel and run at a constant 30 mA until the dye front ran to the bottom.

Protein was transferred ont0 PVDF membrane in 1 X CAPS (10 mM) solution in 10%

methanol at LOO V for 50 min using the Bio-Rad Trans-blot Western Blot apparatus.

Membranes were stained with O. 1 % Coomassie blue dissolved in 40% methanol for 5

min , destained with 50% rnethanol, rinsed 3 times with ddH20 and dned on Whatman

paper and sent to the HSC Biotechnology Center for N-terminal sequencing.

M. Activity of SspB on Benzoyl-Ro-Phe-Arg p-nitroanilide

Puxified precursor and mature SspB were assessed for their proteoiytic activity on

the synthetic chromogenic peptide Benzoyl-Pro-Phe-Arg pnitroanilide that has k e n

shown to be processed by the cysteine protease, SpeB of S. pyogenes (72). In a microtitre

plate, 0.5 pg and 1 pg each of precursor and mature SspB was mixed with 1 mM

chromogenic su bseate (Sigma) in 2 0 fl of 0.2 M Tris-HCI, pH 7.4,O. 1 M NaCl 5 mM

CaC12 containing 10 m M cysteine. The same was done with 1 pg of proteinase K as a --- LpL- - -

positive control and a set containing no protease was done as a blank. Al1 samples were

done in triplicate and the reaction was allowed to proceed for 2 h at 37' C. The amount

of chromogen liberated was assessed by measuring the absorbance at 405 nm at both time

points using a microtitre plate reader

X. Treatment of Rimary Human Keratinocytes with SspB

X. (i) Harvesting of Kerstinocytes

Primary cultures were prepared fiom newbom foreskins courtesy of the

Dermatology lab, S-123, SWCHSC. Skin samples were washed in phosphate-buffer

saline (PBS) and connective tissue was trimmed. The foresicin samples were then treated

with 1 % dispase II (Boehringer Mannheim, Mannheim, Germany) in Eagle's minimum

essential medium (MEM) with 10 % heat-inactivated fetal bovine serum (FBS) (Gibco) at

4°C ovemight. Epidermal sheets were peeled h m the demis and stirred in 0.05%

trypsin and 0.53 mM ethylendiarnine tetraaceticacid (EDTA) solution (trypsin-EDTA) for

20 min at room temperature. Ce11 suspensions were filtered through 40 Fm nylon mesh

and centrihiged at 1000 X g for 10 min. Cell pellets were resuspended in Eagle's MEM

with 10 % FBS, plated at 2 X 106 cells per IO-cm dish, and cultured at 37' C in a

humidified atmosphere of 5 % C02. After 2-3 days of growth, culture medium was

changed to complete keratinocyte serum-free medium (K-SFM) contai ning bovine

pituitary extract (BPE) aml recombinant epidermal growth factor (rEGF; Gibco BRL,

Burlington, Ont.) Cultures were fed every third day and before confluency they were L - - -

passaged.

X. @) Passaging of Keratinocytes

Keratinocytes at 80-90 % confluency were washed 2 times with PBS and then

treated with pre-warmed 0.05 % trypsin for 2- 3 min at 37" C or until virtually al1 of the

attached cells were detached and seen floating. Trypsin was diluted 1 5 with fresh K-

SFM and cells were collected by centrifugation at 1 1,000 rpm for 6 min. Cells were

resuspended in K-SFM, counted with a heamocytometer and re-plated to a density of 2 X

i d cells per IO-cm dish. Cells were not passaged beyond passage four. Final passage

cells were plated in 24-weil microtitre plates at a density of 2.5 x 104 and grown to 80-90

% confluency. These cells were used in the experiments with purified SspB.

X. (di) Treatment of Keratinocytes with SspB

Keratinocytes grown to 80-90 % confluency in 24-well rnicrotiee plates were

washed twice with PBS and subsequently grown in K-SFM (+ 10 mM cysteine),

containing either precursor or mature SspB at a concentration of 10 Ccglml, for 7 h and

24 h. For inhibition experiments, SspB was pn-incubated with 28 pM E-64 at 37OC for

30 min and then added to K-SFM supplemented with 28 pM E-64 in place of cysteine.

In samples containing the broad-range matrix metallopmtease inhibitor (MMP inhibitor)

(GM6001. Chernicon) precursor and mature SspB was CO-incubated with 25 pM MMP

inhibitor. Control cells were treated with K-SFM containing both 10 mM cysteine and

28 ph4 864. In a second set of experiments ketatinocytes were treated with mature SspB - &-A-'?. -. z - - -

(but not precursor SspB) and this set of experiments included treatment with recombinant

human TNF-a at a concentration of 100 nglml in place of the MMP treatment done in the

first experiment. Ail treatments were done in triplicate at each time point for each set of

experiments.

X. (iv) Ce11 Viabiiity

To examine whether ce11 detachment was a result of keratinocyte necrosis, the

viability of cells treated with mature SspB, SspB + E-64 and media alone for 7 h was

assayed. Keratinocytes present in the culture supematant were collec ted by

centrifugation at 11,000 rpm for 6 min. Keratinocytes that were still attached to the

microtitre wells were trypsinized, collected by centrifugation, resuspended in 100 ul of

fnsh media and ndded to the cells collected h m the supematant. 20 pl of ce11

suspension was added to an equal volume of 0.4% trypan blue. At least 1 0 cells were

counted and the number of white (viable) cells and blue (dead) cells was determined such

that percent viability could be calculated. Al1 samples were done in triplicate.

XI. Pmteolysis of Protein Substrates by SspB

To assay the proteolytic activity of the precursor and mature fom of SspB, 10 pg

each of Bovine Serum Albumin, (BSA) Rabbit Immunoglobulin G, IgG, (Sigma),

Human Fibrinogen, Fbg, (Sigma), and Human Fibronectin Fn, (Gibco) was treated with

1 pg of either precursor or mature SspB at 37OC in PBS containing 10 rnM cysteine as a

reducing agent. Similar experiments were conducted with 5 pg of Human Vitronectin,

(Gibco) but Vn was treated with only 0.25 pg of SspB in 0.1 M Tris-HCI pH 7.4 -- - -- -

containing 0.15 M NaCl and 10 m M cysteine. For inhibition experiments, buffer was

supplemented with 28 AM E-64 in the place of cystcine. Samples were allowed to

incubate at 37OC for various amounts of time and then analyzed by SDS-PAGE (1ûûV for

- 1.5 tus). Samples were first boiled in non-reducing sample buffer in the presence of

inhibitor E-64 (28 w) and then boiled again in the presence of dithiothreitol (Dm.

This process of boiling was done to ensure that any proteolysis of substrates observed

would not be due to degradation during boiling in reducing buffer as was reported for

Porphyromonas gingivalis degradation of coliagen by the cysteine protease gingipain

(156).

To evaluate the proteolytic activity of SspB on casein substmte, resorufin labeled

casein was emloyed. Btiefly, 50 pl of 0.2% resorufin-labeled casein was treated with

100 pl of 500ng/ml of either pSspB or m-SspB and 250 ng/ml of V8 protease in

Incubation Buffer (0.2 M Tris, 0.02 M CaCI2, pH 7.8 +1 mM cysteine) at 37O C for 18 h.

The reaction was stopped by adding 480 pl of 5% TCA and allowed to incubate an

additional 10 min at 37' C. Undigested casein was pelleted by centrifugation at 12000

rpm for Smin. 400 pl of the resulting supernatant was added to 600 pl Assay Buffer (0.5

M Tris, pH 8.8) in a cuvette and the ODS74 was determined immediately.

W. Western Blotthg

To examine the integrity of Fn in the culture supernatant of keratinocytes

incubateâ with mature SspB, mature SspB + E-64, N a , and media alone, Fn was

detected by Westem Blotting with anti-Fn antibody. 30 pl sarnples of the culhue

. -

63

superatant coilected fiom each of the four samples were subjected to 10 % SDS-PAGE as ----- 6 - - .- -

described above, and then transferred ont0 ImmobilonP membrane (Millipon) using the

Bio-Rad Trans-lot apparatus and transfer buffer systern of Towbin (183). Transfer was

canied out at lOOV for 1 h. The membrane was then blocked with 3 % BSA (wlv)

BSAIPBSI0.02 % NaN3 for 1 h at rmm temperature on an orbital shaker. The blocked

membrane was then washed with PBSiO.05 % Tween-20 (PBST) (3 X, 5 min each), and

incubated with goat anti-Fn antibody (Sigma) diluted 500-fold in antibody dilution

buffer. Incubation was allowed to proceed for 1 h at room temperature with orbital

motion. After washing with PBST, the membrane was incubated with alkaiine

phosphatase- conjugated rabbit anti-goat secondary antibody diluted in antibody dilution

buffer (I:5000X) for an additional hour at room temperature. After washing with PBST,

the membrane was developed with BCIPMBT substrate (Bio-Rad) in an AP-âeveloping

buffer until the appropriate protein bands became visible. Finally, to stop the reaction the

membranes wen rinsed with ddH20, and air-dried on filter paper.

1. Construction of Staphyloeoreal Expression Vector pIMLl

In an attempt to purify the precursor fonn of SspB, an expression vector was

constructed. The strategy used for construction of a Staphylococcal expression vector

with the potential for high level expression took advantage of the availability of the agr-

nul1 strain RN691 1. The agr locus in RN691 1 is deleted such that it exhibits enhanced

expression of adhesive factors, such as Rotein A, and a pleiotropic defect in the

expression of secreted virulence factors. It was hypothesized that if the sspB gene could

be cloned downstrearn of the protein A promoter and leader sequence and transfected into

the RN691 1 agr-negative genetic background, that constitutive and elevated expression

of SspB precursor would be achieved, such that it would be the only protease present in

the culture supernatant. Vector construction was accomplished by incorporating the

Staphylococcal Rotein A promoter and leader sequence into a Staphylococcal shunle

vector, pMLl (composed of pC194 and pBKS) that contains both the S. aureus and E.

coli origin of replication as well as chloramphenicol and ampicillin resistance markers.

This resulted in the 6.3 kb vector designated pIMLl which was used in subsequent

cloning of sspB and sspBC.

65

ïI. Expression of sspB and sspBC --LA= La - -

The sspB gene was cloned in frame with the Rotein A promoter and leader

sequence of pIML1, creating pIMB and d o r m e d into ugr-nul1 strain RN69 1 1, creating

strain SP69 13. By expressing sspB from the Rotein A promoter, w hich is de-repressed in

an ugr-nul1 genetic background, it was anticipated that high level expression and

secretion of the 40 kDa precursor f o n would be achieved. Unexpectedly, this multi-

copy plasmid construct (pIM 1B) resulted in less expression of the 40 kDa precursor

(Fig. 3, Lane 4) than was achieved from a single copy of sspB under control of the ennAB

cassette in the SP6391 strain (Fig. 3, Lane 2). Furthemore, the precursor form of SspB

expressed fiom pIMlB showed a slight decrease in electrophoretic mobility when

compared to either SP6912 (Fig. 3, Lane 3) or SP6391 (Lane 2) in which both sspB and

sspC are transcribed from the ermAB cassette inserted in sspA. This suggested that the

altered mobility of SspB was due to the absence of SspC. To test this hypothesis, a

consnuci was made in which both sspB and sspC were expressed in tandem from the

Pmtein A promoter (pIM IBC) and transformed into RN69 1 1 creating strain SP69 14.

Although this resulted in reduced expression (Fig. 3, Lane 5) compared to pIMlB (Lane

4), the secreted SspB precursor now exhibited normal electrophoretic mobility. When the

proteolytic activities of the secreted SspB precursoa were compared by gelatin

zymography, SspB expressed fiom pMlB exhibited greatly reduced activity (Fig. 4,

Lane 4) compared to that expressed fiom pIMlBC (Lane 5). This was observed even

though the protein itself was more abundant in the culture supernatant and despite the fact

that the amount of secreted SspB was less in the strain harboring pIMBC.

SspB precursor

Figure 3: 7.5 % SDS PAGE of 8 h culture supernatant proteins. Culture supematants from

S. aureus strains RN6390, SP639 1, SP69 12, SP69l3, SP69 14 and SP69l5 (negative control)

(Lanes 1 , 2 , 3 , 4 , S and 6 respectively) grown for 8 h in P.A.M were precipitated with 20%

(vlv) TC A. boiled in 1 xRB and loaded to equivalent ce11 densities according to ODm values

taken at time of harvest.

Precursor SspB

Mature SspB

Figure 4: Gelatin Zymography of 8 h culture supernatant proteins. Volumes of culture

superanatant representing equivalent ce11 densities of S. aureus strains RN6390, SP639 1,

SP6N 2, SP69l3 (piMB/RN69 1 1) SP69 14 (pIMBCIRN691 l), and SP69 15 (pIMLI/RN6911)

(Lanes 1,2, 3,4,5 and 6 respectively) grown for 8 h in P.A.M were mixed with 2 volumes of

zymogram sample buffer and incubated for 5 min at room temperature and loaded on 12 %

SDS-PAGE containing 1mgM of gelatin. Gels were nin at 100 V for 2 h and incubated

ovemight at 37OC in development buffer. Zones of cledng, after staining witb Coornassie

blue and destainhg with Coornassie destain, represent gelatinase activity.

L .

68

----- - Cumulatively, the slight decrease in mobility that is observed when SspB is - . -

exptessed without SspC, combined with its reduced protease activity, suggests that SspC

rnay confer a chaperone function that assists in the secretion or proper folding of the

SspB precursor.

III. Protease Purification and N-terminal Sequencing

Because the expression of SspB precursor from pIMB was not as expected and

was not adequate for purification, both the precursor and mature form of SspB were

purified from SP6391 and RN6390 culture supematant, respectively. Both purified

precursor and mature SspB were subjected to SDS-PAGE (Figure 5 A, Lane 4 and 2

respectively). The purified precursor fom of SspB CO-migrated with the 40 kDa band

that is seen to accumulate in the culture supematant of the sspA mutant, SP6391 (Figure

5A. Lane 3), previously confirmed to be the SspB precursor by N-terminal sequencing.

Lane 1 shows the 8 h culture supematant protein profile of wildtype S. aureus strain

RN6390B fkom which the 22 kDa mature form of SspB was purified. The 22 kDa

purified protein was subjected to N-terminal sequencing, and yielded the sequence

DQVQYN which matches the N-terminal sequence of the protein species that would

result after cleavage of of the SspB precursor after glutamic acid 219. Both purified

precursor and mature SspB were subjected to 12 % gelatin zymography (Fig SB). The

gelatinase activity representing the mature 22 kDa purified protease (Fig SB, Lane 2) co-

migrated with that present in the culture supematant of RN6390B (Fig 5B, Lane l). The

40 kDa precursor form of SspB which accumulates in the culture supernatant of strain

SP6391 (Fig SB, Lane 3) as the only protease active on gelatin zymography displayed a

gelatinase activity which was identical to that of the purified 40 kDa precursor (Fig SB, - ! A - - - - - . - - -

Lane 4). When gelatin zymograpy was repeated with cysteine protease inhibitor, E-64 in

the development buffer, al1 gelatinase activities representing cysteine protease activity

were no longer visible (data not shown).

IV. Pmcessing of SspB Precursor by SspA

The sspA mutant SP6391 shows the accumulation of SspB precursor, which is not

present in the wildtype culture supematant. This accumulation is accompanied by a loss

of protein species in the 20 kDa range which are present in the wildtype supematant.

When culture supematant fiom SP6391 is analyzed by gelatin zyrnography, the zone of

gelatinase activity that represents the precursor is completely converted to that

representing the mature fonn of the protease when supernatant is incubated with purified

SspA (sspA). Moreover, SspA is a member of a family of proteases known as the

glutamyl endopeptidases that cleave after the carboxyl side of glutamic acid. This led to

the hypothesis that SspA is responsible for the conversion of SspB precursor to its mature

fonn. To confirm the assumption that SspA proteolyticdly converts the SspB precursor

into its mature fom, SspA was CO-incubated with purified SspB precursor. After 2 h of

treatment with SspA, the 40 I<DA precursor form (Figure 6, Lane 1) was completely

converted to two lower molecular weight species of 18 and 22 kDa (Figure 6, Lane 2)

which correspond to the size of protein products that would result after cleavage of the

SspB precursor at glutamic acid 2 19. We have thus d k t l y shown that SspA is

responsible for the conversion of SspB precursor into a mahm cysteine protease.

Figure 5: SDS-PAGE and Gelatin Zyrnography of purified SspB. A and B show

SDS-PAGE and gelatin zymography respectively, of TCA precipitated culture

supematant and purified proteins. Lane I and 3 show the protein profile of wildtype

Saureus RN6390, and V8 protease mutant SP639I respectively. Lane 2 and 4 show

10 pg of purified m-SspB and pSspB, respectively.

Figure 6: Processing of SspB by V8 protease. 0.1 pg of purified V8 protease

was incubated with 10 pg of purified SspB precursor in 1 X PBS for 2 h at

37OC and subjected to 12 % SDS-PAGE (Lane 2). After incubation with V8

protease, SspB precursor (Lane 1) was completely converted to two smaller

molecular weight species of approximately 18 and 22 kDa (Lane 2).

V. Activitg of SspB on Bemoyl-Phe-Ro-Arg gnitroaniiide -A-=--- -. - - -

The protease activity of both the precursor and mature form of SspB was assessed

using the chromogenic substrate Benzoyl-Phe-Ro-Arg pnitroanilide. Both 0.5 pg and 1

pg each of precursor and mature SspB were incubated with 1 mM substrate for 2 h at

37' C. Table 3 shows the mean absorbance at 405 nm of triplicate samples. The results

of this assay indicate that the precursor form of SspB was relatively inactive on this

substrate compared to the mature form of SspB which displayed approximately 10-fold

the activity of the precursor. Furthemore, the arnount of mature protease was positively

conelated wi th the absorbance measured (0.307/ 0.5 pg and O.78Ul pg of mature

protease) whenas that of the precursor remained relatively constant at 0.059 and 0.043

as the amount of protease changed fkom 0.5 to 1.0 pg, respectively.

Therefore, although the 40 kDa fonn of SspB is active on gelatin in a zyrnogram

assay, these data suggest that this form of the protein is an inactive precursor.

--d-:L--" L- . -

Table 3: Activity of SspB on Benzoyl-Ro-Phe-Arg p-nitroanilide

* Mean absorbance at 405 nm of triplicate samples after 2 h incubation at 37' C.

VI. Mature SspB pmmotes detachment of primary humin keratinocytes. - - - - - - - - - - - - - - - - - - - .

S. aureus is a major cause of serious skin infections and is also associated with

inflammatory skin diseases such as psoriasis and atopic dermatitis. Although the clinical

significance of S.aureus as a cause of skin disease is well established, the role of specific

vimlence factors and their contribution to pathogenesis remains poorly understood.

To begin elucidating the specific hinction of SspB, we proceeded to evaluate its effect on

primary human keratinocytes. Keratinocytes grown to 80 % confluency were incubated

with ce11 culture media containing either mature SspB (m-SspB) or precursor SspB

(p-Ssp). After 7 h of incubation. a portion of the keratinocytes that had been treated with

m-SspB appeared to round-up and detach from the substratum (Figure 7 (i) Panel A).

This effect was enhanced after 24 h where vimially al1 cells were detached and observed

floating in the ce11 culture media. In contrast, cells treated with p-SspB appeared normal

and maintained their attachment to the substratum, such that they appeared identical to

control cells that were treated with ce11 culture media alone. Concurrent experiments

were included wherein ce11 culture media contained either m-SspB or p-SspB plus

cysteine protease inhibitor E-64 (28 pM). (Figure 7 (i)/(ii), Panel B). Cell detachment

associated with exposure to m-SspB was no longer visible when E-64 was CO-incubated

with m-SspB (Figure 7 (i), Panel B). These results indicate that ce11 detachment is in

fact comlated with the activity of the mature cysteine protease and support the relative

inactivity of the precursor that was seen when both precursor and mature SspB were

tested on chromogenic substrate (Part V).

Streptococcal cysteine protease, SpeB. has ken shown to cause a cytopathic

effect on Human Umbilicai Vein Endothelial Cells after 6 h of incubation (92).

This effed is associated with the activation of a 66 kDa maaix-metalloprotease (MMP) - - -- -

which appeared as a novel gelatinase activity in the ce11 culture supernatant revealed by

zymography (18). To determine whether the ce11 detachment that we observed was

involved with the activation of MMPs, m-SspB was CO-incubated with with a broad-

range MMP inhibitor. If MMP activation is a factor contributing to ce11 detachment

through the action of m-SspB, it would be expected that CO-incubatioin with MMP

inhibitor would attenuate ce11 detachment. However, after 7 (Figure 7 (i), Panel C) and

24 h, m-SspB was still able to promote keratinocyte detachment in the presence of MMP

inhibitor and there appeared to be no visible difference fiom the detachment associated

with m-SspB alone. Moreover, analysis of keratinocyte ce11 culture supematants by

gelatin zyrnography did not reveal any novel gelatinase activity (Figure 8 A and B). Al1

supernatants, except those containing media alone (Figure 8 A and B, Lane 3) showed the

usual gelatinase activity attributed to the activity of m-SspB (Figure 8 A and B, Lanes 1,

2 and 4). Tumor Necrosis Factor Alpha (TNF-alpha) has been reported to activate MMP-

9 in human oral keratinocytes (1 13). In an effort to compare ce11 culture supematants

from keratinocytes exposed to m-SspB and those treated with TNF-alpha, keratinocytes

were incubated with 100 n g h l of recombinant TNF-alpha. No ce11 detachment was

observed and exposure to TNF-alpha was not associated with activation of any new

gelatinase or caseinase activity as revealed by zymography (data not shown).

ii) p-SspB

Figure 7: Treatrnent of primary human keratinocyte ce11 culture with SspB. mSspBIp-SspB

was incorporated into keratinocyte cell culture media containing 10 m M cysteine at a

concentration of 10 pghl and allowed to incubate for 7 h at 37' C (Panel A). Similar

experiments were done withlO pg/ml m-SspBlpSspB + 28 @l E-64, (Panel B), MMP

inhibitor 25 ng/ml (Panel C). Panel D shows keratinocytes after treatrnent with media

containing only 10 mM cysteine and 28 E-64. Cells treated with mSspB and mSspB +

MMP inhibitor were seen to lose their elongated morphology. round up, and detach h m the

substratum. Whereas, those incubated with m-SspB + E-64 or pSspB retained their nomal

morphology.

Figure 8: Gelatin zymography of keratinocyte culture supernatant. 12 % gelatin

zymography of ce11 culture supematants ftom keratinocytes trrated with m-SspB

(Lane l), m-SspB + 28 ph4 8 6 4 (Lane 2) media alone (Lane 3), and m-SspB +

25 ph4 MMP inhibitor (Lane 4) for 7 and 24 h (Panel A and B respectively) were

analyzed by. No new gelatinase activity was detected in any of the culture

supematants afier 7 or 24 h. Al1 samples, except the sarnples containing media

alone, showed the typical gelatinase activity of m-SspB.

*A. .

W. CeU Viability

We assessed the viability of the keratinocytes treated with m-SspB and compared it

with that of those treated with m-SspB + E64, TNF-alpha, and media alone. Ce11 culture

supernatants were collected and spun down to collect any keratinocytes that were floating

in the supernatant. Pelleted cells were then resuspended and added to remaining

trypsinized keratinocytes. Cells were stained with trypan blue and at least 100 cells (blue

and white) were counted using a haemocytometer such that percent ce11 viability could be

calculated. (Table 4) The results of this expriment revealed that most of the cells

remained viable in al1 samples and that there was no difference in cell viability between

sarnples (p-value = 0.468, one way ANOVA, Table 5). This result suggests that m-SspB

does not have a direct necrotic function. It is probable that if the detached keratinocytes

were allowed to remain in culture for an extended period of time that they would undergo

apoptotic cascade after having lost attachment, a process known as anoikis.

Table 4: Ceii Viability Results - ...---. - . - - --- - *L - - -

Table 5 : Statistical Analysis of Ce11 Viablility

Anova: Single Factor

SUMMARY Otwps Count Sum Av8lgg8 Variance

m-SspB 3 282 94 1 E-64 3 279 93 1 media 3 277 92.33333333 5.333333333

ANOVA Source of Variation SS df MS F P-value

Behinreen Gmups 4.222222222 2 2.1 1 1 1 1 1 1 1 1 0.863636364 0.4681 39222 W ithin Groups 1 4.66666667 6 2.-

F cnt Total 18.88880009 8 5.14

Vm. hteolysis of Extracellular Matrix Proteins - - * -

The above results appear to indicate that keratinocyte detachment associated with

the treabnent of m-SspB is not associated with the activation of MMP activity and is not

a result of a direct apoptotic function. To hirther investigate the mechanism by which m-

SspB promotes keratinocyte detachment we tumed Our attention toward direct proteolysis

of extracellular matnx proteins that possess a role in cell-matrix adhesion. We therefore

proceeded to evaluate the proteolytic activity of SspB on human fibronectin and

vitronectin (Gibco), both of which mediate ce11 attachment by binding to integrins.

10 pg of fibronectin and 5 pg of vitronectin were treated with 1pg and 0.125 pg

respectively, of either p-SspB or m-SspB in PBS containing 10 m M cysteine. After

incubation at 37 '~ . proteins were visualized by by SDS-PAGE. m-SspB, but not p-SspB

(Figure 9, Lane 5). was able to cleave fibronectin to completion after 20 h (Figure 9.

Lane 3). Vitronectin degradation by m-SspB (Figure 10. Lanes 1-5). but not pSspB

(Figure 10, Lane 8), was seen to commence after lhr, and was almost completely

degraded after 4 h of exposure to mSspB (Figure 10, Lane 5). This degradation was

completely inhibited when m-SspB was CO-incubated with E-64 (Figure 10, Lane 6).

In an attempt to establish a relationship between extracellular matrix proteolysis

and keratinocyte ce11 detachment, the integrity of fibronectin from ceIl culture

supematants that were exposed to m-SspB, m-SspB +E-64, TNF-alpha and media alone

for 24 h was analyzed by Western Mot (Figure Il). Culture supematants were analyzed

by SDS-PAGE and then bloned with primary antibody against human fibronectin. Bands

were visualized using Alkaîine phosphatase conjugated secondary antibody. Culnue

supematants h m cells treated with m-SspB + 864, TNF-alpha, and media alone (Fig

11, Lane 3.4,s respectively) carried intact fibronectin that CO-migrated with undigested

fibronectin (Fig 11, Lane 1). In contrast, fibronectin in the supernatant from cells treated

with rn-SspB was at least partially degraded. The degradation pattern, however, was not

identical to that which was seen after fibronectin was digested by m-SspB for 24 h in

PBS + 10 mM cysteine (Fig 1 1). Instead, it appeared to mimic the pattem of partial

degradation seen after 2 h of digestion with m-SspB (Fig 9, Lane 1). This result can be

explained by several factors. Firstiy, fibronectin is not the only substrate for mSspB. We

have established that m-SspB is also capable of degrading vitronectin and other

extracellular matrix protein substrates could also be possible. Thus, d l the proteas

activity of m-SspB will not be devoted only to the degradation of fibronectin as is the

case when m-SspB is incubated directly with fibronectin. Moreover, fibronectin in

keratinocyte cell culture is part of a complex network of extracellular matrix proteins and

also binds integrins on the keratinocyte ce11 surface. In this environment, which may be

more representative of the scenario in vivo, access of m-SspB to fibronectin may be far

more restricted than when they are directly incubated together. We, therefore, have

found that mSspB is capable of cleaving fibronectin and degrading vitronectin, whereas

the pSspB is not, and that degradation of fibronectin is associated with keratinocyte ce11

detachment. Degradation of extracellular matrix proteins by m-SspB is an important and

interesting finding and may implicate an important virulence function for m-SspB.

Figure 9: Degradation of Fn by m-SspB. 10 pg of human Fn was treated with either

1 pg of m-SspB or pSspB at 37' C in PBS containing 10 mM cysteine for various

amounts of time. The resulting protein sarnples were analyzed by 12 % SDS-PAGE

(100 V, 1.5 h). Fn treatments with m-SspB after 2.7 and 20 h are shown in Lanes 1,

2 and 3 respectively. Fn treated with pSspB for 20 h is shown in Lane 5 and Fn

incubated in PBS containing 10 mM cysteine and no protease is shown in Lane 4.

Figure H: Degradation of Vn by m-SspB. 5 pg of Vn was treated with 0.25 w m-

SspB or pSspB in O. l M Tris-HCl pH 7.4'0. 15 M NaCl containing 10 mM cysteine

at 37' C for various amounts of time. The resulting protein samples were first boiled

for 5 min in non-reducing containing 28 E-64 and then reboiled in 1xRB for 5

min. The result of Vn aeamient with m-SspB after 15 min, 30 min, 1,2, and 4 h is

shown in Lanes 1,2,3,4, and 5 respectively. Treatment of Vn with m-SspB after 4 h

in the presence of 28 pM E-64 is shown in Lane 6. Vn migrates at 65 kDa and 75

kDa and this was the pattern that was observed after Vn was incubated at 37' C in 10

mM cysteine alone for 4h (Lane 7). Lane 8 displays the result of treating Vn with p-

SspB for 4 h.

Figure 11: Western Blot of fibronectin in keratinocyte culture supematants. Protein

samples wen Loaded on 12 % SDS-PAGE and transfered to nitrocellulose membrane.

Blotting with polycional antibody to human fihnection. Bands detected by aikaline

phosphatase conjugated secondary antibody. Fibronectin is shown in Lane 1.

Fibronectin after treatment with mSspB for 24 h is shown in Lane 2. Lanes 3-6

represent fibronectin detected in culture supematants coliected fkom keratinocytes

tteated with m-SspB, m-SspB +E-64, TNF-alpha and media alone for 24 h respectively.

EX. m-SspB Substrate Selectivity A-.- - -

To ensure that m-SspB has a specific proteolytic hinction and is not a general,

broad-range degradative protease, we tested the activity of SspB on other protein

substrates that are not specific to the extracellular matrix. 10 pg of Bovine Serum

Albumin (BSA), rabbit IgG, and human fibnnogen were treated with lpg of either m-

SspB or p-SspB in PBS containing 10 mM cysteine at 37OC for 2.4.8 and 20 h. Protein

sarnples were analyzed by SDS-PAGE and revealed that even after 20 h of treatment,

neither p-SspB nor m-SspB has any significant activity on any of the substrates (Figure

12).

Revious work in our lab has shown that the precursor and mature form of SspB

lack activity on casein zyrnography. To confirm this result, we tested the activity of the

purified p-SspB and m-SspB on resorufin-labeled casein. Briefly, 500 ngirnl of p-

SspB/m-SspB were incubated with nsorfin-lakled casein for 18 h at 37' C and the

was taken after the reaction was stopped. 250 n g h l of V8 protease was included as a

positive control. Relative to V8 protease, neither form of SspB showed significant

activity toward casein by this assay. Samples containing SspB gave ODS4, close to those

that were obtained h m blank readings, confirming the lack of activity of SspB against

casein (Table 6). This apparent substrate selectivity supports the idea that SspB possesses

a specific role in virulence. The relative inactivity of the precursor form of SspB may

indicate that the 40 kDa protein is in fact a zyrnogen, (detectable by gelatin zyrnography)

which upon cleavage by V8 protease is converted to its active form. This type of p s t -

translational regdation of SspB may be important to ensure that proteme activity does

not occur within the bacterial ce11 before the pmtease is secreted.

BSA IgG Fbg kDa

1 2 3 1 2 3 1 2 3

Figure 12: m-SspB Substrate Selectivity. 10 pg each of BSA, IgG and Fbg were

treated with 1 pg of p-SspB (Lane 1) or m-SspB (Lane 2) in PBS containing 10

mM cysteine for 20 h at 37OC. The resulting sarnples were boiled for 5 min in

lxRB and analyzed by 12 % SDS-PAGE (100 V, 1.5 h). Lane 3 shows the

protein substrate after 20 h of incubation at 37' C in PBS containing 10 mM

cysteine and no protease.

22kDa Cysteine Protease

*ODs74nm as a measure of digested resonifin-labeled casein after 18 h exposure to SspB

and V8 protease.

4OkDa Cysteine Protease

V8 Protease

Blank (water)

0.07 10 0.0707

0.0282

0.3090

0.0430

0.0346

0.2682

0.0424

-~ .

---- - - -

PART IV: DISCUSSION -- - -

1. Expression of SspB precursor

Results of this work involving the expression of the precursor form of SspB has

suggested that SspC may confer a chaperone function, assisting in the proper folding of

the SspB precursor. This is supported by the observation that SspB expressed in the

absence of SspC exhibited decreased electrophoretic mobility and a significant loss of

protease activity. There are several examples of bacterial proteins that are secreted as

precursors where proper secretion and folding is facilitated through a chaperone hinction.

Staphylococcal lipases are organized as pre-pro-enzymes that are secreted in the pro-

lipase fonn and processed to the mature fom by an extracellular protease. There is

evidence that the pro region acts as an intramolecular chaperone facilitating the

translocation of the native lipase, and also protecting the proteins from degradation (56).

The proregion of the Bombyx mori cysteine protease also functions as an intermolecular

chaperone to promote the proper folding of the mature enzyrne(l93). The prosequence of

themolysin, produced by Bacillus themwproteolyticus has also been shown to act as an

intramolecular chaperone and is requind to obtain active themolysin (1 14). The

possibility that the pre-propeptide N-terminal domain of SspB acts in this manner must

also be considered and should be investigated through purification of the 18 kDa N-

terminal region. However, our finding that sspC follows sspB in an operon structure, and

may act as a chaperone to facilitate its secretion appears to be a unique arrangement, and

is woah of more detailed analysis. Staphopain, another of the cysteine proteases secreted

by S. aureus, is also followed by downstrearn genes. One such gene codes for a protein -- -- - . - 2

that is similar in structure to the predicted SspC protein structure.

IL SspB Activity on Benzoyl-Pro-Phe-Arg p-nitroenilide

The activity of mSspB and p-SspB was determined using the synthetic

chromogenic substrate, Benzoyl-Pro-Phe-Arg p-nitroanilide. mSspB was able to cleave

this chromogenic substrate, while pSspB displayed approximately one tenth of the

activity of m-SspB on the same substrate. The fact that m-SspB is capable of cleaving

this peptide is a significant one given that it npresents a substrate for plasma kallikrein

function. Plasma kallikrein, the active enzyme, is f o m d as a consequence of the

cleavage of prekallikrein by factor Xna or factor W (89). Human prekallibein is a

single chah g-globulin synthesized and secreted by hepatocytes ( 195). Plasma kalli krein

releases bradykinin from high molecular weight kininogen by hydrolysis (195). Factor

W, and prourokinase are also two major protein substrates of plasma kallikrein (195).

Bradykinin exerts powerful biological activities, and at the site of infectiodinflarnmation

is responsible for pain and local extravasation leading to edema white, at a systemic level,

it enhances the development of hypotension and shock (195). This potent mediator is -

released from high-molecular-weight kininogen by plasma kallikrein (89). Despite the

tight regulation of this pathway, bradykinin generation by pathogenic proteinases is a

universal event occumng during most bacterial infections, (1 1 1; 112) and can greatly

enhance pathogen dissemination h m the local site of infection into the systemic

circulation . Interestingly, SpeB of S. pyogenes has been shown to convert H-kininogen to

biologically active kinin and release of kinins was suggested to be an important potential

virulence mechanism of S. pyogenes (62). The ability of P. gingivulis proteinases to -=-- A- - - - - - ... -- - - - - -

activate the kallikreidkinin pathway was first described by Hinode et al. (65) and

Kaminishi et al., (88) and later studied in detail by hamura et al. (74;75). It was found

that gingipains R are very potent vasnilar permeability enhancement factors, inducing

this activity thrwgh plasma prekallikrein activation and subsequent bradykinin release.

In crude bacterial extracts the vascular permeability enhancement activity was completely

removed by the gingipains R-specific inhibitor, leupeptin, and by anti-RgpB antibodies.

indicating that gingipains R are exclusively responsible for prekallikrein activation (75).

In contrast, gingipain K by itself was not able to induce vascular permeability

enhancement in human plasma. However, working synergistically aith gingipains R, the

pair efficiently released bradykinin directly from high-molecular-weight kininogeii, thus

mimicking the action of kallikrein (74).

The possibility that m-SspB mimics kallikrein fûnction is an important one and

could point to a major virulence function for m-SspB; one that promotes dissemination

and sepsis. It is therefore plausible that m-SspB could be factor contributing to the

development of senous deep tissue and metastatic infections of S. aureus. Thus, the

possible kallikrein mimicry of m-SspB warrants further investigation.

m. Detachment of Keratiweytes and m-SspB

Given that S. aureus is major cause of skin infection and disease, the finding that

mSspB promotes detachment of keratinocytes, the major constituents of the epidennis, is

a meaningfùl one. Several other staphyiococcal factors (including a-toxin) have been

implicated in causing keratinocyte damage, and S. aureus has also been shown to cause

apoptosis in keratinocytes. Furthemore, cysteine protease activity in P. gingivalis has -.- * - - -- - - - - - - - -

been implicated in promoting the detachment and apoptosis of keratinocytes. The

cysteine protease, SpeB has k e n shown to cause a cytopathic effect on endothelial cells,

which was accompanied by the activation of a MME? Our data show that keratinocyte

detachment associated with the cysteine protease activity of mSspB is not mediated by

the activation of MMPs, and keratinocyte detachment was correlated with the degradation

of fibronectin present in the ce11 culture. Whether mSspB causes keratinocyte

detachment indirectly by degrading fibronectin (and vitronectin) or directly causes

keratinocyte detachment independently of its proteolytic activity on ECM proteins is not

known. However, ce11 viability experiments show that there is no significant difference

in the viability of cells that have been treated with mSspB and those that have not,

indicating that SspB does not have a direct necrotic function thereby causing ce11

detachment. Nonetheless, it is plausible that the promotion of keratinocyte detachment

may represent a means for the bacteria to gain access to the extracellular matrix, where

degradation of ECM components could facilitate invasion of the basement membrane and

m e r dissemination. It may also be a contributing factor to the skin damage and

destruction seen in a variety of S. aureus skin infections. The lack of keratinocyte

detachment in the presence of the SspB precursor supports the relative inactivity of the

precursor observed on Benzoyl-Pm-Phe-Arg p-nitroanilide. Also, the precursor has

shown little or no activity on the ECM substrates tested, whereas degradation of

fibronectin in the culture fluid by mature SspB was in fact correlated with keratinocyte

detachment (see below). Together, these results may suggest that pSspB is an inactive

zymogen, as is the case with the precursor form of SpeB. SpeB undergoes autocatalytic

conversion to its mature fonn. We have confirmed that p-SspB undergoes catalytic --.da - - - * -- - --- - - -. - - - - - - -

conversion to its mature fonn, m-SspB through proteolytic action by SspA (V8 protease).

IV. Degradation of Fibronectin and Vitmnecüa by m-SspB

This shidy has assayed the proteolytic activity of SspB and the results have shown

that m-SspB is capable of cleaving fibronectin and degrading vitronectin, whereas the p-

SspB is not, and that degradation of fibronectin is associated with keratinocyte ce11

detachment. Degradation of extracellular rnatrix proteins by m-SspB is a significant and

meaningfbl finding and may suggest that this enzyme is involved in host-pathogen

interactions, and suggest that the protease possesses an important virulence function. We

note that among the îùnctions of the established virulence factor, SpeB, of S. pyogenes, is

its cleavage fibronectin and degradation of vitronectin (92). To hirther elucidate the

specific role in pathogenesis of SspB, it is important to understand the hinctions of those

proteins on which it is proteolyticdly active. Fibromctin is a high-molecular weight

glycoprotein present in plasma and most other body fluids, which is essential for the

adhesion of almost al1 types of marnmalian cells (28;7 1;157). The molecule is associated

with ce11 surfaces and interstitial connective tissue and participates in diverse processes

such as hemostasis, host defense, and ce11 adhesion, migration and differentiation (49).

in addition, fibronectin assists in wound healing by mediating migration and attachment

of monocytes, fibroblasu and epithelial cells to the area of injury (49). Cleavage of

fibronectin by m-SspB in vivo may considerably impair the migration and attachment of

fibroblasts, epithelid cells and monocytes that participate in wound npair and resolution.

Fibronectin also mediates attachment of S. aureus to host tissue through FnBP and

internalization of S. aureus by epitheliai cells is also dependent on FnBP (45;97; 126). . . - --. -..-A-.- - . -2 * - - - - -+- -- -

The proteoiysis of fibronectin caused by m-SspB could therefore also serve to aid in the

reiease of S. aureus cells that are bound to host tissue, once bacterial growth has reached

the invasive phase of infection, thereby facilitating the spread of S. aureus and

potentiating its invasive capacity. This function has also been suggested for the cysteine

protease SpeB of S. pyogenes. SpeB has been characterized as an important virulence

factor for S-pyogenes infections and is capable of degrading fibronectin (92). It was

suggested that the fibronectin-degrading ability of SpeB could theoretically allow S.

pyogenes to detach ftom the host by abrogating the interaction between fibronectin and

the fibmnectin binding protein of S. pyogenes (92). Moreover, SpeB has been shown to

abrogate fibronectin-dependent intemalization of S. pyogenes by cultured mammalian

cells (26). The phenornenon of bacterial detachment from host tissue through the action

of secreted proteases was also reported in Vibrio cholera. This work showed that an

extracellular protease secreted by V. Cholera was able to cause detachment of the

bacteria from intestinal cells through the digestion of putative receptors V. Cholera

adhesins. These data may suggest a "detachase" hinction for m-SspB, in which m-SspB

allows S. aureus to free itself from host tissue targets, namely fibronectin, facilitating

dissemination and invasion within the host or transmission to a new host.

Vitroneetin is a 75 kDa protein found both in the extracellular matrix and in

plasma It is anchored to the extracellular matrix via its collagen-binding and

glycosaminoglycan-binding domain. and it promotes ce11 adhesion and migration by

interacting with integrins and the urokinase receptor (170). The detachment of

keratinocytes and degradation of vitronectin caused by m-SspB may be comlated given

that vitronectin is an important cell-adhesion molecule. Moreover, vitronectin is an - -

important modulator of the immune response involved in recruitment and migration of

polymorphonucleocytes (PMNs) to the site of infection (t 70). Thus, m-SspB may play a

role in enabling S. aureus to evade host defenses. Plasma Vn is found in two forms; a

single chah (75kDa) and a clipped form of two chahs: 65 and 10 kDa (170). In plasma,

vitronectin binds to plasminogen activator inhibitor -1 (PAI-1), and stabilizes its

inhibitory activity giving it an anti-fibrinolytic property (170). Plasminogen activation is

observed in the human epiderrnis dunng nepithelialization of epidemal defects and

under certain pathological conditions. The activation reaction depends on keratinocyte-

associated plasminogen activators (PAS), which convert the ubiquitous proenzyme

plasminogen into the active trypsin-like senne proteinase plasmin. The PAS are

controllcd by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-

2. In vitro and in vivo keratinocytes express both PAIs. Plasminogen activation by human

keratinocytes is thought to play a major role in generating pericellular proteolytic activity

in the epidermis (37;57;79; 125). Plasmin or pericellular proteolysis may be generated by

secreted PAS in the pericellular Buids, as well as by cell-bound PAS, in particular by uPA

while it is bound to a specific ce11 surface receptor (uPA-R) (3737; 1 19). Keratinocytes

are known to express the PA inhibitors PAI-1 and PAL2 in vivo and in

vitro(60;6 1 ;63;64; 80). Cleavage of vitronectin by m-SspB, therefore, may represen t a

mechanism by which mSspB promotes detachment of keratinocytes through pericellular

proteolysis by abrogating the PAI-stabilizing function of vitronectin. Furthemore, in a

more general sense, m-SspB may promote the invasiveness of S. aureus by abolishing the

95

anti-fibrinolytic properties of vitronectin, tipping the balance toward fibnnolysis and - -- -- - - - ---- - -

bacterial dissemination.

The use of a host-denved plasminogen activating system by invasive bacteria is

an increasingly recognized mechanism for acquisition of extracellular proteolytic activity

(29). Staphylokinase, a 15.5-kDa S. aureus protein, ( S M ) expresses plasminogen

activator (PA) activity by forming a complex with plasmin, which in turn activates other

plesminogen molecules to plasmin (81). It is conceivable that mSspB, by degrading

vitronectin, could act in concert with SAK by making more plasmin available for

complex formation. Plasminogen activation has been recently reported for Enterobacteria

(2). This study devised meihods to assess in vitro the role of plasminogen activation in

Enterobacterial degradation of extracellular matrices and their protein components, as

well as in penetration through basement membrane. Development of these methods was

initiated after the findings that Enterobacterial surface structures function in plasminogen

activation, as well as in laminin- andor fibronectin-specific adhesion (2). Enterobacteria

with these properties degrade radiolabeled laminin as well as metabolically labeled

extracellular matrix fiom cultured endothelial or epithelial cells (2). Plasmin-coated

bacteria also penetrate through the reconstituted basement membrane preparation

Matrigel (2). The processes w e n dependent on plasminogen activation by the invasive

bacteria and suggested a pathogenic similarity between Enterobacteria and Nmor cells in

cellular metastasis through tissue barriers (2). Borrelia burgdorferi, the spirochetai agent

of Lyme disease. binds plasminogen in vitro. Exogenously provided urokinase-type

plasminogen (PLG) activator @PA) convem surface-bound PLG to enzymatically active

plasmin. A recent study investigated the capacity of a B. burgdorferi human isolate, once

r,

96

-- -A

complexed with plasmin, to degrade purified extnicellular matrix (KM) components and - - - - - - - - -

an interstitial ECM (30). Plasmin-coated B. burgdorfcn' degraded fibronectin, laminin,

and vitronectin but not collagen (30). incubation of plasminsoated organisms with

biosynthetically radiolabeled native ECM resulted in breakdown of insoluble

glycoprotein, other noncollagenous proteins and collagen (30). B. burgdoiferi is an

invasive bacterial pathogen that may benefit by use of the host's plasminogen activation

system (30). The results of this study have identified mechanisms in which the spirochete

cm use this borrowed proteolytic activity to enhance invasiveness.

Cleavage of extnicellular matrix proteins by m-SspB suggests that this protease is

important in host-pathogen interactions and provides evidence that this enzyme may play

a role in bacterial dissemination, invasion and inhibition of wound healing. Then are

several examples of bacterial pathogens that either directly or indirectly cleave or degrade

ECM proteins. The adhesiveness of three strains of Haemophilus influenzae to the ECM

was examined in a recent study (189). Al1 strains exhibiteà efficient adhesiveness to

reconstituted basement membrane and to ECM h m cultureci human endothelial cells

(189). Two strains efficiently adhered to immobilized laminin, fibronectin, and various

collagens and another strain adhered efficiently to fibronectin and type I and III

collagens, but with low efficiency to laminin (189) . With al1 3 strains, plasmin generated

on H. infzuenzae plasminogen receptors degraded laminin and fibronectin as well as ECM

from human endothelial cells (189). Plasmin bound on H. influenzae cells also

potentiated penetration of bacteria through a basement membrane preparation

nconstituted on membrane filters (189). These results gave evidence for a role of ECM

adherence and plasminogen activation in the spread of H. influenzae through tissue

barriers. The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly ---L-- - . A .

understd but it is ceasonable to assume that there is a contribution from proteases

capable of disnipting the extracellular matrix of the pulmonary epithelium and the blood

vessels. A study was undertaken to identify and characterize collagen-degrading activity

of M. tuberculosis (123). It was shown that a Mycobucterium. tuberculosis strain showed

collagenolytic activity that was four times higher than that of the avirulent strain (123).

The 75 kDa enzyme responsible was divalent cation dependent (123). Other

mycobacterial species and those isolated from patients with tubercutosis also had

collagen degrading activity (1 23).

Cleavage of these extracellular matrix proteins by m-SspB suggests that this

protease is important in host-parasite interactions and provides evidence that this enzyme

may play a role in bacterial dissemination, invasion and inhibition of wound healing.

There are many examples of bactecial pathogens that possess virulence factors that cleave

or degrade ECM proteins.

V. Substrate Selectivity of m-SspB

In an attempt to discem whether mSspB possesses a distinct and selective

protease fûnction rather than a general. broad-range proteolytic activity serving to break

down host proteins and acquire nutrients, the activity of SspB on protein substrates that

are not specific to the extracellular matrix was tested. The results demonstrated that

neither precursor nor mature SspB exhibited proteolytic activity against each of BSA,

IgG, Fbg or casein. The substrate selectivity that is demonstrated by m-SspB may be

indicative of a specific, defined role in virulence and thus malces it's ability to degrade ---- - - . * -. - - -- - - -

fibronectin and vitronectin more interesthg and worthy of further study.

The fmdings pnsented in this work represent a significant step forward in the

charactenzation of the Staphylococcal cysteine protease SspB. As such they represent a

stepping Stone for future examination into the contribution that SspB makes to S. aureus

pathogenesis.

The expression of sspB in tandem with sspC resulted in greater proteolytic

activity and decreased electrophoretic mobility of the SspB precursor. It appears that

SspC may confer a chaperone hrnction to the SspB protein, ensuring the proper folding

and secretion of the Ssp precursor. Subsequent work should invoive hirther studies to

demonstrate the binding of SspC to SspB. Further insight into the interaction between

these two proteins rnight aiso be gained by the generation of a SspC-deficient mutant of

S. aureus.

The mature form of SspB promotes detachment of pnmary human keratinocytes.

This phenornenon was not observed in the presence of cysteine protease inhibitor and

detachment was not observed in presence of the precursor form of SspB. Keratinocytes

exposed to mature SspB did not show a significant difference in ce11 viability, as

deterrnined by trypan blue staining, when compared to cells that retained a normal,

attached morphology. In addition, detachment did not appear to involve the activation of

MMPs as revealed by zyrnography and by the incorporation of MMP inhibitor into the

culture fluid. Furiher studies are necessary to ascertain whether keratinocytes treated with

mature SspB undergo apoptosis.

The detachment of keratinocytes was positively comtated with the ability of

mature SspB to degrade ce11 culture fibronectin. The proteolytic activity of SspB was

also assessed with purified fibronectin and vitronectin, both of which represent major

components of the ECM. In these assays, only mature SspB, and not precursor SspB.

was able to cleave fibronectin and vitmnectin and cleavage was abolished in the presence

of cysteine protease inhibitor. It would prove useful to determine the N-terminal

sequence of the fibronectin cleavage products so as to gain insight into the substrate

specificity of mature SspB.

To assess the substrate selectivity of SspB its proteolytic activity against BS A,

Fbg and rabbit IgG was examined. hterestingly neither form of SspB demonstrated

visible amounts of proteolytic activity on these substrates, which may be indicative of a

specific virulence role for SspB. The mature form of SspB was also able to cleave the

chromogenic substrate Benzoyl-Ro-Phe-Arg pnitroanilide that represents a kalliluein

substrate. The precursor showed relatively no activity on this substrate. Further insight in

ternis of SspB function will be gained thmugh proteolytic assays with other ECM

components as well as kallikrein substrates. Given that mature SspB is able to promote

detachment of keratinocytes, degrade ECM components and possibly mimic kallikrein

hinction it is reasonable to assume that this protease may possess a substantiai virulence

function. It would therefore be very useful to examine clinical isolates of S. aureus, ftom

infections ranging in severity, for the expression of SspB. This would allow for the

discovery of any comlation between virulence of S. aureus strains and SspB expression.

The contribution of SspB to the pathogenesis of S. aureus infections would be effectively

elucidated through an in vivo model. This wiil be accomplished through the use of a

murine tissue abscess mode1 whewin purified SspB will be injected subcutaneously and --- = = - -- ---

any lesions that result will be analysed for histopathology.

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