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The role of toxic shock syndrome Toxin-1 and Staphylococcal Enterotoxin A in the pathogenesis of toxic… Chang, Alex Hongsheng 1992

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THE ROLE OF TOXIC SHOCK SYNDROME TOXIN-1 AND STAPHYLOCOCCALENTEROTOXIN A IN THE PATHOGENESIS OF TOXIC SHOCK SYNDROMEbyALEX HONGSHENG CHANGM. D. Capital Institute of Medicine (Beijing, China)A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMaster of ScienceinTHE FACULTY OF GRADUATE STUDIESEXPERIMENTAL MEDICINEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1992(c) Alex Hongsheng Chang, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) IiDepartment of ^L- x. >ert vv,e41-- c-k/Cr AA ect, vofThe University of British ColumbiaVancouver, CanadaDate ^A. 2.), le 12DE-6 (2188)ABSTRACTToxic shock syndrome (TSS) is a multisystem diseaseassociated with S. aureus infection. Toxic shock syndrometoxin-1 (TSST-1) is implicated in the majority but not allcases. We have previously demonstrated that seroconversionto staphylococcal enterotoxin A (SEA) is also more prevalentin TSS than in non-TSS cases, suggesting its possible rolein TSS. Previous studies have demonstrated that TSST-1 andSEA are superantigens, which bind directly to class IImolecules on antigen presenting cells, and stimulates Tcells bearing specific VB sequences. Whether there aredirect binding sites for TSST-1 and SEA on human resting Tcells remains unclear. Three approaches were taken tofurther examine the role of TSST-1 and SEA in thepathogenesis of TSS: a) to detect TSST-1 and staphylococcalenterotoxin A (SEA) in the culture supernatants of S. aureus by ELISA method, in order to study the production of TSST-1and SEA among 350 isolates of S. aureus collected from TSSpatients and other individuals; b) to characterize thedistribution of these isolates by multilocus enzymeelectrophoresis at 19 enzyme chromosomal loci; and c) tocharacterize receptor-mediated binding of TSST-1 and SEA tohuman T cells, to confirm whether there are direct TSST-1and SEA binding sites on resting T lymphocytes, to study theiiAbstractinteraction between toxin-monocyte membrane fragment complexwith T cells, and the requirement of T cell-monocyte contactin the activation of T cells by TSST-1 or SEA.A sensitive and specific non-competitive enzyme-linkedimmunosorbent assay (ELISA) capable of detecting SEA atconcentrations from 1 to 128 ng/ml was developed. TSST-1 wasdetected using a similar sensitive and reproducible ELISAalready developed in our laboratory. We examined theproduction of TSST-1 and SEA among 350 S. aureus isolatesfrom urogenital and non-urogenital TSS patients, individualswith non-TSS S. aureus infection, and asymptomatic carriersin Vancouver. The clonal distribution of these isolates wasstudied by multilocus enzyme electrophoresis at 19chromosomal enzyme loci in collaboration with Dr. Robert K.Selander in the Department of Biology, Pennsylvania StateUniversity. SEA was frequently produced in TSS, primarily inurogenital TSS isolates compared to non-TSS isolates. 63distinctive electrophoretic types (ETs) were identified. Asingle clone of S. aureus (designated as ET 4) accounted for79% of urogenital TSS isolates and 47% of non-urogenital TSSisolates, as compared to 27% of non-TSS isolates.Furthermore, this clone was less hemolytic (hemolyticactivity: 1.70 ± 0.25 vs other ETs 3.16 + 0.19, p < 0.001);and most likely to co-produce both TSST-1 and SEA (49% ofiiiAbstract116 isolates of ET 4 vs 3% of 234 isolates of other ETs, p <0.01).To enable the characterization of receptor-mediatedbinding of TSST-1 and SEA to human T cells, purified TSST-1and SEA were radiolabeled by the chloramine T procedure.Highly purified human resting T cells and monocytes wereused for the binding assays.Binding assays were performed as previously described.10 7 T cells were incubated with 0 - 40 nM of 125 I-TSST-1 or125 I-SEA for at least 1.5 hours at 4 °C. In contrast tomonocytes there was no detectable binding of 125 I-TSST-1 or125 I-SEA to T cells. However, in the presence of monocytemembrane fragments, specific binding of 125 I-TSST-1 to Tcells was demonstrated in 4 of 7 donors. Similarly, specificbinding of 125 I-SEA to T cells was observed in 3 of the 4donors. The necessity of T cell-monocyte contact for theinduction of T cell activation was demonstrated while Tcells separated from monocytes by a semipermeable membranewhich allowed free diffusion of macromolecules failed tostimulate T cell proliferation by TSST-1 or SEA.We conclude that: 1) the majority of the isolatesrecovered from TSS patients with a urogenital focus co-produce both TSST-1 and SEA; and a single clone of S.aureus, characterized by multilocus enzyme electrophoresis,ivAbstractwhich produces both TSST-1 and SEA causes the majority ofurogenital TSS cases; 2) our study of 125I-TSST-1 and 125 I-SEA binding to highly purified human resting T cellsresolved a controversy over whether there is direct bindingof TSST-1 to human resting T cells. Our results demonstratedthat TSST-1 or SEA alone does not bind to the T cellreceptor and that the presence of monocyte membranecomponents (presumably MHC class II molecules) is needed toenable the efficient binding of these toxins to the T cellreceptor. Furthermore, direct T cell-monocyte contact isrequired for the induction of resting T cell proliferationby TSST-1 or SEA. Further studies are required to clarifythe genetic expression and regulation of staphylococcalexotoxins, the environmental factors which influenceexotoxin expression, and the clonal evolution of S. aureus isolates which cause TSS. Our method of binding assay isvaluable in approaching the interaction of staphylococcalexotoxin-class II molecule complex with the TcR. Howeverfurther modification may be required to decrease the non-specific binding activity and increase the sensitivity ofthe assay.vTABLE OF CONTENTSAbstract^ iiTable of Contents^ viList of Tables ixList of Figures^List of Abbreviations^ xiAcknowledgement^ xiiiCHAPTER 1INTRODUCTION AND OBJECTIVES^ 1CHAPTER 2LITERATURE REVIEW^ 52.1 ETIOLOGY OF TSS 52.2 TYPING OF TSS-ASSOCIATED S. aureus^92.3 GENETICS OF TOXIN PRODUCTION^ 122.3.1 TSST-1^ 122.3.2 Staphylococcal enterotoxins 142.4 TSS-ASSOCIATED TOXINS^ 162.4.1 Structure of staphylococcal toxins^ 162.4.2 Interaction of staphylococcal toxins with themajor histocompatibility complex 182.4.3 Interaction of staphylococcal toxins with the Tcell receptor^ 202.4.4 Biological activities of Staphylococcal toxins^ 22CHAPTER 3MATERIALS AND METHODS^ 293.1 TSST-1 AND SEA DETECTION BY ELISA^ 293.1.1 Preparation of bacterial cultural filtrates ^ 293.1.2 Purification of anti-SEA IgG from anti-SEAantiserum with Protein A-Sepharose CL-4B^ 293.1.3 TSST-1 and staphylococcal enterotoxins 303.1.4 Conjugation of rabbit anti-SEA to alkalinephosphatase^ 313.1.5 Quantitation of TSST-1 by noncompetitive ELISA^ 32viTable of Contents3.1.6 Quantitation of SEA by noncompetitive ELISA ^ 333.2 HEMOLYTIC ACTIVITY^ 343.3 ELECTROPHORESIS OF ENZYMES^ 353.4 STATISTICAL METHODS 363.5 PURIFICATION OF HUMAN T CELLS AND MONOCYTES^ 373.5.1 Fractionation of human peripheral bloodmononuclear cells^ 373.5.2 AET treated sheep red blood cell rosetting toseparate T cells from non-T cells^ 383.5.3 Treatment of mononuclear cells with AET treatedSRBC^ 383.5.4 Purification of monocytes from non-rosettedcells 393.5.5 Purification of T cells from rosetted cells ^ 393.6 IODINATION OF TSST-1 AND SEA BY THE CHLORAMINE TMETHOD^ 403.7 BINDING ASSAY^ , ^ 403.7.1 Direct binding of Ut I-TSST-1 or itI-SEA to Tcells and monocytes^ 413.7.2 Binding of labeled toxin-monocyte membranefragment complex to T cells^ 413.8 T CELL MITOGENICITY ASSAY 42CHAPTER 4RESULTS^ 444.1 SEA DETECTION BY ELISA^ 444.2 TSST-1 AND SEA PRODUCTION AMONG 350 ISOLATES OF S.aureus^444.3 ELECTROPHORETIC TYPES 484.4 TSST-1 AND SEA PRODUCTION IN ET 4 ISOLATES^ 554.5 HEMOLYTIC ACTIVITY^ 584.6 CHARACTERIZATION OF RADIOLABELED TOXIN 584.7 BINDING OF RADIOLABELED TOXIN TO PURIFIED T CELLS ANDMONOCYTES^ 614.8 BINDING OF RADIOLABELED TOXIN-MONOCYTE MEMBRANEFRAGMENTS TO T CELLS^ 634.9 T CELL MITOGENICITY ASSAY USING TRANSWELL FILTERINSERTS^ 65CHAPTER 5DISCUSSION^ 68CHAPTER 6CONCLUSION^ 83viiTable of ContentsBIBLIOGRAPHY ^ 8 6viiiLIST OF TABLESTable 1. Case definition of toxic shock syndrome^ 6Table 2. Diseases caused by the staphylococcal enterotoxin-like toxins^ 9Table 3. Genetic basis for staphylococcal toxins^ 12Table 4. Classification of staphylococcal exoproteinexpression (agr) ^ 13Table 5. Biochemical properties of staphylococcal toxins ^ 17Table 6. VB specificity of staphylococcal toxins^ 21Table 7. Biological activities of staphylococcal toxins ^ 22Table 8. TSST-1 and SEA production in 350 isolates of S.aureus^ 47Table 9. Allele profiles of 350 S. aureus isolates^ 50Table 10. The genetic diversity at 19 enzyme loci and clonaldistribution of 350 isolates of S. aureus^ 51Table 11. Properties of 350 isolates of S. aureusrepresenting 63 ETs^ 54Table 12. TSST-1 and SEA production in ET 4 S. aureus isolates compared with other ETs^ 57Table 13. Hemolytic activity of urogenital and non-urogenital S. aureus isolates 59Table 14. Hemolytic activity of ET 4 S. aureus isolatescompared with isolates of other ETs^ 59Table 15. Binding of 1251-TSST-1-MMF complex with T cells ^ 64Table 16. Binding of 125 1-SEA-MMF complex with T cells^ 64ixLIST OF FIGURESFigure 1. Hypothetical structure for the complex of MHCclass II, T cell receptor, and superantigen^ 16Figure 2. Typical standard curve and regression analysis ofthe non-competitive ELISA method for quantitation ofextracellular SEA in the supernatant of S. aureus^ 46Figure 3. Dendrogram showing relationship of 63 ETs of 350isolates of S. aureus^ 52Figure 4. Autoradiography of 125 I-TSST-1 and 125I-SEA^ 60Figure 5. Repvgentative specific binding curve of 125I-TSST-1 or -L" I-SEA with monocytes and T cells^ 62Figure 6. Proliferative responses of staphylococcal toxin-treated T cells separated from monocytes by asemipermeable membrane^  66xLIST OF ABBREVIATIONSagr^ Accessory gene regulatorAPC(s)^ Antigen presenting cell(s)CD Cluster of differentiationELISA^ Enzyme-linked immunosorbent assayentA The gene for SEAentB^ The gene for SEBentC The gene for SECentD^ The gene for SEDentE The gene for SEEET(s) ^ Electrophoretic type(s)FCS Fetal calf serumHLA^ Human lymphocyte associated Antigen(s)Ia MHC class II moleculesIFN^ InterferonIgG Immunoglobulin GIL-1^ Interleukin-1IL-2 Interleukin-2Mab^ Monoclonal antibodyMHC Major histocompatibility complexMls Ag(s) ^ Minor lymphocyte stimulating AntigensMMF^ Monocyte membrane fragmentsMMTV Murine mammary tumor virusMW^ Molecular weightx iList of AbbreviationsPBMC^ Peripheral blood mononuclear cellPCR Polymerase chain reactionPEC^ Pyrogenic exotoxin type C(TSST-i)PGE Prostaglandin ERES^ Reticular endothelial systemRFLP Restrictive fragments length polymorphismSDS-PAGE^ Sodium dodecyl sulfate-polyacrylamide gelelectrophoresisSEA^ Staphylococcal enterotoxin ASEB Staphylococcal enterotoxin BSEC^ Staphylococcal enterotoxin CSED Staphylococcal enterotoxin DSEE^ Staphylococcal enterotoxin ESEF Staphylococcal enterotoxin F (TSST-1)SPE(s) ^ Streptococcal pyrogenic exotoxin(s)SRBC Sheep red blood cellTcR^ T cell receptorTNF Tumor necrosis factorTSS^ Toxic shock syndromeTSST-1^ Toxic shock syndrome toxin-1tst The gene for TSST-1VB^ VB region of T cell receptorxiiACKNOWLEDGEMENTI gratefully acknowledge the guidance, support andencouragement of Dr. Anthony W. chow throughout the courseof this study. I am also very thankful to Dr. Grant Stiverand Dr. Donna Hogge for their effort in co-supervising mystudy.This study could not complete without theelectophoretic typing work previously done in Dr. Robert K.Selander's Laboratory in Department of Biology, PennsylvaniaState University, and the weekly supply of precious blood bythe staff headed by director Dr. Donna Hogge in CellSeparator Unit, Vancouver General Hospital.I also wish to thank Raymond H. See and Dr. Winnie Kumfor their greatly appreciated technical advice and inspiringdiscussions.This Master's project was carried out as part of theCanada-China University Linkage Program sponsored by theCanadian International Development Agency (project No. 908-282/14218, to Dr. H. G. Stiver).This thesis is dedicated to my wife YanboCHAPTER 1INTRODUCTION AND OBJECTIVESStaphylococcal toxic shock syndrome (TSS) is adevastating illness which affects menstruating women who usetampons exclusively, but other forms of the illness in menand children are not uncommon (2-4, 8, 10-13, 15-18). Thepathogenesis of the illness remains obscure, although toxicshock toxin-1 (TSST-1) is the primary but not the only toxinimplicated in TSS. Staphylococcal enterotoxins (SEA, SEB,SEC, SED, SEE) and streptococcal pyrogenic exotoxins (SPE A,B, C) are also implicated in TSS(21-24, 34, 35).Numerous investigations have attempted to identifyunique phenotypic and genotypic characteristics of TSS-associated S. aureus isolates. Among them, multilocus enzymeelectrophoresis, which yields estimates of diversity in thechromosomal genome and the genetic relatedness of isolatesis proven to be a powerful method to elucidate the clonalpopulation structure and genetic relationships of pathogenicTSS isolates (33, 58, 59).TSST-1 and staphylococcal enterotoxins have manydistinctive physiochemical and biological characteristics.They are intermediately sized, single-chain proteins with MWof approximately 22-30 kd (72). They are the most potent Tcell mitogens known. The ability of staphylococcal toxins to1INTRODUCTION AND OBJECTIVESstimulate large number of T cells bearing specific VBsequences in the presence of MHC class II molecules is thebasis for categorizing them as superantigens (97).These toxins have significant binding affinities forMHC class II molecules on the antigen-presenting cells (80-84). There is no MHC species restriction in binding and noprior processing is required for their presentation (86,87). In addition to the class II receptor on antigen-presenting cells, considerable insight has been gained intothe nature of the staphylococcal toxin receptor on T cells.There is a controversy about whether there are directbinding receptors for TSST-1 on T cells. Schlievert's group(101) demonstrated that there were TSST-1 receptors on humanT cells and reported that they were similarly distributed inCD4+ and CD8+ T cells; while Scholl et al. and Uchiyama etal. (83, 102, 103) did not find direct TSST-1 binding to Tcells, but noted that class II molecules on antigenpresenting cells were required for T cell activation byTSST-l. Until recently, there was no definitive evidence fordirect binding of Staphylococcal toxins to TcR.Based on these studies, we believe that: 1) TSST-1,either alone or in combination with other staphylococcalexotoxins, is the primary cause of TSS; 2) mutilocus enzymeelectrophoresis may be a useful method in studying genetic2INTRODUCTION AND OBJECTIVESrelationship of toxic shock syndrome-associated S. aureus isolates; 3) the receptor mediated binding of staphylococcaltoxins to monocytes and T cells may be crucial for thepathogenesis of TSS; 4) effective treatment and preventionwill require better understanding of the molecular basis andthe biological interaction of these toxins with their targetcells.My Specific aims were: 1) to develop a noncompetitiveELISA to detect staphylococcal enterotoxin A (SEA) inculture supernatants of S. aureus, and to study theproduction of TSST-1 and SEA among S. aureus isolates fromTSS and non-TSS individuals; 2) to characterize the geneticdistribution of these isolates by multilocus enzymeelectrophoresis at 19 chromosomal loci; 3) to characterizereceptor-mediated binding of TSST-1 and SEA to human Tcells, to confirm whether there are direct TSST-1 bindingsites on resting T lymphocytes, to study the interactionbetween toxin-monocyte membrane fragment complex with Tcells and to determine the requirement of T cell-monocytecontact in the induction of T cell proliferation by TSST-1or SEA.These studies will provide new insights into the roleof TSST-1 and SEA in the pathogenesis of TSS, the clonalstructure and genetic relationships of TSS-associated and3INTRODUCTION AND OBJECTIVESnon-TSS-associated S. aureus isolates, as well as theassociation of toxins production with specific S. aureus clusters; also, the interaction of TSST-1 and SEA with the Tcell receptor and the requirement of T cell-monocytescontact in the induction of T cell activation by TSST-1 andSEA will be examined.4CHAPTER 2LITERATURE REVIEW2.1 ETIOLOGY OF TSSToxic shock syndrome (TSS) is an acute onsetmultisystem illness characterized by fever, hypotensionor dizziness, scarlet fever like rash, desquamation ofthe skin upon recovery, and a variable multiorganinvolvement(1-14) (Table 1). TSS was brought to theattention of the medical community by Todd et al. in 1978(1), who also first reported the association of TSS withS. aureus. Use of tampons during menstruation wassubsequently found to be a risk factor for TSS (2-4, 8,10, 12, 13). Major reports of nonmenstrual TSS whichoriginated from wound infection by S. aureus became moreprevalent after 1982 (11, 15-18).Most recently, streptococcal TSS, possibly a severeform of scarlet fever, is now recognized as associatedwith group A streptococcal infections (19).In many cases of TSS, the causative organisms remainlocalized despite the presence of systemic manifestations(19). Toxins produced by S. aureus and group Astreptococci are strongly implicated as etiologicalagents in the pathogenesis of the illness (20-22).5LITERATURE REVIEWTable 1. Case definition of toxic shock syndrome (19)1. Fever, temperature >38.9 °C2. Rash3. Desquamation upon recovery4. Hypotension5. Involvement of three or more organ systems:a. gastrointestinalb. muscularc. mucous membrane, reddeningd. hepatice. renalf. cardiovascularg. central nervous systemThe first toxin shown to be involved in TSS is nowreferred to as toxic shock syndrome toxin 1 (TSST-1).This toxin was first identified and characterized in 1981through the independent investigations of Schlievert etal. and Bergdoll et al. (23, 24) and was named aspyrogenic exotoxin type C (PEC) and staphylococcalenterotoxin F, respectively, by these two groups ofresearchers.TSST-1 production can be demonstrated in over 90% ofS. aureus isolates associated with menstrual TSS, 50-60%of isolates from nonmenstrual cases and 5-25% of isolatescausing other diseases (15, 16, 23-26). Crass et al. (17,27) in their studies of the production of TSST-1 andenterotoxins by staphylococcal isolates of TSS patientsshowed that apart from TSST-1, staphylococcalenterotoxins SEA, SEB, SEC were also involved in TSS. SEA6LITERATURE REVIEWand SEC tend to be co-produced with TSST-1, while SEBproduction is more independent of TSST-1 or otherenterotoxins. SEB production was also significantly morecommon in nonmenstrual than menstrual cases (50% vs 15%,respectively). Experiments in animal models have alsoshown that purified TSST-1, or enterotoxins produce manysigns and symptoms similar to those observed in human TSS(28-32).The reason for the differences in toxin productionbetween menstrual (vaginal) and nonmenstrual (nonvaginal)isolates is not known. One hypothesis is that TSST-1production may be linked to other phenotypiccharacteristics that enable the organism to colonize thevagina (19). TSST-1 positive isolates, such as thoselikely to cause menstrual TSS, produce less hemolysin,lipase and nuclease than isolates which colonize theskin. Another hypothesis is that isolates from a specificclone of S. aureus which is more adapted to thecervicovaginal milieu may regulate expression of thetoxin gene differently from other clones, perhaps as aconsequence of allelic variation in the tst gene itselfor in the accessory gene regulator (agr), a gene locuscoding for a trans-activator of many exoprotein genes(33).7LITERATURE REVIEWThe streptococal toxins have long been known toinduce the symptoms of scarlet fever (34, 35). Thesetoxins are known as streptococcal pyrogenic exotoxins(SPEs). There are three antigenically distinct SPEs, SPEA, B and C, respectively (35-37).Although the clinical similarities of TSS tostreptococcal scarlet fever have been noted since theinitial description of TSS (Table 2), it was not until1987 that a description of two well-defined case reportsled to the recognition of a severe toxic shock syndrome-like streptococcal illness, called streptococcal TSS(39).The role of SEA in TSS is not very wellcharacterized, although SEA was shown to have a tendencyto co-produce with TSST-1 (17, 27). It is especiallyinteresting for us to study SEA production in TSS as wellas non-TSS isolates, since earlier findings in ourlaboratory had demonstrated that significantly morefrequent seroconversions to SEA as well as TSST-1 amongTSS patients as compared to patients with non-TSSassociated S. aureus infections (159).8LITERATURE REVIEWTable 2. Diseases caused by the staphylococcalenterotoxin-like toxins (71)ToxinToxic shock syndrome toxin-1Staphylococcal enteroltoxinsA, B, Cl, C2, C3, D, EGroup A streptococcal pyrogenicexotoxins (SPEs) A, B, and CM. arthritis mitogenDisease AssociatedToxic shock syndromeFood poisoning,Toxic shock syndromeScarlet fever, Toxicshock like syndromeShock, arthritis inrodents2.2 TYPING OF TSS-ASSOCIATED S. AUREUSNumerous investigations have attempted to identifyunique phenotypic characteristics of TSS-associatedisolates. As a group, these isolates more frequentlyexhibit proteolytic activity in vitro (40, 41), produceless hemolysis on sheep-blood agar (40-43), and lessfrequently harbor plasmids than do control isolates (41,44). TSS isolates also differ from other S. aureus isolates in having a higher frequency of resistance toheavy metal and antibiotics (40, 45), as well as in otherphenotypic characteristics (46-48).Phage typing, widely practiced for epidemiologicalmonitoring, is based on strain variations insusceptibility to a standard set of typing phages, all of9LITERATURE REVIEWwhich are naturally occurring temperate phages or clear-plaque mutants thereof. It has been recognized for anumber of years that many cases of menstrual TSS arecaused by S. aureus strains of bacteriophage lytic groupI (45, 49-51), but 30-40% of isolates are nontypeablewith the panel of phages in the International Basic Setfor phage typing S. aureus (50). Strain discriminationrecently has been achieved by analysis of restrictionfragment length polymorphism (RFLP) patterns of genomicDNA, with a segment of the TSST-1 structural gene (tst)used as a probe (44, 52, 53). However, neither phagesubtyping nor RFLP analysis provided a basis forunderstanding the genetic structure of populations orassociations of chromosomal background and virulencefactor expression (33).Bacterial population genetics has emerged as an areaof considerable activity and interest in microbiology(54-57). Bacteria is unique and attractive candidates forboth descriptive and experimental studies of evolutionaryprocesses, because of their extensive genetic andphenotypic diversity, short generation times, haploidchromosomal genomes, and accessory plasmids, phages, andtransposable genetic elements.10LITERATURE REVIEWA primarily empirical method currently used inbacterial population genetics research is multilocusenzyme electrophoresis, which yields estimates of geneticdiversity in the chromosomal genome and the geneticrelatedness of different strains (58). The key concept ofthis method is that electromorphs (mobility variants) ofthe bacterial cellular enzyme can be directly equatedwith alleles of the corresponding structural gene andthat electromorph profiles over a sample of differentenzymes (electrophoretic types or ETs) thereforecorrespond to multilocus chromosomal genotypes (58, 59).In a study of multilocus genotypic variation of 20enzymes among 315 isolates of S. aureus that expressedtoxic shock syndrome toxin-1 (TSST-1) and were recoveredprimarily from humans with toxic shock syndrome (TSS),Musser et al. (33) identified 49 distinctive ETs, orclones. Cluster analysis revealed two major phylogeneticdivisions separated at a genetic distance of 0.35 (theproportion of enzyme loci at which dissimilar allelesoccur). A single clone (ET 41) belonging to the smallerof the two divisions accounted for 88% of cases of TSSwith a female urogenital focus, and 53% of total TSScases involving non-urogenital infections. Thisobservation and the recovery of isolates of ET 41 from11LITERATURE REVIEWthe genital tract of a large proportion of asymptomaticcarriers strongly suggests that this clone is especiallyadapted for colonization of the female urogenital site.Further studies are needed to examine the clonalstructure and genetic relationships of TSS and non-TSSstrains and the expression of TSST-1 and staphylococcalenterotoxins, such as SEA, in order to clarify the roleof TSST-1 and staphylococcal enterotoxins in thepathogenesis of TSS, and the clonal origin of the strainscausing TSS.Table 3. Genetic basis for staphylococcal toxins (19)Exoproteins^Genetic determinants^ReferencesTSST-1 (61)SEA^Phages^ (65)SEBSEC1SED^penicillinase plasmid^(66)SEE Phage-like element?a. TSST-1 is not associated with either bacteriophage orplasmid DNA.b. The current data suggest SEE may be carried by phage.c. The current data suggest that most of staphylococcalexotoxins are capable of heterologous, but limitedchromosomal insertion.2.3 GENETICS OF TOXIN PRODUCTION2.3.1 TSST-112LITERATURE REVIEWTable 4. Classification of staphylococcal exoproteinexpression (agr)^(180)Class^ExoproteinI^a-hemolysinserine protease(V8)TSST-16-hemolysinGenehlasprtsthidExpression inagr+^agr-High^Undetectabe"^II"^II"^IIII nuclease nuc High Low Moderate13-hymolysin hlb " IIenterotoxin B entB " IIIII protein A spa Low Highcoagulasefibronectin-bindingcoa II 'Iprotein fbn II IIIV B-lactamase bia no effects no effectsenterotoxin A entA " 11The tst gene is located on a genetic determinantthat is capable of heterologous chromosomal insertion andlysogeny is not involved in TSST-1 production (60, 61)(Table 3). The TSST-1 genetic element is absent innontoxigenic S. aureus strains (61). There appears to bemultiple, but a limited number, of integration sites forthe element in the staphylococcal chromosome. Based uponthe unusually high rate of tryptophan auxotypy observedfor TSST-1 producing strains, it has been suggested thatthis operon may be a preferred insertion site (47).Studies on the genetic regulation of TSST-1 hasresulted in the demonstration of one or more trans-acting13LITERATURE REVIEWregulatory elements which control expression of severalS. aureus exoproteins (62, 63). The regulatory elementwas designated agr (accessory gene regulator). Thepleotrophic effect was shown to act at the level oftranscription (63) (Table 4).2.3.2 Staphylococcal enterotoxinsGenetic systems for the staphylococcal enterotoxinsare diverse. For instance, the gene for SEA (entA) isharbored on a family of heterogeneous phages that appearcapable of integration at several chromosomal loci (64,65). Probing experiments for SEA revealed considerablepolymorphyism in lengths of restriction fragmentscontaining the gene. Most entA containing phages appearto be defective and cannot be integrated into the S.aureus chromosome. In contrast, entD is contained on aplasmid that also encodes a penicillinase (66). Althoughstudies with entB and entC1 have demonstrated somesimilarities with the genetic systems for SEA and SED,several differences are evident. Work done on SEB-producers suggests that entB is transferred by ahitchhiking transposon (67). In these systems, the mobilegene on a site-specific element has a high transpositionfrequency. Transposition onto a carrier plasmid is14LITERATURE REVIEWrequired for mobilization. Evidence suggests that theSEC1 element is under similar regulation. Other evidencesuggests that entB may be harbored on either thechromosome or a plasmid (68).Southern hybridization using toxin gene probesreveals a high degree of restriction length polymorphismfor entB and entC1 (69). Human strains that were TSST-1negative had extensive restriction length polymorphismfor entC1. In contrast, strains that co-produced TSST-1and SEC1 displayed little variability, although human andanimal strains had clearly distinct hybridizationpatterns. In a very rare strain which co-produces SEB andSEC1, both genes are harbored on the same plasmid (70).This plasmid, which also encodes penicillin resistance,occasionally integrates into the chromosome.Collectively the existing evidence suggests thatTSST-1 and staphylococcal enterotoxins may be containedon mobile genetic elements, similar to hitchhikingtransposons described previously (67). Presumably, theseelements have a limited number of preferred chromosomalintegration sites, which could explain the restrictionlength variability, the influence of tst on entC1 probingprofiles, and also the mutually exclusive occurrence or154niupertigensAntigenPresceentingllLITERATURE REVIEWrarity of some toxin combinations (TSST-1 and SEB; SEC1and SEB).2.4 TSS-ASSOCIATED TOXINS2.4.1 Structure of staphylococcal toxinsTSST-1 and staphylococcal enterotoxins are alsoknown as microbial superantigens because of their abilityto stimulate large number of T cells bearing specific VBT oellFigure 1. Hypothetical structure for the complex of MHCclass II molecule, T cell receptor, and superantigen. Thediagram shows a MHC class II molecule, in contact with aT cell receptor and a staphylococcal toxin or Mlsproduct. Ag, the probable site of binding of aconventional antigenic peptide (71).16LITERATURE REVIEWTable 5.(72)Biochemical Properties of staphylococcal toxinsMolecularToxin mass (kd) pI DisulfideTSST-1 22.0 7.2 NoSEA 27.8 7.3 YesSEB 28.3 8.6 YesSEC1 27.5 8.6 YesSED 27.3 7.4 YesSEE 29.6 7.0 Yessequences in the presence of MHC Class II molecules(Figure 1).Staphylococcal enterotoxins are intermediatelysized, single-chain proteins with MW of approximately 22-30 kd (Table 5). They are charged (isoelectric pointsfrom 7.0 to 8.6); acid and heat stable; and rich inserine, threonine, and aspartic acid residues, whichtogether account for about 25-30% of their amino acids.With the exception of TSST-1, staphylococcal enterotoxinshave a centrally located disulfide loop.Toxins associated with TSS share many biological andphysiochemical properties as a direct consequence ofsequence homology at the amino acid and nucleotide levels(19, 71). SEA is related in amino acid sequence to SEEand SED, whereas SEB has greater homology with SEC (71).The two streptococcal toxins, SPE A and C, are about assimilar to each of the staphylococcal toxin groups as17LITERATURE REVIEWthey are to each other (71). The homology among thestaphylococcal enterotoxins has been suggested to bedescended from a common gene (71). TSST-1 shares minimalsequence homology with other toxins. Thus, the functionalsimilarity of TSST-1 to other enterotoxins may depend onan active site maintained at the level of secondary ortertiary structure (72).2.4.2 Interaction of staphylococcal toxins with the majorhistocompatibility complexMany of these toxins have significant bindingaffinities for Ia (MHC class II) molecules on theantigen-presenting cells that are involved with theimmune response and stimulation of T cells (80-84).Presentation of these toxins differs in two importantways from conventional protein antigen presentation.First, presentation of TSST-1 and staphylococcalenterotoxins is not MHC restricted, so that human cellscan present enterotoxin to mouse T cells as effectivelyas mouse antigen-presenting cells. However, thispresentation capability varies among different MHC classII haplotypes (85). Second, the prior processing orproteolysis to peptide fragments that is required forconventional antigens is not necessary for these toxins18LITERATURE REVIEW(86, 87). In the mouse, staphylococcal enterotoxins bindpreferentially to the I-E isotype, whereas others bindboth I-A and I-E (73). In human, they typically bind tohuman leukocyte antigens HLA-DR and -DQ, but not -DP.Direct measurements of binding affinities and of T cellreactivity suggest that the S. aureus toxins bind moreefficiently to human class II molecules than to mouse Iamolecules (71).The structure of class II molecules was deduced fromthe solution of the structure of MHC class I molecules(89, 90) and is thought to consist of two immunoglobulin-like domains, located closely to the cell membrane, whichsupport a structure constructed from the NH2-terminalregions of both polypeptides of the protein and comprisean extended 13 pleated sheet supporting two a helices,separated by a cleft. It is believed that peptidesderived from conventional antigens normally lie in thisgroove, and that the complex of MHC and peptidestimulates T cells bearing a13 receptors (91-93).Staphylococcal toxin binding sites are likely located onthe 13 chain side of the class II molecules, as suggestedby Janeway and co-workers and Fleisher on theoreticalgrounds (71, 73, 94). Using the synthetic peptideapproach (72), it was shown that SEA can bind either the19LITERATURE REVIEWa or 13 chain of the Ia molecule. Whereas the a chain ofIa is not necessary for SEA binding, the 13 chain is bothnecessary and sufficient for SEA binding. SEA bindingprobably occurs outside the antigen-binding groove (95,96). One molecule of SEA may bind at the same time toboth a and 13 chain of an Ia molecule, although it is alsopossible that more than one SEA molecules bind to one Iamolecule.2.4.3 Interaction of staphylococcal toxins with the Tcell receptorThere exists a controversy about whether there aredirect TSST-1 binding receptors on T cells. Schlievert'sgroup (101) demonstrated that there were TSST-1 bindingreceptors on human T cells and reported that they weresimilarly distributed in CD4+ and CD8+ T cells; whileScholl et al. and Uchiyama et al. (83, 102, 103) did notfind direct TSST-1 binding to T cells, but that class IImolecules on antigen presenting cells were required forthe activation of T cells by TSST-1.It was shown that the TcR for antigenic peptides isalso the receptor for staphylococcal enterotoxins (73,97, 98). Further, the ability of a given enterotoxin toactivate a particular T cell depended on the amino acid20LITERATURE REVIEWTable 6. V13 specificity of staphylococcal toxins (71)VB specificityToxin^Human^ MouseTSST-1^2 3,15,17SEA 1,2,10,11,17SEB^3,12,14,15,17,20^3,7,8.1-3,17SEC1 12,?^ 3,8.2,8.3,11,17SEC2^12,13.1,13.2,14,15,17,20^3,8.2,10,17SEC3 5,12,? 3,7,8.1,8.2SED^5,12,?^ 3,7,8.1-3,11,17SEE 5.1,6.1-3,8,18^11,15,17sequence of the VB region of the TcR (73, 97); differententerotoxins could activate different T cells, dependingon the VB type of the TcR (Table 6). The latter propertyis the basis for coining the name superantigens forstaphylococcal toxins (97).There is a functional similarity betweensuperantigens such as the staphylococcal toxins andputative products of the mouse minor lymphocytestimulating (Mls) antigen locus (73, 71) (Table 2). Mlsantigens were discovered when it was shown that T cellsfrom some strains of mice could be stimulated by spleencells from other mice even though the mice were identicalwith respect to MHC (74). The functional response of Tcells to Mls antigens is essentially the same as that tomicrobial superantigens, except that the microbialsuperantigens are relatively well characterized but21LITERATURE REVIEWTable 7. Biological activities of staphylococcal toxins1. Pyrogenicity2. Mitogenicity for T lymphocytes3. Induction of monokines: IL-1,TNF from monocytes4. Immunosuppression5. Enhancement of delayed hypersensitivity6. Enhancement of lethal endotoxic shock7. Causation of TSS manifestations in Rabbit animal modelnothing is known about the nature of the Mls antigens.Genetic data have shown a linkage between the endogenousprovirus murine mammary tumor virus (MMTV) and Mlsantigens (75-79). These findings suggest that Mls andpossibly other "endogenous" superantigens are reallyvirus-derived microbial superantigens.2.4.4 Biological activities of Staphylococcal toxins2.4.4.1. Pyrogenicity.The rapid onset of a high fever (>38.9 °C) is aprominent characteristic of TSS-like illness, and theassociated exotoxins all induce a potent fever responsewhen injected into rabbits (23, 104-106). TSST-i has beenshown to induce both IL-1 and TNF which may contribute tofever (107, 108)). Both IL-1 and TNF are thought toinduce fever by stimulating the production of PGE2 in thepreoptic area of the anterior hypothalamus (109, 110).22LITERATURE REVIEWThe increased rate of PGE2 synthesis is thought to be dueto an increased rate of oxidation of arachidonic acid bythe cyclooxygenase pathway. Inhibition of this enzyme,such as indomethacin and ibuprofen, inhibits fever causedby purified IL-1 and TNF, (109,110) and as well, feverinduced by TSST-1 and SPEs (111).2.4.4.2. Mitogenicity for T lymphocytes.The staphylococcal toxins are powerful T cellmitogens. (23, 112, 118). The toxins do not act asconventional mitogens in the sense that they do notactivate T cell clones nonspecifically, but selectivelyaccording to the VB specificity of the T cell receptor(73, 87, 97, 119-121). Antigen presenting cells withclass II molecules, such as monocytes are required forthe activation. IL-1 by itself is not sufficient totrigger a proliferative response of purified lymphocytesto TSST-1 in the absence of APC (122). This againsuggests, but does not prove, that some contact withclass II molecules and toxin complex, in addition to IL-1stimulation is necessary for a mitogenic response (Figure1).2.4.4.3. Effects on monocytes/macrophages.23LITERATURE REVIEWStaphylococcal toxins also act on mononuclearphagocytes and induce the release of IL-1 and TNF.Monokines may play an important role in the pathogenesisof TSS since IL-1 and TNF have been shown to havemultisystem effects (123-125). Work done recently in ourlaboratory (126) indicated that highly purified monocytesor T cells alone did not induce TNF or IL-1 in responseto TSST-1. However, the addition of 10 ug/ml TSST-1 to a1:1 mixture of monocytes and T cells resulted insignificant TNF and IL-1 production at 24 h, 37 °C. It wasalso shown by using a semipermeable membrane to separateT cells and monocytes that direct contact between themwas required for cytokine induction by TSST-1.2.4.4.4. Immunosuppression.Staphylococcal toxins also have the capacity tononspecifically suppress the production ofimmunoglobulins, indirectly by stimulating a populationof T suppressor cells. (23, 128, 130). Spleen cells frommice immunized with SRBC do not secrete immunoglobulinswhen cultured with nanogram quantities of toxins asmeasured in a 4-d direct Jerne plaque assay. TSST-1 alsoinhibits the production of all immunoglobulin classes bypokeweed mitogen stimulated human peripheral blood cells.24LITERATURE REVIEW(130). This suppression is independent of class IIantigen restriction in that supernatant fluid from toxinstimulated cells suppresses immunoglobulin production byheterologous as well as homologous lymphocytes. In vivoprimed T lymphocytes from mice injected with 1 ug dosesof TSST-1 will also cause suppression when incubated withnaive splenocytes and SRBC in the Jerne plaque assay.The inhibition of immunoglobulin production by TSST-1 may explain why some TSS patients do not make antibodyagainst the toxin despite exposure to the toxin duringthe illness. Cases have ocurred in which patients do notbecome seropositive to TSST-1 even after repeatedepisodes of the disease (24). TSS patients also havedecreased titers to the staphylococcal enterotoxinscompared to the healthy controls (24). Potentialpolyclonal inhibition of immunoglobulin synthesis isindicated by the fact the TSS patients have decreasedlevels of total serum IgG (131).The suppressive effect for immunoglobulin synthesismay be due to toxin stimulated activation of a T cellsubpopulation which results in the production of IFN-r, alymphokine known to inhibit polyclonal Ig production(130, 132-137).25LITERATURE REVIEWThe evidences reviewed here support the hypothesisthat staphylococcal toxins induce a general state ofantibody immunosuppression in TSS patients resulting inthe failure to produce anti-toxin antibodies. Theimportance of protective antibody is shown in thatrabbits with systemic immunity to TSST-1 are protectedagainst lethal challenge by a combination of TSST-1 andendotoxin (138). Toxin induced immunosuppression alsoallows growth of opportunistic Gram-negative bacteria, aproposed source of endotoxin in TSS patients (138, 139).2.4.4.5. Enhancement of delayed hypersensitivityOther effects on the immune system by thestaphylococcal toxins have been described. One isenhancement of delayed type hypersensitivity in rabbitsthat have been given repeated subcutaneous injections ofstaphylococcal toxins followed by a challenge dose after4 to 6 weeks. (113). These rabbits develop highlyerythematous and edematous skin reactions that may beexplained in terms of an enhanced hypersensitivityresponse mediated by an activated T cell subpopulation.This effect may in part explain the rash and mucosalsurface reddening in TSS and scarlet fever illnesses.26LITERATURE REVIEW2.4.4.6 Enhancement of lethal endotoxic shockAnother effect that TSST-1 has on the immune systemis alteration of the function of the reticuloendothelialsystem (RES) after intravenous exposure to both thestaphylococcal toxins and endotoxin (113, 128-141). Uponinjection of both types of toxin, a blockade of RESfunction was demonstrated by the reduced ability to clearcolloidal carbon or endotoxin.The staphylococcal toxins enhance the susceptibilityof rabbits to lethal endotoxin shock by up to 100,000-fold (138, 142), probably as a result of alteration ofRES clearance function. Rabbits are protected fromenhancement to endotoxin shock when immunized withstaphylococcal toxin or endotoxin. Increased levels ofsystemic endotoxin have been shown in sera fromconvalescent TSS patients and also in rabbits when givenSEB intravenously (139, 143).The enhancement of endotoxin shock by TSST-1 can beblocked by the administration of pooled human IgG andmethylprednisolone (144), probably as a passiveimmunization and anti-inflammatory effects.The renal tubular cells are another target site whenthey are exposed to TSST-1 and endotoxin (145). When therenal cells are exposed to TSST-1 for 20 min and then to27LITERATURE REVIEWendotoxin, there is a significant degree of cytotoxicity.But neither TSST-1 nor endotoxin alone causes significantcytotoxicity.2.4.4.7 Animal modelsVarious rabbit TSS animal models have beenestablished, e.g. i.v. injection of staphylococcaltoxins; constant release of toxin by utilizing chambersto localize toxin producing staphylococci (31, 146, 147);isogenic strains of staphylococci in which a TSST-1negative strain was converted to a TSST-1 positive strainusing a bacteriophage containing the tst gene (146); oruse of a subcutaneous infusion pump to administer TSST-1at constant rate (148). The results were consistentlyshown that introducing staphylococcal toxins into rabbitscould reproduce most of the symptoms of TSS with theexception of rash and desquamation.28CHAPTER 3MATERIALS AND METHODS3.1 TSST-1 AND SEA DETECTION BY ELISA3.1.1 Preparation of bacterial cultural filtratesThe 350 isolates of S. aureus were collected frompatients and volunteers in Vancouver during the period 1974to 1988. Among them, 84 isolates were from blood, 166 fromurogenital sources, 26 from nasal and throat, 74 from woundand other sites.The isolates of S. aureus were examined in a codedfasion. Overnight cultures of S. aureus were inoculated into30 ml of dialyzed brain heart infusion broth (DifcoLaboratories, Detroit, Mich.) and incubated with shaking for18 h at 37 °C. Cells were removed by centrifugation (9,000 xg for 5 min), and supernatants were filter sterilized andfrozen at -70 °C until testing (149).3.1.2 Purification of anti-SEA IgG from anti-SEA antiserumwith Protein A-Sephadex CL-4BSEA specific IgG was purified on a protein A-Sepharosecolumn (Parmacia Fine Chemicals, Uppsala, Sweden) asdescribed by Hardy (150). Briefly, the protein A column was29MATERIALS AND METHODScycled with cycling buffer (0.1 M glycine, pH 2.4) andequilibrated with running buffer (Tris/saline, pH 8.0)first. Then 1 ml of polyclonal SEA rabbit antiserum (ToxinTechnology Inc., Madison, WI) diluted with 1 ml Tris/salinebuffer was applied to the protein A column and chased with 1ml of Tris/saline buffer. After incubation for 1 hour,albumin and proteins other than IgG in the serum were washedaway with 10 column volumes of Tris/saline buffer, pH 8.0.IgG was eluted from the column by adding 15 ml of elutingbuffer (0.1 glycine, pH 3.2) and collected at 2 min perfraction (flow rate 0.5 ml/min), monitored by a UV monitorat O.D. 280 (UV-1, Parmacia). The column was washed bycycling buffer again and equilibrated with running bufferuntil the pH value of the column was raised up to 8.0. TheIgG eluate was dialysed at 4 °C against PBS, pH 8.0,overnight with at least 2 changes of buffer. The proteinconcentration of the IgG was determined by the Bradfordmethod. Purity was greater than 95% as determined by silverstaining after sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) (156).3.1.3 TSST-1 and staphylococcal enterotoxinsFor the ELISA assay, reference TSST-1 was purified fromculture supernatant of S. aureus MN8 in our laboratory, by30MATERIALS AND METHODSion-exchange chromatography, chromatophocusing, and gelfiltration, as previously described (151, 152). In addition,purified staphylococcal enterotoxins A, B, Cl, C2, C3, D, Eas well as their respective rabbit antisera, were obtainedfrom commercial sources (Toxin Technology Inc., Madison,WI).For binding and mitogenicity assay further purifiedtoxins were used. TSST-1 was purified by preparativeisoelectrofocusing followed by chromatofocusing. SEA fromToxin Technology was further purified by chromatofocusing.Both TSST-1 and SEA were highly pure, as evaluated by silverstaining after SDS-PAGE and by Western blot analysis withpolyclonal toxin-specific antibodies purified fromcommercial sources (Toxin Technology Inc., Madison, WI).3.1.4 Conjugation of rabbit anti-SEA to alkaline phosphatasePurified anti-SEA rabbit IgG was conjugated withalkaline phosphatase (Sigma) according to Voller et al.(153). Firstly, 1 mg of purified rabbit anti-SEA IgG wasadded to 2.5 mg alkaline phosphatase (Sigma). Glutaraldehyde(25%) was added to make a 0.2% concentration of the mixture,which was incubated at room temperature for 2 hours withconstant stirring. The conjugate was dialysed against PBS,pH 8.0, at 4 °C overnight with 2 changes of buffer, then31MATERIALS AND METHODSdialysed against 0.05 M Tris-HC1 buffer (pH 8.0), at 4 °Cwith 2 changes of buffer. Finally the conjugate was dilutedto 2 ml with Tris-HC1 buffer, pH 8.0, containing 1.0% bovineserum albumin and 0.02% sodium azide. The conjugate wasstored in the dark at 4 °C.3.1.5 Quantitation of TSST-1 by noncompetitive ELISAS. aureus culture supernatants were quantitated by anELISA as previously described (154). Polyclonal TSST-1antiserum was from Toxin Technology Inc., Madison, WI. TSST-1 specific IgG was purified on a protein A-Sepharose column(Pharmacia Fine Chemicals, Uppsala, Sweden) as described byHardy (150). Culture filtrates were pretreated with 10%(vol/vol) normal rabbit serum to remove protein A beforetesting. The total protein content of the culture filtrateswas measured by the Bradford assay (155). The sensitivity ofthe ELISA for TSST-1 was 0.5 ng/ml, and the interassayvariability was less than 10% (coefficient of variation). Nocross-reactivity was observed with S. aureus enterotoxins A,B, Cl, C2, C3, D and E at concentrations ranging from 10 to1,000 ng/ml.3.1.6 Quantitation of SEA by noncompetitive ELISA32MATERIALS AND METHODSS. aureus was quantitated by a noncompetitive ELISAmethod as modified from that described by Rosten et al.(154). Polyclonal SEA rabbit antiserum was from ToxinTechnology Inc., Madison, WI. SEA specific IgG was purifiedon a protein A-Sepharose column (Parmacia Fine Chemicals,Uppsala, sweden) as described by Hardy (151). Purity wasgreater than 95% as determined by silver staining aftersodium dodecyl sulfate-polyacrylamide gel electrophoresis(156). Wells of microdilution plates (Immulon I; DynatechLaboratories Inc., Alexandria, Va.) were coated with thepurified rabbit anti-SEA IgG (100 ul per well: 1 ug/ml in0.05 M carbonate buffer, pH 9.6) optimized by checkerboardtitration. After absorption overnight at 20 °C, plates werewashed with phosphate-buffered saline (PBS, pH 7.4)containing 0.05% Tween 20 (Sigma Chemical Co., St. Louis,Mo.) (PBS-T). SEA standards (1 to 128ng/m1 in PBS-T; ToxinTechnology, Madison, WI) or culture filtrates diluted inPBS-T were added (100 ul per well). The SEA positive strain(ATCC13565) and SEA negative strain confirmed by Westernblotwere used as controls. All samples were tested intriplicates. Culture filtrates were pretreated with 10%(vol/vol) normal rabbit serum to remove protein A beforetesting (149). Plates were incubated for 1.5 h at 37 °C andwashed as described above. Rabbit Anti-SEA IgG was33MATERIALS AND METHODSconjugated with alkaline phosphatase (Sigma) according toVoller et al. (153) as described above. The conjugate,diluted 1 : 2,400 in PBS-T (optimized by checkerboardtitration), was added in 100 ul volume to each well, andplates were incubated again at 37 °C for 1.5 h. Afterthorough washing to remove unbound conjugate, 100 ul of p-nitrophenyl phosphate (1 mg/ml in 10% diethanolamine buffer,pH 9.8) was added to the wells. Plates were read at 405 nmafter incubation at 37 °C for 1 h. The total protein contentof the culture filtrates was measured by the Bradford assay(155). The sensitivity of the ELISA for SEA was 1 ng/ml, andthe interassay variability was less than 12% (coefficient ofvariation). No cross-reactivity was observed with TSST-1, S.aureus enterotoxins B, Cl, C2, C3, D and E at concentrationof 100 ng/ml.3.2 HEMOLYTIC ACTIVITYHemolytic activity was studied on infusion agar (bloodagar; BBL Microbiology Systems, Cockeyville, Md.) containing5% sheep blood. Standard inocula (2 x 10E5 CFU) of isolateswere incubated at 37 °C under 30% CO2 as previously described(42). The zonal radius of complete hemolysis after 48 hincubation was measured from the edge of the uniformmacrocolony to the outer periphery of the zone of hemolysis.34MATERIALS AND METHODS3.3 ELECTROPHORESIS OF ENZYMESTyping of S. aureus isolates by multilocus enzymeelectrophoresis at 19 chromosomal loci was performed in thelaboratory of Dr. Robert K. Selander in the Department ofBiology, Pennsylvania State University. Methods of lysatepreparation, protein electrophoresis, and selective enzymestaining have been described by Selander et al. (58).Briefly, isolates were grown at 37 °C overnight in 150 ml oftryptic soy broth (Difco) on an orbital shaker (250 rpm) andharvested by centrifugation at 6000 x g for 10 min at 4 °C.After suspension in 2 ml of 50 mM Tris-HC1 buffer containing5 mM EDTA (pH 7.5), lysostaphin (Sigma) was added to a finalconcentration of 100 ug/ml, and the cells were incubated at37-40°C for 30 min in a water bath. The bacteria weresonicated with a Branson model 200 sonifier-cell disruptorequipted with a microtip for 30 sec at 50% pulse, with ice-water cooling, and were centrifuged at 20,000 x g for 20 minat 4 °C. The clear supernatant (lysate) was stored at -70 °C.Cell lysates were electrophoresed on starch gels andselectively stained for 19 metabolic enzymes by methodsdescribed by Selander et al. (58). Each isolate wascharacterized by its combinations of allele mobilityvariants at the 19 enzyme loci. The 19 enzymes assayed wereaconitase (aco), phenylalanylleucine peptidase (pip),35MATERIALS AND METHODSphosphoglucose isomerase (pgi), carbamylate kinase (cak),glucose 6 phosphate dehydrogenase (g6p), malatedehydrogenase (mdh), mannitol-l-phosphate dehydrogenase(mlp), 6-phospholuconate dehydrogenase (6ph), glutamatedehydrogenase (gld), nucleoside phosphorylase (nsp),catalase (cat), esterase (est), lactate dehydrogenase-1(1d1), lactate dehydrogenase-2 (1d2), lactate dehydrogenase-3 (1d3), alcohol dehydrogenase (adh), maltose dehydrogenase(map), indophenol oxidase (ipo), shikimate dehydrogenase(shk). Distinctive electromorphs (mobility variants) of eachenzyme, numbered in order of decreasing rate of anodalmigration, were equated with alleles at the correspondingstructural gene locus. Isolates that lacked activity for aspecific enzyme were assigned a null allelic state at thelocus in question. Each isolate was characterized by itscombination of alleles at the 19 enzyme loci, anddistinctive multilocus enzyme profiles were designated aselectrophoretic types (58).3.4 STATISTICAL METHODSThe categorical data were analyzed by means of chi-square statistics with Yates correction, the hemolyticactivity was analyzed by Wilcoxon rank sum test. SAScomputer program was use to do the statistical analysis. The36MATERIALS AND METHODSstatistical difference was interpreted as significant if pvalue was below 0.05. Single-locus genetic diversity amongETs was calculated from allele frequencies as h = (1-Exi2 )(n/(n-1)], where xi is the frequency of the ith alleleand n is the number of ETs; mean diversity per locus (H) isthe arithmetic average of h values over all loci. Geneticdistance between pairs of ETs was expressed as theproportion of enzyme loci at which dissimilar alleles occur(58).3.5 PURIFICATION OF HUMAN T CELLS AND MONOCYTES3.5.1 Fractionation of human peripheral blood mononuclearcellsFresh human PBMC were obtained by centrifugation ofplateletpheresis buffy coats from healthy adult donors overHistopaque 1.077 (Sigma Chemical Co., St. Louis, Mo.).Basically, buffy coats were separated into several 30 mlaliquots, and Histopaque 1.077 was underlayered with thetube at 45 ° angle. The tubes were centrifuged at 800 x g for15 minutes with brake off. Cells were pipetted from theinterface and put into 3 tubes. PBMC were washed 5 timesusing Hank's salt to delete platelets.37MATERIALS AND METHODS3.5.2 AET treated sheep red blood cell rosetting to separateT cells from non-T cells30-40 ml of SRBC (ubc, 20 ml SRBC with 20 ml Alseversolution) were washed 3 times with Hank's salt at 225 x gfor 10 minutes. 0.28 M AET (5-2 aminoethylisothiouroniumbromide hydrobromide, Sigma)) was prepared, pH was adjustedto 9.0, and sterilized. 25 ml of AET solution was added to25 ml washed and packed SRBC in a sterilized 50 mlcentrifuge tube. After mixing gently, it was incubated at37 °C for 15 min. Then it was washed 3 times with Hank'ssalt, centrifuged at 225 x g for 10 min.3.5.3 Treatment of mononuclear cells with AET treated SRBCFractioned PBMC were divided into 3 x 15 ml aliquots, 2ml of FCS and 10 times of AET-SRBC were added to each of thethree aliquots, then they were incubated for 5 minutes at37 °C. They were centrifuged at 100 x g for 5 min, thenincubated in ice for 10 minutes. The cells were resuspended, 10 ml of Histopaque 1.077 was underlayered, andcentrifuged at 800 x g for 15 minutes with brake off. Thenon-rosetted interface cells were pipetted out, and washed 3times at 250 x g with Hank's salt.3.5.4 Purification of monocytes from non-rosetted cells38MATERIALS AND METHODSNon-rosetted cells were resuspended in 16 ml of RPMI1640 with 10% FCS. 1 ml of RPMI with 10% FCS was layered ontop of the mixture of 4 ml of the resuspended cells and 3.3ml Percoll (d 1.1262, 278 mOsm) to give a final specificgravity of 1.062 g per ml. The tubes were centrifuged at 800x g for 15 minutes without brake. The interface cells weretaken out, and washed 3 times at 225 x g with Hank's salt.The purity was greater than 90% as determined by non-specific esterase assay.3.5.5 Purification of T cells from rosetted cellsE-rosetted cells were treated with ammonium chloride toremove sheep red blood cells, washed three times andsubjected to antibody directed complement lysis usingantibodies to the HLA-DR antigen, L243, and to the monocyteantigen OKM1 and pooled rabbit complement as described(157). Monoclonal antibodies L243 and OKM1 were purified byprotein G Mab Trap Kit (Pharmacia) from ascites fluidobtained by injecting hybridomas (ATCC, Rockville, MD) intopristine-primed BALB/c mice. Purified human T cells were >99% CD2+ and < 1% HLA-DR+ as determined by flow cytometryanalysis.3.6 IODINATION OF TSST-1 AND SEA BY THE CHLORAMINE T METHOD39MATERIALS AND METHODSTSST-1 and SEA were iodinated by a modified chloramineT procedure, as previously described (158). Basically, 100ug of TSST-1 or SEA was incubated with 2.0 mCi of Na 125 I(carrier-free NaI; 100 mCi/m1; ICN Radiochemicals, Irvine,CA.) and 10 ul (1 mg/ml) of chloramine T (Sigma chemicalCo., St. Louis, Mo.) in iodination buffer containing 10%dimethyl sulfoxide and 100 g/ml Polyethylene glycol 4000(Sigma Chemical Co., St. Louis, Mo.) in 0.1 M sodiumphosphate buffer, pH 7.2. The total volume was 155 ul. Afterincubation at room temperature for 20 min, 10 ul (3.0 mg/ml)of sodium metabisulfite (Sigma chemical Co., St. Louis, Mo.)was added and the mixture was put on crushed ice for 5 minto stop the reaction. Iodinated toxins were separated fromfree iodine by gel filtration through 10 ml Sephadex G-25columns (Pharmacia Fine Chemicals, Doral, Quebec, Canada)equilibrated with phosphate-buffered saline containing 0.25%gelatin. Pooled fractions corresponding to the protein peakwere adjusted to 0.1% with bovine serum albumin and storedin 200 ul aliquots at -70 °C.3.7 BINDING ASSAY1253.7.1 Direct binding of^I-TSST-1 or 125I-SEA to T cellsand monocytes40MATERIALS AND METHODSBinding assays were performed as described (158).Basically, human monocytes or T cells were suspended at 3 x108 cells per ml in binding buffer of RPMI 1640 with 1%bovine serum albumin. All procedures were performed at 4 °C.Approximately 3 x 10 7 cells were incubated with variousconcentration of 125I-TSST-1 or 125I-SEA in final volumes of300 ul. The cell suspensions were gently rotated for atleast one hour and unbound radiolabeled toxin was thenseparated from the cells by transferring triplicate 80 ulportions of assay mixture to precooled microcentrifuge tubescontaining 200 ul of 1.1:1 mixture of dibutyl phthalate anddioctyl phthalate oils (BDH, Vancouver, B.C.). The tubeswere centrifuged at 16,000 x g for 1 min, immediately frozenat -70°C and the tube tips containing the cell pellets wereexcised. Cell-associated radioactivity was determined with aSearle 1185 gamma counter. Non-specific binding wasdetermined by adding a 100-fold or greater molar excess ofunlabeled toxin. All data were corrected for nonspecificbinding.3.7.2 Binding of labeled toxin-monocyte membrane fragmentcomplex to T cellsMonocyte membrane fragments (MMF) prepared from morethan 1 x 10 8 monocytes were preincubated with 5 nM of 1251-41MATERIALS AND METHODSTSST-1 or 125I-SEA for at least 1 h at 4 °C in 200 ulvolumes. For preparing MMF, monocytes were incubated in 10mM Tris-HC1 hypotonic buffer with protease inhibitors formore than 10 min, then cells were sonicated using SonifierCells Disruptor 350 for 30 sec on crushed ice. MMF werepelleted by spinning supernatant, after low speedcentrifugation at 800 x g for 5 min, at high speed 16,000 xg for 30 min. 5 x 10 7 T cells in 100 ul of volumes wereadded to the toxin-MMF complex. After overnight incubationwith rotating, unbound toxin-MMF were separated from thecells by the same method as above, except centrifugation wasat low speed 800 x g for 5 min.3.8 T CELL MITOGENICITY ASSAYT cell mitogenicity assays were performed as previouslydescribed (157). Briefly, T cells isolated from human PBMCwere suspended at a concentration of 5 x 10 6 cells per ml,monocytes were suspended at a concentration of 5 x 10 5 perml, in complete RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mM HEPES, 2 mM L-glutamine,and 20 ug per ml polymyxin B sulfate. 5 x 10 5 T cells werecultured in 0.5 ml culture medium with 1 ug/ml of TSST-1 andSEA in 24-well tissue culture plates (Falcon 3074, BectonDickinson, Lincoln Park, NJ) or culture plate inserts(0.4542MATERIALS AND METHODSum, 12 mm, Millicell-HA, Millipore Corp., Bedford, MA),either separately or jointly as indicated. The culture wasincubated for 3 days at 37 °C in 5% CO2. At 48 h, cells werepulsed with 1 uCi of 3H-thymidine (5 Ci/mmole; Amersham,Arlington Heights, Ont.). Eighteen hours after pulsing, theculture was harvested using Titertek Harvester (Skatron,Norway). 3H-thymidine incorporated into DNA was measured bycounting in a liquid scintilation counter (Beckman LS 1800).All assays were carried out in triplicate and controlsincluded T cells alone, T cells plus monocytes with orwithout toxin.Monocyte conditioned media were used, alternatively tostimulate T cell proliferation. It is prepared by culturingmonocytes (5 x 10 5 cells per ml) in complete medium for 24 hwith or without activation with TSST-1 or SEA. Later, themedium was harvested by centrifugation at 800 x g. Thesupernatant (either concentrated or 1:10 diluted) was addedto T cells and cultured as above.43CHAPTER 4RESULTS4.1 SEA DETECTION BY ELISAThe noncompetitive ELISA for quantitation of SEA had asensitivity of 1 ng/ml and was reproducible with acoefficient of variation less than 12%, as determined byrepeated examination of culture supernatant of S. aureus ATCC13565 (a SEA producing strain) or brain heart infusionbroth spiked with known amounts of SEA. A representativestandard curve and regression analysis are shown in Fig-2.4.2 TSST-1 AND SEA PRODUCTION AMONG 350 ISOLATES OF S.AUREUSThe TSST-1 and SEA production in the 350 isolates of S.aureus was first examined (Table 8). TSST-1 and SEA wereboth produced significantly more frequently in TSS (68%,48%, respectively) than in non-TSS isolates (26%, 23%,respectively) (both p < 0.01). Production of TSST-1 and SEAwere most frequent in urogenital TSS isolates (86%, 69%,respectively) (both p<0.001) as compared to urogenital non-TSS isolates. SEA was co-produced with TSST-1 morefrequently among TSS (42%) compared to non-TSS isolates(13%) (p<0.001). Co-production of TSST-1 and SEA was more44RESULTSfrequent in urogenital TSS isolates (66%) compared to othergroup (p<0.05). No significant difference in co-productionof TSST-1 and SEA was found between non-urogenital TSS andnon-TSS isolates.45RESULTSFigure 2. Typical standard curve and regression analysiswith 95% confidence interval (dotted curve) of the non-competitive ELISA method for quantitation of extracellularSEA in the culture supernatant of S. aureus.1.5E 1 '0cin0vcici 0.50.01^10^100^1000SEA (ng/ml)46RESULTSTable 8. TSST-1 and SEA production inTSST -1350 strains ofSEAS. aureusNEITHERBOTHUrogenital TSS(29) 25 (86%) a 20(69%) a 19(66%) a ' b 3(10%)Urogenital Non-TSS(137) 40(29%) 28(20%) 18(13%) 87(64%)Non-urogenital TSS(36) 19(53t) a 11(31%) NS 8(22%) NS 14(39%)Non-urogenital Non-TSS(148) 35(24%) 37(25%) 19(131) 95(64%)Total TSS(65) 44(68%) a 31(48t) a 27(42%) a 17(26%)Total Non-TSS(285) 75(26%) 65(23%) 37(13%) 182(64%)Total(350) 119(34%) 96(27%) 64(18%) 199(57%)a: P < 0.001 compared with non-TSS strainsb: P < 0.01 compared with non-urogenital TSS strainsNS : not significantly difference compared with non-TSS strains*p: Chi-square with Yates correction47RESULTS4.3 ELECTROPHORETIC TYPESThe 350 S. aureus isolates represented 63 distinctelectrophoretic types. Allele profiles of the ETs are shownin Table 9. Among these 63 ETs, 36 were represented bysingle isolates and 27 by more than one isolates (range, 2-116). Eighteen out of 19 enzyme loci were polymorphic, withan average of 3.58 alleles per locus. Table 10 shows thegenetic diversity of the 350 stains of S. aureus, the meangenetic diversity per locus (H) was 0.272. A single clonedesignated as ET 4 accounted for 79% of urogenital TSS, 62%of total TSS isolates, and 33% of the total S. aureuspopulations.The genetic relationship of the 63 ETs, based onallelic variation at 19 enzyme loci, are shown in thedendrogram in Fig-3. The smallest observed genetic distance(0.05) between ETs corresponds to a single-locus difference,and the largest (0.45) corresponds to differences at 11 ofthe 19 loci assayed. At genetic distance of 0.20, there aresix branches designated A through F; cluster A which wascomposed of 9 ETs was separated from clusters B-E at adistance of 0.35 and 0.45 from cluster F. These threedivisions are designated as I, II and III. Cluster A wascomposed of 9 ETs, eight of them were represented by singleisolates, only ET 4 which accounted for the majority of TSS48RESULTSisolates was represented by multiple isolates (116 isolates,33% of total isolates). Cluster B was represented by 3 ETsand Cluster F was represented by a single ET. The propertiesof the isolates of the corresponding ETs are shown in Table11.490.• 0, 0, 0, IA VI %AI V* VI VI LA LA VI VI 4".. 4, 4., 4, 4, 4, 4, 4" 4, 4, 4 1 1,4 U/ LAI IN 03 1.4010101NhIrvhorVIVIV/VIVIV^41 N •-• 4:24 .0 C ^-4 O. VIA 4.44 IV -• 0 .0 100 ,4 0, tit 4,  4 o 4 N -. O•O 10 -4 O. Vs 4, 01 N W. 0 .0 03 ..4 0, VI 4". 41 IV -1 0 .4:1 OI N 0. VI 4.`  44 IV -+0 • 0 CA '4 Os VI 4". 44 I.V -.III-4IV^ -..^....^.r.^ NI^ -a^-.•^ --.-., .... ..... .... vi, 0.,,,, .., r,, _. t,i,,,i ,_,, s., -.N ... 0, 4-, VI N 4,4 A_. -. -a iv .... .-.. vi .... -a _. -a 0) -. -a ..4. 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Os vl Vs Vi .0 04 01 I.4 1.4 04 04 0.31 -4 NN .0 IV N N NN VI VI -4 43 01 1.4 01 01 01 01 04 04 04 44 41 A A 4 4 4 1 4144 44 44 CO 44 4 1 0, 0" 0, 0. 0• 0.. 0..0 0,......... U1 IA VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI LA VIP 0 VI VI 4.11 VI LA VI VI VI VI VI 1.11 U1 VI VI VI VI VI VI VI VI Ul VI Ul VI VI UI VI VI VI VI VI VI V1 VI VI VI VI VI0.wa.VIN A]0 CI 0 0 0 CO 0 CI a 0 CI Ct 0 CI CI 0 CI 0 0 01 CI Ol 0 a Iv Ks iv iv r..) 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VI 1.01 VI VIP Lo vo VI VI VI 0 VI vi VI g'0VI VI VI VI V1 VI VI U1 V1 VI VI VI VI VI VI VI VI VI VI VI LA VI VI VI VI VI VI VI VI VI VI VI 1/1 VI VI Ul VI VI VI VI VI VI VI VI VI VI VI VI VI VI VI -.4 v) vo VI vo vl -4 vi VI VI VI VI "S41O VI ...n SA VI 04 %A VIN VI VI VI VI VI ....i -4 -.4 4.1  W vi 04 1..n .1 VIN IA V4 IV 41 VI 1.11 VI LA VI VI VI VI vi VIP vi VI Lit VI Lit Lit VI VI vi IA IA vi -.; -4 vi vi vl vi Vs vs VI VI vi 7ICRESULTSTable 10. The genetic diversity at 19 enzyme loci and genotypicdistribution of 350 strains of S. aureus Source^Isolates No.^ETs^No. of ET 4^HisolatesUrogenital TSS^29^7^23(79%)^0.287Urogenital Non-TSS^137 36^37(27%)^0.246Non-urogenital TSS^36^15^17(47%)^0.246Non-urogenital Non-TSS^148 39^39(26%)^0.268Total^ 350^63^116(33%)^0.272H: genetic diversity per locus among ETs.51RESULTSFigure 3. Dendrogram showing relationship of 63 ETs of 350isolates of S. aureus. There are three primary divisions (I,II ans III) and six major lineages (A-F). Most of theurogenital TSS isolates are in cluster A.52I^A II^ET ^No. ofisolates^4 ^116S 16 ^1^7 19 ^2^10 111^312 113^11415^1016 217^3I8 11920^12212223^224 225^226 427^12329^130 2031^I^32 I3334^13536^37 ^18 ^239 140^141 I^4 2^443 4344^245 ISRESULTS46 416I474824950451522753254552565735855960III F 6116263 110.5^0.4 0.3^0.2^0.1^053Toxin production^Urogenital NonurogenitalETNo.^ofstrains TSST-1onlySEAonlyBoth Neither TSS Non-TSS TSS^Non-TSS1 1 1 12 1 1 13 1 1 14 116 31 9 57 19 23 37 17^395 1 1 16 1 1 17 1 1 18 1 1 19 2 1 1 1 110 1 1 111 3 1 2 2^112 1 1 113 1 1 114 1 1 115 10 1 9 5 1^416 2 2 217 3 3 318 1 1 119 1 1 120 12 1 1 10 6 621 1 1 122 1 1 123 2 2 1 124 2 1 1 1 125 2 2 226 4 1 2 1 2 1^127 1 1 128 1 1 129 1 1 130 20 4 6 10 6 3^1131 1 1 132 1 1 133 1 1 134 1 1 135 5 5 1 1^336 1 1 137 1 1 138 2 2 1 139 1 ,^1 140 1 1 141 1 1 142 4 4 443 43 1 42 1 13 3^2644 2 1 1 1 145 15 10 5 8 1^646 4 2 2 2 1^147 16 1 1 14 7 1^848 1 1 149 2 1 1 250 1 1 151 4 4 1 1 1^152 1 1 153 27 1 26 17 1054 2 2 1^155 1 1 156 2 2 257 1 1 158 3 3 2 159 5 5 3 260 1 1 161 1 1 162 1 1 163 1 1 1RESULTSTable 11. Properties of 350 strains of S. aureus representing63 ETsTotal 350^119^96^64^199^29^137^36^14854RESULTS4.4 TSST-1 AND SEA PRODUCTION IN ET 4 ISOLATESThe production of TSST-1 and SEA among ET 4 isolateswas compared with isolates in other ETs (Table 12). BothTSST-1 (76%) and SEA (57%) were produced significantly morefrequently in ET 4 isolates than in other ETs (13%, 13%,respectively) (both p < 0.001). Among ET 4 isolates,production of TSST-1 was significantly more frequent in TSS(93%) than in non-TSS isolates (67%) (p < 0.01), and wasmost frequent in urogenital TSS isolates (96%) compared tourogenital non-TSS isolates (76%) (p < 0.05). Production ofSEA was also significantly more frequent in urogenital TSSisolates (83%) than in urogenital non-TSS isolates (49%) (p< 0.05). No significant difference was found in SEAproduction between total TSS (68%) vs total non-TSS isolates(51%) (p = 0.18). The co-production of TSST-1 and SEA wassignificantly more frequent in ET 4 (49%) than in other ETs(only 3%) (p < 0.001). Among ET 4 isolates, co-productionwas significantly more frequent in TSS isolates (63%) thanin non-TSS isolates (42%) (p < 0.05). Significantly morefrequent co-production was also found in urogenital TSSisolates (78%) vs urogenital non-TSS or total non-TSSisolates among ET 4 isolates (both p < 0.05). No significantdifference was found in non-urogenital TSS isolates vs non-55RESULTSurogenital non-TSS or total non-TSS isolates among ET 4isolates.56RESULTSTable 12. TSST-1 and SEA production in ET 4 S. aureus strains compared with other El'sStrainsET 4 OTHER ETsM TSST-1 SEA 80TH N TSST-1 SEA BOTHUrogenital TSS (29) 23 22(96%) 0 19(83%) 0 18(78%)b6 3(50%) 1(17%) 1(17%)Urogenital Non-TSS (137) 37 28(76%) 18(49%) 17(46%) 100 12(12%) 10(10%) 1(1%)Nonurogenital TSS (36) 17 15(88%)NS8(47%)NS7(41%) N5 19 4(21%) 3(16%) 1(5%)Nonurogenital Non-TSS (148) 39 23(59%) 21(54%) 15(38%) 109 12(11%) 16(15%) 4(4%)TSS (65) 40 37(93%)b27(68%)N525(63%) 0 25 7(28%) 4(16%) 2(8%)Non-TSS (285) 76 51(67%) 39(51%) 32(42%) 209 24(11%) 26(12%) 5(2%)Total (350) 116 88(76%) a 66(57%) 8 57(49%) 8 234 31(13%) 30(13%) 7(3%)a: p < 0.01 compared with strains of other ETsb p < 0.01 compared with non-TSS strainsc: p < 0.05 compared with non-TSS strainsNS: not significantly difference compared with non-TSS strains'p: Chi-square with Yates correction57RESULTS4.5 HEMOLYTIC ACTIVITYThe hemolytic activity of urogenital and non-urogenitalS. aureus isolates recovered from individuals with orwithout TSS were compared (Table 13). The urogenitalisolates associated with TSS had significantly lesshemolytic activity than urogenital non-TSS isolates(p<0.01). There were no significant differences in non-TSSvs TSS, and non-urogenital non-TSS vs non-urogenital TSSisolates. The relationship of electrophoretic type withhemolytic activity was examined (Table 8). Significantlyless hemolytic activity was found among ET 4 isolates (1.70± 0.25) than in other ETs (3.16 ± 0.19) (p<0.001).4.6 CHARACTERIZATION OF RADIOLABELED TOXINTypically, the specific activity of radioiodinatedTSST-1 or SEA ranged from 69.7 to 11.56 uCi/ug and 43.3 to23.4 uCi/ug of protein, respectively; with < 10% loss ofbiological activity after iodination, as determined bymitogenic effect on human PBMC. The precipitable countsdetermined by trichloral acetic acid precipitation wasgreater than 95%. Analysis of 1251 -TSST-1 and 125I-SEA bysodium dodecyl sulfate-polyacrylamide gel electrophoresisand autoradiography indicated bands at 22 and 28kilodaltons, respectively (Fig-4).58RESULTSTable 13. Hemolytic activity of urogenital and non-urogenital S. aureus strainsZonal radius (mm) of S. aureusIsolates^ p (rank sum)TSS associated^Non-TSS associated(Mean + S.E./No.)^(Mean + S.E./No.)Urogenital^1.85 + 0.576/13^3.71 + 0.250/82^< 0.01Non-urogenital^3.00 + 0.601/9^2.44 + 0.215/64^NS* p: Wilcoxon rank sum testNS: not significantly differenceTable 14. Hemolytic activity of ET 4 S. aureus strains comparedwith strains of ETsStrains^Zonal radius (mm) of S. aureus(Mean + S.E./No.)ET 4^1.70 + 0.25/50Other ETs^3.16 + 0.19/118p (rank sum)^< 0.001* p: Wilcoxon rank sum test59RESULTSFigure 4. Autoradiography of 125 I-TSST-1 and 125 I-SEA97694630 "11-1251-SEAI-TSST-160RESULTS4.7 BINDING OF RADIOLABELED TOXIN TO PURIFIED T CELLS ANDMONOCYTESSpecific binding of 125 I-TSST-1 and 125I-SEA to 10 7human monocytes at 4 °C was demonstrated to be concentrationdependent and saturable (Fig-5). The optimum conditions forbinding were adopted from previous studies in our laboratory(158). Nonspecific binding was typically < 25% of totalbound activity, and radioactivity associated with excisedmicrocentrifuge tips in the absence of cells was negligible.However, no specific binding activity of 125I-TSST-1and 125I-SEA to highly purified human T cells was found(Fig-5).61RESULTSFigure 5. Mpresentative specific binding curve of 125 I-TSST-1 or J."I-SEA with monocytes and T cells. Datarepresent mean ± S.E. of triplicates for one donor (whereS.E. bars were not indicated, they were smaller thansymbols). Cells were incubated with 0-40 nm of radiolabeledtoxins and processed as described in the text.E0CO4J7C 80...4 2Oa_00^10^20^30^40^50125 1-TSST-1 (nM)■ T cells^0 MonocytesE QSOcoto Q0O e0^10^20^ 40125 1-SEA (nM)■ T cells^Q monocytes62RESULTS4.8 BINDING OF RADIOLABELED TOXIN-MONOCYTE MEMBRANE FRAGMENTCOMPLEX TO T CELLSAfter pre-incubation of 1251-TSST-1 with MMF for 1 h,the specific binding of the 125 I-TSST-1-MMF complex to Tcells was demonstrated in 4 of the 7 donors (Table 15).Similarly, the specific binding of the 1251-SEA-MMF complexto T cells was demonstrated in 3 of the 4 donors (Table 16).The non-specific binding activities (expressed as percentageof total activities) of 1251 -TSST-1 and 125 I-SEA wereunderstandably high (59% and 60%, respectively), probablydue to the use of MMF. Despite the high non-specific bindingactivities, the total binding activities of the 125I-TSST-1-MMF complex to T cells were significantly higher than thecorresponding non-specific binding activities (p < 0.05,one-tailed paired t test) (Table 15). Similarly, the totalbinding activities of the 125 I-SEA-MMF complex to T cellswere significantly higher than the corresponding non-specific binding activities (p = 0.05, one-tailed paired ttest) (Table 16). These results provide statistical evidencethat even though our method to study specific bindingactivity is relatively insensitive, the conclusion thatspecific binding can be demonstrated is sound.63RESULTSTable 15. Binding 125I-TSST-1-MMF complex with T cellsBinding activity (kcpm) aDonor ^No.^Totalc^Non-specific Specificb1 54.2 + 2.9 32.7 + 0.8 21.5 + 1.92 11.3 T 0.6 5.6 + 0.3 5.7 + 0.5_3 7.4 + 0.2 8.1 T 0.3 04 8.7 T 0.5 8.4 T 0.4 05 30.8 + 1.2 16.5 + 1.0 14.3 + 1.1_6 21.8 T 1.8 22.2 T 2.5 07 20.6 T 0.1 14.3 + 0.7 6.3 + 0.4_a: the cell-associated radioactivity determined with aSearle 1185 gamma counterb: the total binding activity substracts the correspondingnonspecific binding activity of each assayc: p < 0.05 (one-tailed paired t test) compared withnon-specific binding activityTable 16. Binding of 125I-SEA-MMF complex with T cellsBinding activity (kcpm) aDonor ^No.^Totalc Non-specific Specificb1 6.9 + 0.4 7.8 + 0.1 02 50.3 + 3.9 21.7 +^1.8 28.6 + 2.93 57.5 + 10 34.8 T 2.5 22.7 + 6.34 47.8 +2.6 37.2 + 5.2 10.6 + 3.9a: the cell-associated radioactivity determined with aSearle 1185 gamma counterb: the total binding activity substracts the correspondingnonspecific binding activity of each assayc: p = 0.05 (one-tailed paired t test) compared withnon-specific binding activity64RESULTS4.9 T CELL MITOGENICITY ASSAY USING TRANSWELL FILTER INSERTST cell activation by staphylococcal toxins has beenshown to require antigen presenting cells; neither IL-1 norIL-2, alone or in combination, can substitute for monocytesin supporting TSST-1-induced T cell proliferation (122).This prompted us to test whether T cell-monocyte contact isrequired for supporting T cell activation and whether othersoluble monokines could replace monocytes in supporting Tcell proliferation. We did two approaches, as follows (Fig-6). First, T cells and monocytes were co-cultured incompartments separated by a semipermeable membrane (0.45 umof diameter), and stimulated in the presence of TSST-1 orSEA. This was achieved by utilizing Millicell-HA cultureplate inserts (Millipore). Figure 6 demonstrates that Tcells failed to proliferate in response to TSST-1 or SEAwhen the physical contact of T cell-monocyte was preventedby a semipermeable membrane. Second, conditioned mediaderived from cultures of unstimulated or toxin-stimulatedmonocytes were tested for their ability to induce T cellproliferation. The results were negative. The conditionedmedia containing soluble factors, such as monokines, butwithout monocytes, failed to support T cell proliferationefficiently.65RESULTSFigure 6. Proliferative responses of staphylococcal toxin-treated T cells separated from monocytes by a semipermeablemembrane. Each bar represents the mean + S.E. of triplicatesfor one donor. T, T cells; Mo, monocytes; *, within cultureinsert; RPMI, complete media; (-)CM, the supernatant ofconditioned media prepared by culturing monocytes incomplete media for 24 h without toxin stimulation; (+)CM,the supernatant of conditioned media prepared by culturingmonocyte in complete media for 24 h with TSST-1 or SEA.66T+Mo+RPMIT+TSST 111T+Mo+TSST 1Mo*/T+TSST 1TSST-1 * /T+Mo(-)CM+T(+)CM T3H-Thymidine Incorporated (kcpm)0 20 40 60 80 1003H-Thymidine-Incorporated (kcpm)0 10 20 30 40 50T+Mo+RPMIT+SEAT+Mo+SEAMo*/T+SEASEA*/T+Mo(-)CM+T(+)CM T67CHAPTER 5DISCUSSIONEarlier studies in our laboratory demonstratedsignificantly more frequent seroconversions to TSST-1 andSEA among TSS patients (159). Our data of TSST-1 productionamong TSS-associated and non-TSS-associated isolates gavefurther support to the etiological role of S. aureus in thepathogenesis of both urogenital and non-urogenital TSS.Furthermore, since SEA production was also significantlymore frequent among TSS isolates (primarily in urogenitalisolates) compared with non-TSS-associated control isolates,this staphylococcal enterotoxin may also be implicated inTSS, and primarily in urogenital TSS cases. Since 26%isolates produce neither TSST-1 nor SEA, other enterotoxinsbesides TSST-1 and SEA may be involved in TSS. Studies ofthe role of staphylococcal enterotoxin B (SEB) in TSS byCrass et al.(17, 27), and Lee et al. in our laboratory(paper submmitted) indicate that SEB may also be animportant factor of TSS where TSST-1 is not involved,especially in non-menstrual cases.Parsonnet et al. (160) found that purified SEA alsoinduced interleukin-1 and was more potent than TSST-1 incausing lethality in the rabbit following constantsubcutaneous infusion. Fisher et al. (161) found that SEA at68DISCUSSIONconcentration of less than 1 pg/ml induced significant TNFactivity in human peripheral blood mononuclear cells. Theyand others (108, 160, 161) postulated that induction ofinterleukin-1 or tumor necrosis factor by TSST-1 andenterotoxins may provide a common pathway of TSSpathogenesis. Our finding that SEA was frequently co-produced with TSST-1 in urogenital TSS compared to non-TSSisolates suggested the additive role of SEA in thepathogenesis of TSS.Our study of the electrophoretic typing of 350 S.aureus isolates revealed that a single clone of S. aureus accounted for the majority (79%) of TSST-1-producingisolates recovered from TSS patients with a urogenital focusand this is in accordance with the finding of a singlepredominant clone from urogenital focus among TSS patientsreported by Musser et al. (33). The fact that our isolateswere from a single geographic location (Vancouver), whilethe isolates reported by Musser et al. were from diversegeographic regions (5 countries of 3 continents), yet bothstudies identified a single clone of S. aureus responsiblefor the majority of urogenital TSS cases was very striking.Furthermore, our finding was the first to reveal thatthe same clone (ET 4, the equivalent of ET 41 which wasarbitrarily designated by Musser et al.) accounted for the69DISCUSSIONmajority of TSST-1 (76%) and SEA (66%) producing isolatesfrom urogenital sites of TSS patients, and was most likelyto co-produce both TSST-1 and SEA (49% of 116 ET 4 isolatesvs 3% of 234 other ET isolates).In the current study, we examined the hemolyticactivity of a large sample of TSS-associated and non-TSS-associated S. aureus isolates. Our finding that urogenitalTSS-associated isolates were significantly less hemolyticthan urogenital non-TSS-associated isolates further extendedprevious report from our laboratory involving a small numberof isolates (42), which demonstrated that TSS-associatedisolates were significantly less hemolytic than those non-TSS-associated. However, our results showed no significantdifferences between total TSS and non-TSS isolates, or non-urogenital TSS and non-urogenital non-TSS isolates,indicating that this reduced hemolytic activity is unique tothe urogenital TSS isolates.ET 4 isolates were found to be less hemolytic thanother ETs. The genetic and biological basis of this findingremains to be elucidated.There are two major findings in this study. One is thatthe majority of the isolates recovered from TSS patientswith a urogenital focus co-produce both TSST-1 and SEA andare less hemolytic. It is already well known that many70DISCUSSIONexoproteins of S. aureus are coordinately regulated by theaccessory gene regulator (agr) (Table 4). However, althoughit was shown by Recscei et al. (62, 63) that TSST-1 and thehemolysins are both up-regulated by activation of agr, wefound that the majority of urogenital TSS isolates produceTSST-1 and are less hemolytic, while SEA which is notaffected by agr tends to be co-produced with TSST-1. Apossible explanation of the co-production of TSST-1 and SEAmay be that the genes for expressing TSST-1 and SEA are bothcarried on mobile elements which are capable of multiple butlimited number of insertions on the chromosome of S. aureus.Another explanation may be that urogenital isolates areadapted to a different niche than non-urogenital isolates,such that the genes of S. aureus exoproteins may expressdifferently in isolates from a urogenital site versus non-urogenital sites. The gene expression and regulation of S.aureus is still poorly understood, Further studies areneeded to clarify these unanswered questions.The second major finding of our study is that the greatmajority of the isolates recovered from a urogenital focusof TSS patients which co-produce TSST-1 and SEA, areidentical in electrophoretic type. They most likelyrepresent a single clone which are derived, but still sharelinear descent from a common precursor cell, because71DISCUSSIONevolution convergence to the same ET from differentancestors over a short time span is very unlikely (167). Arecent study in our laboratory by Goh et al. (unpublisheddata), using polymerase chain reaction (PCR) method toexamine the DNA fragment length polymorphic types ofcoagulase among S. aureus isolates, indicated that there aresubgenotypes within this clone. But the PCR analysis canneither tell the clonal structure nor the geneticrelationships among them. This further indicates thatmultilocus enzyme electrophoresis is a powerful tool toindex levels of genetic diversity and estimate geneticrelationships among strains, while typing by comparativegene sequence or DNA fragment length polymorphism is usefulto supplement electrophoretic typing by giving informationof the sub-genotypes within the same clones determined bymultilocus enzyme electrophoresis.This ET 4 clone may be more adapted than others inclinical specimens, as it is the major clone accounting for33% of the 350 S. aureus isolates and is evenly distributedin urogenital sites and non-urogenital sites (17% vs 16%),while no other single clone accounted for more than 12%. Ittends to produce both TSST-1 and SEA and is less hemolyticthan others. Within this clone, isolates collected fromurogenital sites more frequently co-produce TSST-1 and SEA72DISCUSSIONthan isolates from non-urogenital sites. This unique featureof the urogenital isolates in the same clone may be due toevolutional adaptation of the isolates from this clone tothe urogenital milieu, as mentioned above.The initiative for us to detect binding receptors forTSST-1 and SEA on T cells was an attempt to resolve acontroversy about whether there are direct TSST-1 receptorson T cells. Schlievert's group (101) demonstrated that therewere TSST-1 receptors on human T cells and reported thatthey were similarly distributed in CD4+ and CD8+ T cells;but Scholl et al. and Uchiyama et al. (83, 102, 103) wereunable to detect direct binding of TSST-1 to T cells, whileclass II molecules on antigen presenting cells were requiredfor T cell activation by TSST-1. We believe that furtherelucidation of this question is an important step towardsunderstanding of the pathogenesis of TSS.The results of this study showed that there are nodirect binding sites for TSST-1 and SEA on resting Tlymphocytes, and that the presence of monocyte membranecomponents (presumably class II molecules) was required forTSST-1 and SEA binding to T cells. Using highly purifiedTSST-1, SEA and resting T cell preparation, our data showedthat there was no specific binding of these toxins to Tcells under the conditions of our experiments. However, when73DISCUSSIONmonocyte membrane fragments were added to the toxins priorto the incubation with T cells, the toxin-MMF complex hadspecific binding to T cells.The high degree of non-specific binding activity wasprobably due to methodologic factors using MMF (because itis unlikely that MMF could bind specifically to homologous Tcells), and the less than optimal separation of the cellsand cell-bound proteins from free toxin and MMF bycentrifugation. Despite the high degree of non-specificbinding activities, the total binding activities of the125 I-TSST-1-MMF complex and of the 125 I-SEA-MMF complex to Tcells were significantly higher than the corresponding non-specific binding activities (p < 0.05 and p = 0.05,respectively, by one-tailed paired t test), indicating thatthere was specific binding of the 125I-TSST-1-MMF complexand of the 1251-SEA-MMF complex to T cells. Because of thehigh degree of non-specific binding activity in our assay,it is possible that some donors may have false negativeresults. Further improvement of the binding assay isrequired to generate quantitative data about the receptorson human T cells for TSST-1 or SEA in the presence of themonocyte membrane component.Our findings are in agreement with the hypothesis madeby Marrack and Kappler that the capability of the T cell74DISCUSSIONreceptor for either staphylococcal toxin or class IImolecule alone is low, and that contact of the T cellreceptor with both toxin and class II molecule is requiredto generate efficient binding (71). Recent work by Gascoignewhich demonstrated specific interaction of the SEA-MHCcomplex with the TcR B chain by using a cell binding assay,also supports our findings (88).This further proves that superantigens are similar toconventional antigens, in a way that they both havedetectable binding activity for MHC, but neither antigen norMHC alone can bind the T cell receptor (71, 162, 163). Onlyafter the complex of peptide-MHC class II molecule hasformed can it functionally engage the T cell receptor.Similarly, staphylococcal toxins have detectable bindingaffinities for the class II molecule, but neither class IImolecule nor the toxins separately have detectableaffinities for the T cell receptor, but the combinations oftoxin and class II molecule do. TSST-1 and SEA probably actas bivalent linkers, engage the sides of class II moleculeand VB, and bringing into close contact of the T cellreceptor and MHC.It is very difficult to stimulate T cells with thetoxin in the complete absence of class II molecules,suggesting that binding to MHC may either induce a75DISCUSSIONconformational change in the toxin molecule, uncover a VBregion of T cell receptor, or that a considerable part ofthe binding energy of T cell receptors for the toxin-classII complex is contributed by binding of the T cell receptorto class II molecule.How can the discrepancy between our results and thoseof Schlievert et al. be reconciled? We believe thediscrepancy lies in the purity of resting T cells. It iswell known that staphylococcal toxins can bind to variousregions of MHC class II molecules, and by far class IImolecule is the only receptor for these toxins found amongperipheral blood mononuclear cells (73, 80, 82, 102, 103,164). It is important to stress the purity of the resting Tcells in the binding assay which may be the key todistinguish the direct binding sites for staphylococcaltoxins on T cells. In Schlievert's study (101), the panningmethod and incubation on plastic petri dishes were used todeplete B cells and monocytes in PBMC; the T cellpreparation used had < 2% esterase-positive cells bynonspecific esterase assay and < 10% surface-immunogobulin-positive cells by immunofluoresence staining. However theydid not exclude the presence of other class II molecule-positive cells, including B cells and activated T cells.76DISCUSSIONPrevious studies which demonstrated that TSST-1 bindsto MHC class II molecules on monocytes, but not to resting Tcells (102, 103, and our observation), have used moreefficient T cell purification methods, as well as strictercriteria for purity assessment of cells (>99% CD2-positivecells, <1% class II molecule-positive cells in ourpreparation). Previous studies have shown that theexpression of TSST-1 binding sites on cells closelyparalleled MHC class II antigen expression (102). SeveralMabs to HLA-DR and HLA-DQ inhibited TSST-1 binding to humancells, whereas anti-HLA-DP Mabs failed to inhibit TSST-1binding (102, 103, 158). This does not rule out involvementof HLA-DP in TSST-1 binding, because the epitopes recognizedby the Mabs may be different from those involved in TSST-1binding. However, using mouse L-cell transfectants thatexpressed HLA-DR, HLA-DQ, or HLA-DP gene products in TSST-1binding, it was found that TSST-1 bound to the HLA-DR andHLA-DQ transfectants, but not to the HLA-DP anduntransfected L cells (102). SEA and other enterotoxins havealso been shown by others to interact with MHC class IImolecules, but with no direct binding to T cells (80, 86,122, 158, 164, 165, 166). One study showed that SEA binds tothe purified murine MHC class II molecule I-E d reconstituted77DISCUSSIONin supported planar membranes, indicating no other cellsurface proteins are required for SEA binding (168).TSST-1, SEA and other staphylococcal enterotoxins areknown as microbial superantigens, because of their abilityto bind with class II antigen and stimulate T cells based onVB specificity of the TcR (71, 73). Using the syntheticpeptide approach, it was shown that SEA interacts with the ahelix of the class II B chain, via an NH2-terminal domain ofthe SEA molecule (173, 174). Evidence that the class II achain is also involved in binding of staphylococcal toxinsincludes the result of one study using chimeric a and Bchains of DR and DP expressed on the surface of transfectedcells to show that the al domain of the DR was essential forhigh affinity binding of TSST-1. Using another approach, achain mutation with single alanine substitutions along the ahelix proposed to form one side of the antigen-bindinggroove had negligible effects on staphylococcal enterotoxinpresentation, although drastic effects on peptidepresentation were observed (96). These studies bothconcluded that staphylococcal toxins interact with MHC classII molecules outside the antigen-binding groove (95).Studies have also been carried out with synthetic peptidescorresponding to various regions of the a chain of mouse MHCclass II molecules (72). The data obtained with MHC class II78DISCUSSIONa and B studies suggest that al domain of DR was essentialfor high-affinity binding of TSST-1, while both a heliceswere important for SEA-induced function, although the ahelix of B chain is sufficient for SEA binding to cells, andboth TSST-1 and SEA binding probably occur outside theantigen-binding groove.The presentation of microbial superantigens (likestaphylococcal toxins) differs in two important ways fromconventional antigen presentation. First, presentation ofstaphylococcal toxins is not MHC restricted, althoughpresentation varies among different MHC class II haplotypes(72, 85; 169). Second, the prior processing or proteolysisto peptide fragments that is required for protein antigensis not necessary for superantigen presentation (86, 87, 103,122, 165).It was shown that the TcR for antigenic peptides isalso the receptor for staphylococcal enterotoxins (4, 73,98). One of the major characteristics of superantigens isthat they could activate different T cells, depending on theVB type of the TcR (4, 73, 98). It seems that VB specificityis a quantitative effect rather than a qualitative one,since T cell clones of the same VB specificity vary in theirresponse to microbial superantigens (175). Up to now, theevidence for interaction of staphylococcal toxins with TcRs79DISCUSSIONis based almost entirely on studies with antibodies to theTcR and PCR analysis using VB oligonucleotide primers (73,97, 120, 121, 170, 171). Only one study using a cell bindingassay showed that a secreted, truncated TcR B chain wassufficient to directly bind with SEA complexed to cellsurface MHC class II molecules (88, 99). Using a differentapproach, we have shown that TSST-1 or SEA after complexedwith monocyte membrane fragments (presumably class IImolecules) could bind directly to T cells.We also noticed that for some donors specific bindingcould not be detected when toxin-MMF was incubated with Tcells. We believe there are two possibilities for this. Oneis the high non-specific activity due to the use of MMF;another is that in different donor assays, T cells withdifferent VB specificities in the peripheral blood may bepresent, resulting in the different specific bindingactivity to the toxin-MMF. Because other direct bindingassays and Mabs for these toxins are not available for thetime being, while our method of binding assay is valuable inapproaching the interaction of toxin-class II complex withTcR, further modification is required to decrease thenonspecific binding and increase the sensitivity of thisassay.80DISCUSSIONAlthough there is little similarity between TSST-1 andSEA in amino acid sequence (71, 72), it has been shown thatSEA is the only staphylococcal enterotoxin which can competewith TSST-1 in binding to human monocytes (172, 177). Cross-competition of SEA with TSST-1 in binding assays suggeststhat these toxins may bind to overlapping epitopes on thesame receptor (158). The finding that SEA can compete forTSST-1 receptors may explain the similarity of itsbiological properties to those of TSST-1 and may be thebasis for a possible role of SEA in TSS.To examine the importance of T cell-monocyte contactfor the biological activities of staphylococcal toxins, weused a semipermeable membrane to physically separate T cellsfrom monocytes during toxin stimulation. The results showthat direct T cell-monocyte contact is required for theinduction of resting T cell proliferation in response toTSST-1 or SEA. Culture media from monocytes incubated withtoxin for 24 h could not substitute for antigen presentingcells.One of the first known properties of staphylococcaltoxins was their ability to stimulate many types of T cells,both in human and mice (71, 114, 140, 176, 178, 179). Thetoxins were not acting as mitogens, they did not activate invitro a large proportion of T cells compared to the81DISCUSSIONstimulatory plant lectins, but stimulate only clones of Tcells with specific VB (73, 87, 97, 119-121). The presenceof antigen presenting cells with class II molecules isrequired for this stimulation. IL-1, a monokine released bymonocytes, by itself is not sufficient to trigger aproliferative response of purified T lymphocytes to TSST-1without antigen presenting cells (122). There are no MHCrestrictions: even xenogeneic class II molecules canreconstitute the human T cell response to staphylococcaltoxins (72, 169). Paraformaldehyde fixed antigen presentingcells, irradiated monocytes, even planar membranescontaining only lipid and purified I-E d molecules weresufficient for activation of T cells by staphylococcaltoxins (83, 103, 114, 168). It was also shown that anti-HLA-DR Mab can inhibit binding activity (103). The results ofour study give further support to the notion that T cell-monocyte contact is needed for the induction of T cellactivation by TSST-1 or SEA.82CHAPTER 6CONCLUSIONIn conclusion: 1) Our study of TSST-1 and SEAproduction in 350 isolates from Vancouver gave furthersupport that SEA is a co-virulent factor with TSST-1 inurogenital TSS. 2) The multilocus enzyme electrophoresisprovided a powerful tool for studying the geneticrelationship among the TSS strains. 3) Our studies of theelectrophoretic typing distribution of 350 S. aureus isolates revealed that a single clone of S. aureus accountedfor the majority of TSST-1-producing isolates recovered fromTSS patients with a urogenital site in accordance withearlier reports by Musser et al. (33). Furthermore, ourfinding revealed that the same clone (ET 4) accounted forthe majority of TSST-1 (76%) and SEA (66%) producingisolates from urogenital TSS patients, and was most likelyto co-produce both TSST-1 and SEA (49% of 116 ET 4 isolatescompared with only 3% of 234 isolates of other ETs). 4) ourstudies of 125 I-TSST-1 and 125I-SEA binding to highlypurified human resting T cells demonstrated that there wasno direct binding of TSST-1 and SEA on resting human Tlymphocytes, and that the presence of monocyte membranecomponents (presumably class II molecules) is required forTSST-1 and SEA binding to T cells. Furthermore, direct T83CONCLUSIONcell-monocyte contact is required for the induction of Tcell activation by TSST-1 or SEA.Our finding is in agreement with the hypothesis thatthe capacity of the T cell receptor for eitherstaphylococcal toxin or class II molecule is low, and thatcontact of the T cell receptor with both toxin and class IImolecule is required to generate efficient binding (71).TSST-1 and SEA probably act as bivalent linkers, engagingthe sides of the class II molecule and VB, and bringing intoclose contact of the T cell receptor and MHC. It is verydifficult to stimulate T cells with the toxin in thecomplete absence of class II molecules. 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In vivoeffect of staphylococcal enterotoxin A on peripheralblood lymphocytes. Infect. Immun. 44:401-405.179. Fast, P. M., P. M. Schlievert, R. D. Nelson. 1989.Toxic shock syndrome-associated staphylococcal andstreptococcal pyrogenic toxins are potent inducers oftumor necrosis factor production. Infect. Immun.57:291.180 Kornblum, J., B. N. Kreiswirth, S. J. Projan, H. Ross,and R. P. Novick. 1990. Agr: A polycistronic locusregulating exoprotein synthesis in Staphylococcusaureus. Novick, R.P. ed. In: Molecular biology of thestaphylococci.p. 377.104BIOGRAPHICAL INFORMATION NAME:Alex Hongsheng ChangMAILING ADDRESS:111-360 E. 14th AvenueVancouver, B.C. V5T 2M8CanadaPLACE AND DATE OF BIRTH:Beijing, China/ December 16, 1962EDUCATION (Colleges and Universities attended, dates, and degrees):Capital Institute of Medicine, Beijing, China1980-1986, Bachelor of Medicine (M.D.)University of British Columbia, Vancouver, B.C.1990-1992, Master of SciencePOSITIONS HELD:Beijing You An Hospital, Beijing, China1986-1989, Clinical PhysicianVancouver General Hospital, Vancouver, B.C.,1989-1991, Canadian International Development Agency Clinical TraineePUBLICATIONS (if necessary, use a second sheet):ABSTRACTS:1. Alex H. Chang, James M. Musser, and Anthony W. Chow. 1991. A single clone of S.aureus which produces both TSST-1 and SEA causes the majority of menstrual toxicshock syndrome. Clinical Research 39(1):36A.2. Alex H. Chang, Raymond H. See, and Anthony W. Chow. 1992. Binding of TSST-1for receptors on human T lymphocytes and requirement of T cell-monocyte contact inthe induction of resting T cells proliferation. Clinical Research 40(I):102A.(continue next page)AWARDS:1989-1991, Canadian International Development Agency Fellowship1992, American Federation for Clinical Research Membership AwardComplete one biographical form for each copy of a thesis presentedto the Special Collections Division, University Library.3. Raymond H. See, Alex H. Chang, Swee Han Goh, Winnie S. Kum, and Anthony W.Chow. 1992. Induction of TNF and IL-1 by staphylococcal toxic shock syndrome toxin-1(TSST-1) requires the presence of monocytes and T cells. Clinical Research 40(1):53A.MANUSCRIPTS IN PREPARATION:1. Raymond H. See, Winnie S. Kum, Alex H. Chang, Swee Han Goh, and Anthony W.Chow. 1992. Induction of tumor necrosis factor and interleukin- I by staphylococcal toxicshock syndrome toxin-1 requires the presence of monocytes and T cells. (in press,Infection and Immunity).2. Vivian Lee, Alex H. Chang, Anthony W. Chow. The role of staphylococcal enterotoxinB in toxic shock syndrome. (in press, Journal of Infectious Diseases).3. A single clone of Staphylococcus aureus which produces both TSST-1 and SEA causesthe majority of urogenital toxic shock syndrome. (first author).4. The role of toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A(SEA) in Toxic Shock Syndrome. (first author).5. Binding of TSST-1 and SEA for receptors on human T lymphocytes and requirementof T cell-monocytes contact in the induction of resting T cells proliferation. (firstauthor).

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