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Bergdoll, M. S., J. K. Czop, and S. S. Gould. 1974. ...... 0.1 M. SDS 1 %. Solution III Potassium acetate 3 M. Formic acid. 1.8 M. TE buffer Tris-HCl (pH 7.5) 10 mM ...
REGULATION OF ENTEROTOXIN EXPRESSION BY Staphylococcus aureus By CHING WEN TSENG B. S., National Chung Hsin University, Republic of China, Taiwan, 1994 M.S., Emporia State University, Emporia, Kansas, 1999 A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Diagnostic Medicine/ Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2004 Approved by:

Dr. George C. Stewart

Dr. Donald Robertson

Major Professor

Committee member

Dr. Beth Montelone

Dr. Raymond Rowland

Committee member

Committee member

ABSTRACT Staphylococcus aureus is an important human pathogen causing diseases ranging from food poisoning to systemic infection. The pathogenicity of this organism involves the coordinated expression of virulence factors, including exotoxins and surface proteins. The staphylococcal accessory gene regulator (Agr) quorum sensing system, and members of the Sar family of transcriptional regulators, were identified as global regulators that play important roles in regulating virulence gene expression. Agr has been previously shown to be a positive regulator of staphylococcal enterotoxin B and D (SEB, SED) expression. To identify the Agr cis-regulatory region within the seb and sed promoters, the seb and sed promoter elements were fused with a chloramphenicol acetyl transferase reporter gene and expressed in S. aureus strains specifically mutated at the agr or sar loci or that carried combinations of these mutant alleles. The Agr regulation effect of sed and seb expression was identified to be due to Agr regulation of Rot (repressor of toxins) activity. Staphylococcal enterotoxin-like protein R (SElR) was newly identified to be carried by the pIB485 which also carries the sed and sej determinants. The promoter element of ser and the Agr, Rot, and SarS effect of ser expression were investigated. In addition, the regulation effect of Agr, SarA, SarS, and Rot of rot expression, and transcription profiles were also characterized. The rot expression and transcription profiles indicated that a complex network among the transcriptional regulators, the Agr system, and the alternative sigma factor (σB) participate in the expression of this important transcriptional factor in S. aureus.

ACKNOWLEDGEMENT I would like to thank my advisor Dr. George C. Stewart for his patience in teaching me how to be a good scientist and help me to find my goal in the research. I would also like to express be application to Drs. Donald Roberson, Beth Montelone, and Raymond Rowland who have served as the members of my advisory committee and supported my graduate study in many meaningful ways. I would also like to express my gratitude to Dr. Muthukrishnan who served as the outside chair. I would also like to thank my fellow graduate students and coworkers for sharing my up and down time associated with my project. Finally, I would like to express my sincere gratitude to my family and friends for their supports through these years.

TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................i LIST OF TABLES AND FIGURES FOR CHAPTER I ......................................v LIST OF TABLES AND FIGURES FOR CHAPTER II.....................................vi LIST OF TABLES AND FIGURES FOR CHAPTER III....................................vii LIST OF TABLES AND FIGURES FOR CHAPTER IV....................................viii LIST OF TABLES AND FIGURES FOR CHAPTER V.....................................ix

CHAPTER I. The network of two-component regulatory systems and transcriptional regulators in Staphylococcus aureus. ..........1 1. Regulatory components of virulence factors ......................................4 A.) Two component regulatory systems ......................................4 a.) The Agr system ..............................................................6 b.) YycFG..............................................................................9 c.) SrrAB (SrhSR)................................................................10 d.) ArlSR ...............................................................................11 e.) LytSR ...............................................................................12 f.) SaeSR ..............................................................................13 B.) Transcriptional regulators ........................................................14 a.) Staphylococcal accessory regulator (SarA) ..............14 b.) SarR .................................................................................18 c.) SarS (SarH1) ..................................................................19 i

d.) Repressor of toxins (Rot) .............................................21 e.) MgrA (NorR, RAT).........................................................22 f.) Other SarA homologues ................................................23 C.) Other factors involved in the gene regulation.......................24 a.) TRAP/RAP......................................................................24 b.) Staphylococcal virulence regulator (svrA) .................25 D.) Global regulators.......................................................................26 a.) Alternative sigma factor (SigB) in S. aureus .............26 b.) Superantigens as global regulators ............................28 2. Regulations between transcriptional regulators and two component system .....................................................................................................28 3. The enterotoxins produced by S. aureus ............................................30 4. Regulation of enterotoxins by S. aureus .............................................33 REFERENCE...........................................................................................................37

Chapter II Accessory gene regulator (Agr) control of staphylococcal enterotoxin D gene expression....................................................65 ABSTRACT ..............................................................................................................66 INTRODUCTION .....................................................................................................67 METHODS AND MATERIALS ..............................................................................70 RESULTS.................................................................................................................73 DISCUSSION...........................................................................................................81 ACKNOWLEDGMENTS.........................................................................................84 ii

REFERENCES ........................................................................................................85

Chapter III. Regulation of the repressor of toxin promoter (rot) ..............107 ABSTRACT ..............................................................................................................108 INTRODUCTION .....................................................................................................109 METHODS AND MATERIALS ..............................................................................111 RESULTS.................................................................................................................117 DISCUSSION...........................................................................................................127 REFERENCES ........................................................................................................130

Chapter IV. Regulation of staphylococcal enterotoxin B expression.....153 ABSTRACT ..............................................................................................................154 INTRODUCTION .....................................................................................................155 METHODS AND MATERIALS ..............................................................................159 RESULTS.................................................................................................................162 DISCUSSION...........................................................................................................172 REFERENCES ........................................................................................................175

CHAPTER V. Expression of the plasmid-borne enterotoxin determinants of Staphylococcus aureus...............................................................197 ABSTRACT ..............................................................................................................198 TEXTS

...............................................................................................................199

REFERENCES ........................................................................................................205 iii

Appendix I. Nucleic acid isolations..................................................................215 Appendix II. Preparation of competent cells, transformation, electroporation, and transduction...............................................221 Appendix III. Enzyme and protein assays using a microplate spectrophotometer ..........................................................................229 Appendix IV Plasmid construction designs ..................................................240

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TABLES AND FIGURES FOR CHAPTER I

TABLE 1. Some of the virulence determents that reported regulated by the Agr, SarA, SarS, or Rot. ...............................................................................55 Fig 1. The components of the Agr operon...........................................................56 Fig. 2. The regulation between SarA and other regulators...............................63 Fig. 3. The SigB operon. ........................................................................................64

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TABLES AND FIGURES FOR CHAPTER II TABLE 1. Bacterial strains used in this study. ....................................................92 TABLE 2. Primers used for PCR amplification...................................................94 TABLE 3. Plasmids used in this study. ................................................................96 TABLE 4. Regulation of hybrid promoters...........................................................98 TABLE 5. Promoter activities of sea-sed hybrid promoters. ............................99 TABLE 6. Rot and SigB effects on sed, sea, lac, and rot promoter activities. ..................................................................................................................100 TABLE 7. CAT values and fold differences from wild type and agr-rot- hosts with Rot expression plasmids......................................................................102 Fig. 1. The sed promoter element. .......................................................................105 Fig. 2. The protease activity of different genotypes as demonstrated on skim milk agar................................................................................................................106

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TABLES AND FIGURES FOR CHAPTER III TABLE 1. Bacterial strains used in this study ....................................................135 TABLE 2. Primers used for PCR amplification...................................................137 TABLE 3. Plasmids used in this study. ................................................................138 TABLE 4. Bacteria phenotypes of different genetic backgrounds strains ......139 Fig. 1. The urease activiy from different genetic strains. ..................................144 Fig. 2. Multiplex PCR of genotyping of different background strains. ............145 Fig. 3. The rot promoter activity in sigB+ agr+ , sigB+ sigB- agr- , sigB- agr +, and agrstrains from exponential, post-exponential, and stationary growhth phase. ........................................................................................................................146 Fig. 4. Primer extension of the rot promoter containing DNA fragment. .......147 Fig. 5. Promoter activities in post- exponential phase from different mutant strains. ..........................................................................................................149 Fig. 6. Electrophoretic mobility assays. ..............................................................151 Fig. 7. Hypothsized model for interactions between Rot and othe transcriptional regulators in post-exponential phase .......................................................152

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TABLES AND FIGURES FOR CHAPTER IV TABLE 1. Bacterial strains used in this study .....................................................181 TABLE 2. Primers used for PCR amplification...................................................183 Fig. 1. The seb promoter element from –122 to +13 .........................................188 Fig. 2. Rot regulation of seb promoter activity ....................................................189 Fig. 3. The seb promoter activity from different growth phases.......................190 Fig. 4. The seb promoter activity from different genetic background..............191 Fig. 5. The truncated seb and seb-lac hybrid promoter elements and the promoter activity from Agr +, Agr-, Agr+ Rot- , and Agr- Rot- hosts. ........192 Fig. 6. The E. coli bearing pDT112 colonies on L-agar plate containing 100 µg/ml ampicillin.......................................................................................................194 Fig. 7. Electrophoretic mobility shift assays of a 120 bp seb and 210 bp lac promoter containing DNA fragment, incubated with different amount of the recombinant His6-Rot from 2x to 128x of protein to DNA in molar ratios. ........................................................................................................................195 Fig. 8. Electrophoretic mobility shift assay of the wild type 122 bp seb promoter containing DNA fragment, incubated with cell lysates collected from Agr- , Agr- SarA -, and Agr- Rot- of post-exponential phase of cultures ..........196

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TABLES AND FIGURES FOR CHAPTER V TABLE 1. The relative promoter activities of sea, seb, sed, sej, and ser in Agr + backgrounds...........................................................................................210 TABLE 2. The activities of the sed, sej, and ser promoters in post exponential phase of growth. ....................................................................................211 Fig. 1. The sed, sej, and ser on pBI485...............................................................213 Fig. 2. The ser promoter activities in Agr+, Agr- , Agr+ Rot-, Agr- Rot- , Agr + SarS Rot-, and Agr- SarS - Rot- hosts background strains. .............................214

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Chapter I. The network of two-component regulatory systems and transcriptional regulators in Staphylococcus aureus.

Staphylococcus aureus is a member of normal flora on skin and nasal cavity of 25 to 30% of humans. However, it is also a common pathogen, causing a wide range of infections. The diseases it causes range from food poisoning, community acquired infections, to systemic infections (11, 75, 132). Osteomyelitis, and septic arthritis, and endocarditis are the most common deep tissue infections. The diversity of the diseases this organism causes is due to the plethora of virulence factors it produces. Virulence factors of this organism include a variety of exoproteins and cell wall associated components (2). The secreted components include enterotoxins (A-E, G-R), leukotoxin, exofoliative toxins, α-, β-, γ-, and δ- hemolytic toxins, toxic shock syndrome toxin (TSST), coagulase, and secreted enzymes, such as nuclease and proteases (6, 121). Staphylococcal gastroenteritis is the food poisoning caused by the heat resistant staphylococcal enterotoxins. The symptoms include nausea, emesis, and diarrhea (6, 132). The onset of symptoms usually starts within twenty-four hours after consumption of contaminated food, and usually subsides after an additional twenty-four hours. Staphylococcal enterotoxins and TSST are superantigens in nature, which cause polyclonal T cell proliferation and induce inflammatory cytokines (32). The T cell activation and cytokine release cause a disruption of immune system regulation that may lead to systemic toxic shock, and in serious cases, can be fatal. Staphylococcal endocarditis, osteomyelitis, and septic arthritis are the deep tissue infections associated with a bacterial invasion of the blood stream. 2

The most common causes of osteomyelitis in children are infection associated with surgeries to stabilize broken bones (60). The ability of S. aureus to survive intracellularly resulting in an escape from the immune system and antibiotic treatment may play an important role in bone infections (59). Internalization of S. aureus has been reported and the virulence factors associated with this include collagen binding protein and fibronectin binding protein (12, 38, 59). Although in vitro studies suggest that S. aureus is able to escape from the immune system and antibiotic treatment by surviving intracellularly, there has been no report of intracellular S. aureus in clinical samples. However, a study using a mouse staphylococcal mastitis model has shown this organism was internalized in different types of cells, such as epithelial cells, cells of mammary glands, and cells of alveolar lumen, and the internalized bacteria were able to divide (12). Therefore, S. aureus is capable of being an intracellular pathogen Staphylococcal endocarditis is most frequently associated with heart valve replacement procedures. A virulence property for endocarditis is the ability of this organism to form a biofilm around the plastic surgical implant (31). Additional virulence factors associated with biofilm production are α- hemolysin, teichoic acid, polysaccharide intercellular adhesin (PIA/PNAG), the ica ADBC-encoded proteins, and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (31, 55). The global regulators, accessory gene regulator (Agr), staphylococcal accessory regulator (SarA), and alternative sigma factor (SigB) have been reported to be associated with regulation of biofilm 3

formation (126). The production of α-hemolysin has been reported to have positive effect on biofilm formation (15). SigB and SarA are both upregulators of icaR, the repressor for ica operon, expression (63, 100). SarA and SigB mutants would likely be hyper biofilm producing strains because of the down regulation of IcaR. However, SigB is not essential for biofilm formation, and a SarA mutant strain is defective in biofilm formation (126). The mechanisms and factors involved in biofilm formation, and its regulation, are still under investigation. In view of the wide range of diseases caused by S. aureus and the different virulence factors involved in each clinical syndrome, coordination of virulence gene expression is an important feature of the pathogenicity of S. aureus. Understanding the regulation of various virulence factors and the coordination between regulatory networks of this organism might identify targets for possible therapeutic intervention in the treatment of staphylococci infections.

1. Regulatory components of virulence factors A.) Two component regulatory systems Bacteria live in a constantly changing environment that includes variations in temperature, nutrient levels, pH, ionic strength, and the amount of available oxygen. For bacteria to survive in this changing environment, they must be able to adapt to the changing conditions and communicate the changes within the community. To sense the changes and relay the information to other members within their community, they must develop sensory and communication 4

mechanisms. The two-component regulatory systems have evolved and allowed the bacteria to sense environmental changes and/or relay the information to other members of the bacteria community. The two-component regulatory systems consist of a sensory component for responding to environmental signals and a response regulator for converting the information from the signal to a regulated expression of cellular responses. The sensor is a protein with a membrane anchoring domain and a histidine kinase domain, and responds to a specific environmental signal. The response regulator is a transcriptional regulator with a DNA binding domain, which coordinates the regulation of a group of genes that are important for responding to the environmental changes. Regulation of virulence determinants by a two-component regulatory system is an important feature of bacterial survival in different niches. When the environmental signal reaches a certain threshold level, the sensor is activated. Then, the activated sensor transduces the environmental signal to the response regulator, by phosphorylating the response regulator. The activated response regulator, then, activates or represses the transcription of other genes (114). Six putative two component systems have been identified in S. aureus, with the staphylococcal accessory gene regulator (Agr) being the best studied. The Agr system is a quorum-sensing system, which is responsible for regulation of various exoproteins and cell wall proteins. YycFG is a responsible for sensing nutrient levels in the environment and regulates nutrient uptake (35). SrrAB, also known as SrhSR, regulates the genes for metabolic responses under anaerobic 5

conditions (98, 122). ArlSR and LytSR are responsible for the regulation of cell division, and autolysis (13, 39). ArlSR may also play a role in virulence factor regulation (39). SaeSR is a growth phase dependent regulator, responsible for the control of exoprotein expression. SaeSR regulation of other genes is downstream of the Agr system (46, 47, 48, 49, 50, 51, 101). In addition, there are two possible two- component systems, identified by DNA sequence similarity to known two component system, whose functions are unknown.

a.) The Agr system The Agr system was identified in a transposon Tn551 mutagenesis experiment as an insertion resulting in an exoprotein negative phenotype. The Agr mutant was found to be defective in the production of several exoproteins, such as α- toxin, and proteases, and had an elevated level of cell wall associated proteins (103). Virulence factors determents and genes required for regulation of metabolic pathways have been reported to be regulated by the Agr system using gene array analysis, and some of them are listed in Table 1. Nucleotide sequence analysis revealed that the Agr locus consists of two divergent transcriptional units encoding RNAII and RNAIII. RNAII is the messenger RNA encoding the structural proteins of the two- component system quorum sensing system (97). RNAII is translated into AgrB, AgrD, AgrC, and AgrA (67). RNAIII encodes a 26 amino acid peptide, δ-hemolysin. However, the 6

δ-hemolysin is not the regulatory component of the Agr system, but the RNAIII itself is the regulatory species controlling the expression of virulence genes (62, (92). AgrC, a 46 kDa transmembrane protein, is the sensor of this quorum sensing system, with a histidine kinase domain that is activated during the postexponential phase by the auto-inducing peptide (AIP) (71). AIP is a 7-9 amino acid peptide containing a thiolactone ring between a conserved cysteine residue and the C-terminal carboxyl group. This thiolactone bond has been proven to be important for the activation of AgrC, as the linear peptide was shown to be inactive (81). The AIP is processed from the 46 amino acid AgrD polypeptide (129). AgrD is processed and exported from the bacterium by a 26 kDa membrane protein, AgrB (128). Research on AgrD has shown that a N-terminal membrane anchoring domain is required for the interaction between AgrB and AgrD leading to processing and exporting of the AIP (129). When the concentration of the environmental AIP reaches a critical level, AgrC is phosphorylated at a histidine residue, and then, activates AgrA by phosphorylating it at an asparate residue (71). AgrA, a 34 kDa cytoplasmic protein, is the response regulator (67). The activated AgrA then binds to the promoter region of the agr locus and upregulates the transcription from the P2 and P3 promoters, resulting in elevated production of RNAII and RNAIII. The increased level of RNAIII upregulates the expression of several exoproteins and down-regulates production of various cell wall-associated proteins. A schematic 7

of the Agr system and the proteins involved in the regulation is shown in Figure 1 (36). Northern blot analysis of specific mRNA species suggested that the Agr system regulates the expression of virulence factors at the transcriptional level. In addition, RNAIII activates the translation of α-toxin by base pairing with the untranslated hla mRNA, thus, resolving the intramolecular base pairing that blocks the ribosomal binding site of the transcript. This result is also consistent with studies of a mutant bearing a deletion of the 5’ end of the RNAIII lacking the region of RNAIII that pairs with the hla mRNA. This mutant displays reduced levels of α-toxin production (85). Highly conserved RNAIII primary and secondary structures can be found in all staphylococci (5). Thus, the structure of RNAIII appears to be functionally conserved. However, the mechanism by which RNAIII regulates transcription is still under investigation. In addition to the AgrA-mediated upregulation of transcription of the Agr system, transcription of agr from the P2 and P3 promoters is also influenced by other factors, such as SarA, environmental glucose, pH, and the alternative sigma factor (SigB) (8). Within the P2 and P3 promoter region, there are several direct and inverted repeat sequences, and these repeat elements have been predicted as putative regulatory protein binding sites. The sequence between –45 and –66 upstream of the P2 promoter element has shown to be required for AgrA activation (91). However, electrophoretic mobility shift assay using this promoter element failed to demonstrate AgrA binding using cell extracts from 8

wild-type and isogenic agr mutant strains (90). The protein that was shown to bind to this region was purified and was identified as SarA by sequence determination (15). Depending the length of the promoter region, several distinct bands were seen in the mobility shift assay, which suggests that multiple binding sites exists for SarA within the agr P2 and P3 promoter region (91). Using a recombinant SarA protein, Chien and Chung have demonstrated there are three SarA binding sites within the agr promoter region (28). Although the agr locus is found in most staphylococci, sequence variation among AgrB, AgrD, and AgrC have been reported (36). Based on agr sequence analysis, four different Agr groups were identified (35). AIP produced by member of the same Agr group can cross-activate the agr signaling pathway. However, the addition of the AIP from a different Agr group was found to inhibit the AIP signal pathway (81). These observations have suggested a therapeutic method using the synthesized AIP as an inhibitor to block the upregulation of virulence genes (73).

b.) YycFG The yycFG locus is a two-component system first identified in Bacillus subtilis but also found in other gram-positive organisms including Streptococcus pneumoniae and S. aureus (80). It was first identified in S. aureus by UVmutagenesis in which a mutation of yycF was found to be unable to survive at 40oC. This mutant strain was shown to be hypersensitive to clindamycin, 9

erythromycin, lincomycin, and unsaturated long chain fatty acids. YycG, the sensor, has 69% amino acid sequence similarity and 46% identity to the B. subtilis YycG. The sensor possesses two hydrophobic domains at the N-terminus and the C-terminus contains the histidine kinase domain. YycF, the response regulator, has 89% amino acid sequence similarity and 74% identity to YycF found in B. subtilis (80). This two-component regulatory system is important for control of growth in these organisms. Studies of YycFG in B. subtilis found it to be responsible for sensing nutrient levels in the environment and for regulation of the cell division operon ftsAZ (40). In S. aureus, DNA footprinting analyses have shown that YycF binds to a 6 bp directly repeated motif, TGTAAT (34). There are 12 genes that contain this repeat sequence in their promoters (35). DNA footprinting analyses have shown that YycF binds to the promoter region of peptidoglycan hydrolase (lytM), staphylokinase (sak), immunodominant surface antigen A (isa), and staphylococcal secretory antigen (ssaA) (35).

c.) SrrAB (SrhSR) The srrAB operon was identified using a reverse genetics approach and was independently identified as srhSR by another group (98, 122). It is a homolog of the resDE operon found in B. subtilis that upregulates genes required under anaerobic growth conditions (98, 122). Two-dimensional protein gel electrophoresis has shown that srhSR is part of a regulon which can repress and 10

induce a number of genes during aerobic growth conditions, and regulates a different set of genes under anaerobic growth conditions (122). A mutation in the srrAB locus results in an increased level of RNAIII and toxic shock syndrome toxin 1 (TSST-1) transcripts, and reduced transcription of protein A in both microaerobic and aerobic environments (98). SrrAB is upregulated under microaerobic conditions during both postexponential and stationary phase of growth when compared to an aerobic growth condition. A mutation in srrAB results in reduced growth in both aerobic and microaerobic conditions (98). SrrS act as the sensor and SrrR is the response regulator.

d.) ArlSR The arlSR locus was identified by transposon mutagenesis using Tn917 as an insertion that resulted in an increased level of autolysis and altered peptidoglycan hydrolase activity. In addition, the mutant strain also exhibited increased biofilm formation and reduced overall protease activity in culture supernatants relative to the parental strain. However, a subsequent study identified that the mutant strain has an increased level of serine protease activity compared to the parental strain. A mutation in either ArlS (sensor), or ArlR (response regulator) was shown to result in increased levels of expression of many exoproteins including the α- hemolysin, lipase, coagulase, protease, and protein A. The arl mutants were found to have increased levels of RNAII and 11

RNAIII, reduced levels of the SarA transcript, but unchanged sarS and spa transcript levels (39). SarA is an upregulator of the Agr system, and a reduction in SarA would result in the downregulation of the Agr system (26). However, in the arl mutants, the SarA was down regulated and the Agr system is upregulated. Thus, this suggests the arl system is likely to regulate genes upstream of the Agr system.

e.) LytSR LytSR was identified though a database search for putative twocomponent regulatory systems in the genomic sequences of S aureus. A lytS mutant has an increased level of autolysis and an increased level of the lrgAB transcript (13). The upregulation of the lrgAB resulted in a reduced level of extracellular murein hydrolase activity, and increases the tolerance to penicillininduced lysis during stationary phase (54). Thus, the lytSR regulates murein hydrolase activity through its regulation of lrgAB transcript.

f.) SaeSR The sae locus was identified by transposon mutagenesis using Tn551 (51, 95). The sae mutant expresses reduced levels of α-and β- hemolysins, DNase, coagulase, and protein A in the culture supernatant (51). On the other hand, the level of lipase, protease, staphylokinase, and enterotoxin A were not changed (47). 12

In studies of virulence factors that are regulated by saeSR, northern blot analysis has also shown that the sae mutant has a reduced level of coagulase and protein A transcripts in post-exponential phase cultures (48). In an agr and sae double mutant strain, the lipase and α- and β- hemolysin levels were found to be similar to the single mutant level, and the protein A level was similar to the agr mutant strain (50). Furthermore, quantitative RT-PCR identified a marked reduction in the level of α-hemolysin transcript in the sae mutant strain (116). Sequence analysis has shown that the sae locus consists of a histidine kinase sensor (SaeS) and a response regulator (SaeR) (46). The RNAIII expression pattern in the sae mutant was similar to that in the parental strain (52). In further analysis of the sae locus, two additional open reading frames directly upstream of the saeSR transcript were identified and designated as saeP and saeQ (90). The transcript levels of the saePQ were found to be induced during the post-exponential phase of growth and the induction required RNAIII. SaeR is also required for the transcription of these open reading frames (90). In addition, the sae transcripts were presented in reduced amount in the SigBpositive strains. RNAIII and SarA appear to be required for the induction of sae transcription. In addition, the sar locus might also encode an autoinduction signal for responses to other environmental signals (48). Due to the requirement of RNAIII and SarA for the transcriptional induction of the sae locus, the effect of SaeSR on α- and β- hemolysins, lipase, and protein A is possibly downstream of the Agr regulation (90). 13

B.) Transcriptional regulators The Sar family of transcriptional regulators are proteins with conserved winged-helix motifs, and include SarA, SarS (SarH1), SarT, SarU, SarR, SarV, Rot, mgrA (NorR, RAT), SarY, and SarX (27). These proteins consist of either one or two winged-helix domains which would bind to DNA. The proteins with one winged-helix domain dimerizes when binding to its target DNA sequences (26, 72, 111). Crystal structures of SarA and SarR have revealed a dimeric structure for these proteins (26). Because of the sequence similarity among the SarA family proteins, it has been hypothesized proteins from the Sar family have a similar DNA-binding motif for gene regulation (27). These proteins have been reported to be a direct regulators of gene transcription by binding to the promoter region of the target genes, and were also reported to have indirect regulatory effects on gene expression through regulation on other regulatory proteins (27). By understanding the regulatory effects among these regulators, it is possible to understand the regulatory network between two-component regulatory system and transcriptional regulators in regulation of the virulence determents and provide a possible target for blocking virulence factors production.

a.) Staphylococcal accessory regulator (SarA) SarA was originally identified through transposon mutagenesis using Tn917LT1 insertion into a clinical isolate. In this clinical strain, the sarA mutant 14

exhibits increased levels of α- toxin, protease, and lipase, and decreased expression of coagulase, fibronectin binding protein, and fibrinogen binding protein, an expression pattern that is opposite to that of an agr mutant (22). However, when this mutation was introduced into a strain 8325 derivative, the commonly used laboratory strain of S. aureus, the sarA mutant displayed a reduced level of RNAIII transcript and α-toxin production, which is similar to the agr mutant phenotype (21, 25, 65). Analysis of the SarA locus has revealed three overlapping transcripts designated as SarA, SarC, and SarB, which originate from three distinct promoter regions P1, P3, and P2 respectively (Figure 2) (4, 102). P1 and P2 are σA-dependent promoters and P3 is a SigB, the alternative sigma factor, dependent promoter (77). Activation of SigB occurs under stress conditions and results in elevated expression of the SarC transcript (8, 16, 33). Each of the transcript encodes the SarA protein (77). SarA binding studies have revealed that SarA binds to an A+T-rich sequence (16, 20, 29, 102, 117). Chien and coworkers have identified the SarA binding site as ATTTgTATtTAATATTTataTAAtTg, using DNase I footprinting (26). However, Rechtin and coworker identified a narrower SarA binding site with sequence consensus, ATTATAAAATWT (101). A recent study using the SELEX (systematic evolution of ligands by exponential enrichment) procedure identified a 7 bp consensus sequence for SarA binding motif, ATTTTAT (117).

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Although there are two smaller putative coding regions located between the P2 and P3 with the sarA locus, SarA has been reported to be the major regulating molecule of the sarA locus. Complementation study using a vector harboring the open reading frame of SarA restored the phenotype of a sarA mutant (23). The functionality of the two putative coding regions has not yet been demonstrated. SarA was shown to transcriptionally regulate at least 120 genes, including those responsible for upregulation of the expression of α- and βhemolysins, fatty acid modifying enzyme, fibrinogen binding protein, and fibronectin binding protein, in a gene array analysis (37). In addition, western blot analysis revealed staphylococcal enterotoxin B (SEB) and lux reporter gene assays revealed that SEB and TSST are upregulated by SarA (17). SarA down regulates the expression of protein A, collagen binding protein, lipase, V8 protease, cysteine protease, aureolysin, and staphopain (37). A sarA and agr double mutant produced a higher level of protease than did the agr mutant (64). Some of the virulence factors and genes required for metabolic pathways that are regulated by SarA are listed in Table 1. The regulatory effect of SarA may be direct by binding to a specific promoter or indirect resulting from its regulatory effect on the Agr system. Electrophoretic mobility shift analysis has shown that SarA can interact directly with the agr promoter region (57). The reduction of α-toxin expression by the sarA mutant can be restored by an increased expression of RNAIII (29). Thus, the regulation of α-toxin by SarA is due to SarA upregulation regulation of the agr 16

system (28). The SarA binding site of the agr promoter was mapped to a 29 bp region between –73 to –110 of the agr P2 promoter (28). Thus, SarA affects other gene transcriptionally by indirectly though its regulation of the Agr system. The mechanism of SarA regulation has been hypothesized to be accomplished by binding of the SarA directly to the target gene promoter regions (15). In promoter analysis using XylE as a reporter gene, the loss of the putative SarA binding site with the spa promoter region resulted in the lost of the SarA regulation (26). A different study using CAT gene as a reporter gene has shown that the promoter activities of spa promoter, with the deletion of the SarA recognition site, the promoter were no longer influenced by inactivation of the sarA determent (41, 65). Thus, it was hypothesized that SarA regulation of the target gene can be accomplished by direct binding of SarA to the SarA binding site. Crystal structures of SarA and SarA-agr promoter DNA complex have revealed that SarA binds to the agr promoter region in a dimeric form. Each monomer has four α-helical structures and two loops. The helices and loops form a β-hairpin and a C-terminal loop. Two α1 helices of one monomer interact with the same region of the other monomer hydrophobically and associate with each other tightly, while the β-hairpin and the C-terminal loop are loosely fastened to the helical core. This helical core has been identified as an inducible regions, when interacting with DNA, the conformational change between the helical structural and helix incorporates the C-terminal loop and the β-hairpin into helix 17

and engulfs DNA. This conformational change enables the SarA interacting with DNA in favor of electrostatic and hydrophobic fashion. In this case, SarA may bind to DNA without a base-specific contact and SarA possibly to be responsible for the architectural structure of the genomic DNA (110).

b.) SarR SarR was identified by passing a cell lysate of S. aureus through an affinity column containing a 49 bp sarA P2 promoter- containing DNA fragment. This P2 promoter region contains a 7 to 8 bp repeat sequence that is identical to the repeat sequence in the sarA P1 promoter. The 13.6 kDa SarR protein is 51% similar and 28% identical to SarA. SarR binds to the SarA P1, P3, and P2 regions as demonstrated by DNase I footprinting. A sarR mutant display a reduced level of transcription from the sarA P3 promoter, and an enhanced level of transcription from the P1 promoter. In addition, the sarR mutant has increased levels of the agr RNAII and the sarA transcripts throughout different growth phases (76). Thus, SarR is an activator of sarA P3 transcription, and a repressor of sarA P1 activity. In this case, the increased agr RNAII levels in SarR mutant might result from an indirect effect of the SarR regulation of SarA. In addition, SarR might also exhibit regulatory effects to other virulence factors though its regulation of SarA and the Agr system. The crystal structural of SarR revealed a dimeric structure consisting of five α helices, three β-strands and several loops. The strong hydrophobic 18

interactions between the two monomers occurs between two α1 helices of each monomer and between the N-terminus of one monomer and the C-terminus of the other. This strong hydrophobic interaction allows the two monomers to selfassociate, and chaotropic agents cannot disrupt this dimeric structure. The α3 and α4 helices form a typical helix-turn-helix motif forming a DNA binding pocket. Using the Escherichia coli CAP protein as a model, SarR has been predicted to bind to at the major groove, while the β-hairpin binds to the minor groove of the double stranded DNA double helices (72).

c.) SarS (SarH1) SarS, also known as SarH1, was identified by its affinity binding to the agr P3, spa, hla, and ssp promoter DNA fragments (120). Cheng and coworkers also identified SarS using a genomics approach (24). The SarS gene is located immediately upstream of spa, and it encodes a protein of 250 amino acids (29.8 kDa) (2, 27). An amino acid sequence alignment indicated this protein has twodomains, each of which shares sequence similarity to SarA, SarR, and SarT (24). The crystal structure of SarS revealed that each domain of SarS (S1 and S2) consists of five α-helices and three β-sheets. Although the SarS structure is similar to the dimeric SarR, the movement between each domain is constrained comparing to the dimeric SarR because the linker region holds S1 and S2 together, and between S1 and S2, α1, α2, α5, and α1’ form a strong hydrophobic interaction. This strong hydrophobic interaction between the winged helix and β19

hairpins forms a compacted core structure and exposes a strong positively charged concave structure for DNA binding. On the opposite side of the protein, an exposed negatively charged surface has been hypothesized to be the regulation domain (70). Northern blot analysis revealed three SarS-specific transcripts. One of them is initiated from a σA -dependent promoter (P1) located 150 bp upstream of the transcription start site. A second transcript is produced from a SigBdependent promoter (P2) which is 650 bp upstream of P1. The origin of the third transcript is unknown (120). Transcription of SarS was found to be repressed by SarA, Agr, and Rot (107, 109, 120). SarS mutants show a significantly lower level of spa transcripts indicating that SarS regulates spa transcriptionally (41, 120). Therefore, the Agr regulation of spa promoter is likely through the Agr regulation of SarS expression, and the SarA regulation of spa could be both direct binding to the spa promoter region and indirect by affecting Agr and Rot expression, results in regulation of the sarS transcription. Evidence for this included the finding that the sarS and the sarA sarS double mutant strains have a similar level of spa transcripts as the wild-type. However, with the agr- sarA - strain, the level of repression of spa transcription is not restored (2). In addition, when the truncated spa promoter fragment lost the SarS mediated regulation, Rot was also found not to affect the truncated promoter activity (40). Although, SarS binds to the RNAIII promoter DNA fragment, SarS does not affect RNAIII promoter activity.

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d.) Repressor of toxin (Rot) Rot was identified using transposon Tn917 mutagenesis of an agr-null strain with screening for a protease-positive phenotype (83). Rot is a 153 amino acid residue (18 kDa) protein with a pI of 5.08 and displays 50% amino acid sequence similarity with SarA (18). An agr and rot double mutant shows an elevated level of protease and α-toxin expression when compared to the agr strain (83). Thus, the Tn917 inactive locus was named as repressor of toxins. However, a gene array analysis has revealed that Rot is a global regulator and is not only a repressor for gene expression, but it also is an activator of transcription. It was shown to upregulate the expression of 86 genes and downregulate 60 genes (106). Northern blot analysis also has shown that Rot positively regulates sarS expression (107). Promoter analyses using a reporter gene have also shown that Rot represses the transcription of sea, seb, sed, and autoregulates its own transcription (125). The transcript level of Rot was not found to vary significantly between the exponential and post-exponential phases of growth. Thus, transcription of rot is not regulated by the Agr system, although, the activity of the Rot protein is under Agr control. The inactivation of Rot has been hypothesized to resulted from an interaction between Rot and RNAIII (83).

e.) MgrA (NorR, RAT) MgrA was first identified by a Tn917 insertion that has an altered level of expression of type 8 capsular polysaccharide (CP8), which was also identified 21

independently by Hooper and coworkers named NorR, and as RAT by Cheung and coworkers (124). In addition to CP8, effects were also observed on the transcription of α- hemolysin, nuclease, protein A, and coagulase (79). NorR was reported to be an upregulator for the multidrug resistant efflux pump NorA. Overexpression of NorR increased sensitivity to quinolones and ethidium bromide, and increased the level of norA transcripts. In addition, in agr or sarA mutant strains, the overexpression of NorR did not affect either quinolone sensitivity or the norA transcript levels. However, mutations in norR resulted in higher quinolone tolerance, but did not alter the level of norA transcript. Although a gel-shift assay indicated that NorR binds to the promoter region of norA, it is possible there are additional factors involved in the regulation of the norA transcription (124). A mutation in rat has been characterized to result in an increased level of autolysis (61). The rat mutant strain has been reported to be more sensitive to penicillin and Triton-X 100 at the post-exponential phase of growth compared to the parental strain (59). Northern blot analyses have also shown that a mutation in rat resulted in reduced transcript levels of lytSR, lrgAB, arlSR, and genes associated with cell wall synthesis (61). Thus, the increased level of autolysis might result from the reduction of overall cell wall synthesis. On the other hand, the rat mutation has no effect on the Agr system (61).

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f.) Other SarA homologues SarT was identified using a genomics approach by searching for SarA homologs in the S. aureus genome database (110). SarT is a protein of 118 amino acids (16 kDa) and has a pI of 9.55 (18). SarA and the Agr system repress the expression of sarT. SarT represses the expression of α-toxin. Because a sarA and sarT double mutant has a higher level of α- toxin expression than does the sarA mutant, it is possible that SarA promotes α-toxin expression by repressing sarT transcription. In addition, an agr and sarT double mutant expresses low levels of α-toxin, comparable to that of the agr mutant (110). Thus, the Agr system regulates α-toxin in a sarT-dependent manner (18). SarU and SarY each consist of 247 amino acid residues with molecular masses of 29.3 and 29.8 kDa, and a pI of 9.8 and 9.3, respectively (18). Both SarU and SarY have domains similar to SarA identified by amino acid sequence alignments (18). The sarU determinant is adjacent to sarT , and the intergenic sequences comprise the promoter regions for sarU and sarT. SarU has been shown to regulate sarT transcription by binding to the sarT-sarU intergenic region. However, the binding of SarU to this promoter region does not affect sarU transcriptionally, as demonstrated by reporter gene analysis (78). SarV is a protein of 116 amino acid residues (13.9 kDa) (17). Recent research has shown that the sarV expression level is not detectable in the wildtype strain under laboratory growth conditions, that the sarV expression level was significantly increased in sarA and mgrA mutant strains (79). Thus, the 23

expression of SarV is negatively regulated by SarA and MgrA. Electrophoretic mobility shift and DNA footprinting assays have shown that SarA and MgrA bind to the promoter region of the sarV (77). Promoter analysis using a reporter gene has also shown that SarV is an upregulator of the Agr system, arlS, and lytR (79). Thus, SarV might also involved in regulation of other genes through the Agr, ArlSR, and LytSR systems. Because SarV affects the transcription of arlS and lytR, the sarV mutant was found to be resistant to Triton X-100 and penicillin induced lysis, and displays a reduced level of murein hydrolase activity (79). The function of SarY, and SarX are not yet known. SarX is a protein of 141 amino acid residues (16.7 kDa), and SarY is a protein of 247 amino acid residues (29.8 kDa). Although the functions of these proteins are still under investigation, it has been predicted that SarY and SarX are DNA binding proteins and they are transcription regulators (18).

C.) Other factors involved in gene regulation in S. aureus a.) TRAP/RAP TRAP was first reported to be the target of a RNAIII expression activation protein, which is inhibited by RNAIII-inhibiting peptide (RIP) and activated by RAP (RNAIII activation protein) (53). TRAP has been reported to be a sensor for the environmental level of RAP (66). When RAP reaches a critical level, it was proposed to cause the phosphorylation of TRAP at a histidine residue in a growth phase-dependent manner (45). Sequence analysis of RAP revealed that RAP is 24

an ortholog of ribosomal protein L2, and the monoclonal antibody against RAP also interact with proteins found in S. epidermidis, S. xylosus and Escherichia coli. However, only RAP from S. aureus was found in culture supernatant (66). A mutation in TRAP has been reported to result in a decreased level of RNAIII and/or a delayed production of RNAIII (3). However, the AIP can still stimulate the RNAIII production of cells harboring the TRAP mutation, which suggested that AIP and RAP has distinct regulation pathways in the induction of the Agr system (66). TRAP/RAP pathway is still not well understood, and its existence is controversial (89).

b.) Staphylococcal virulence regulator (svrA) Staphylococcal virulence regulator was initially identified using signature tagged mutagenesis and was found to be required for the pathogenesis of S. aureus (84). The svrA ORF is 1,335 bp, encoding a protein of 451 amino acids and a molecular mass of 48.8 kDa. Sequence analysis revealed that SvrA has 22% identity and 46% similarity to a group of putative integral membrane proteins, which have been proposed to be virulence factors of Salmonella typhimurium. However, the function of this protein is a possible efflux pump protein with undetermined function. Hydrophobicity analysis predicts the protein of 12 putative membrane spanning regions. Each membrane spanning region is possible to be separated by a hydrophilic loop. The predicted protein structure consists of two groups of six transmembrane domains. A svrA mutant exhibited a 25

reduced expression level of the α-, β-, and δ- hemolysins and an increased level of protein A expression (42). This is a phenotype similar to that of an agr mutant strain. Northern blot analyses have also shown a reduced level of hla, agrA , and RNAIII transcripts in the svrA mutant strain, and a higher amount of the spa transcript relative to that of an Agr-negative strain (42). Thus, svrA regulation of gene expression is most likely to be upstream of Agr regulation.

D.) Global regulators Several loci have been reported to be global regulators of various virulence genes and genes required for metabolic pathways or cell division. These include sigB, sarA, agr, and rot (10, 37, 89, 107). Some of the Sar family proteins have been reported as global regulators, such as SarA and Rot (37, 107). These global regulators not only affect transcription directly by binding to the target gene promoters, but also influence transcription indirectly by affecting the expression of other transcriptional regulators. In this section, the alternative sigma factor (SigB) and superantigens (SEB and TSST) will be discussed.

a.) Alternative sigma factor (SigB) in S. aureus The alternative sigma factor is a global regulator that coordinates the gene transcription for the stress response. It has also been reported to be a global regulator for virulence factors through its regulatory effect on the Agr system and sarA (10). The activation of SigB is through environmental signals, such as 26

chemical stimuli, stress conditions, and/or energy depletion (8). The production of SigB is regulated by the rsb operon that encodes rsbUVW. The schematic of the rsb operon is shown in Figure 3. Under stimulation conditions, the phosphorylated RsbV is dephosphorylated by RsbU. The dephosphorylated RsbV would than bind to the anti-sigma factor RsbW resulting in the activation of SigB (44). SigB would then recognize and bind to promoter sequences with the consensus sequence GTTT(N 14-17)GGGTAT, and regulate the target genes transcriptionally (43). In S. aureus, SigB acts as a global regulator by regulating other global regulators and transcriptional regulators, resulting in indirect down-regulation of virulence genes (68). Therefore, inactivation of SigB results in an increased expression of virulence factors being produced, such as proteases and staphylococcal enterotoxin B (SEB) (108). Therefore, it is possible that inactivation of SigB would result in increased level of pathogenicity. However, reports have indicated that SigB mutants of clinical isolates do not have an altered level of virulence (87). This may be because SigB plays an important role in upregulation of adhesins to the host cells. As consequences of the decreased adhesion to host cells, SigB mutants is less efficient to establish infection (112). Many laboratory strains of S. aureus are 8325 derivatives, which harbor an 11 bp deletion at the 3’ end of rsbU. The mutant RsbU cannot release SigB from the anti-sigma factor, resulting in a SigB negative phenotype (96).

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b.) Superantigens as global regulators Two of the staphylococcal superantigens, TSST-1 and SEB, have been reported to act as suppressors of exoprotein production (126). Production of these proteins results in a reduced level of other exoproteins in the culture supernatant. In addition, TSST-1 and SEB have been reported to regulate their own transcription, targeting the tsst and seb promoter regions. The C-terminus of each of these superantigens is required for the inhibitory effect on its transcription. The repressive effect of the C-terminus of these superantigens might be an indirect effect, by which these superantigens may affect expression of other transcriptional factors resulting in a reduced level of exoprotein production (127).

2. Relationships between transcriptional regulators and the Agr system SigB operon is a global regulator, which affects virulence gene expression directly through activation of transcription, or indirectly through its effect on the SarA and the Agr systems. SigB positive strains display a decreased level of RNAIII expression and an upregulation of the SarA P3 promoter (77, 108). Because SarA is an upregulator of the agr P3 promoter, SigB also affects the agr transcription through its regulation of SarA. However, SigB positive strains have been shown to display a reduced level of the agr P3 transcript in both promoter analysis using a reporter gene and northern blot analysis (2, 58). This suggests that additional factors are involved in the repression of the Agr system in the 28

SigB positive strains. However, another group of researchers using a microarray approach has reported that the RNAIII expression in S. aureus strain Newman is similar to its isogenic sigB mutant (9). Thus, the influence of SigB in the Agr system is still controversial. On the other hand, SigB has found to be an upregulator of sarS and arlSR transcription in S. aureus COL and Newman (9). Northern blot analysis of the agr RNAIII transcript from the strains with a combination of Agr-, SarA-, and SarS- negative backgrounds has shown that the loss of SarS resulted in no significant change in RNAIII expression level; whereas the loss of SarA resulted in a moderate reduction of RNAIII. In addition, no dramatic change in RNAIII levels was found in the sarA and sarS double mutant (2). Similar results were obtained in reporter gene studies of the agr P3 promoter activity utilizing a reporter gene (19, 28). SigB has been shown to be a down regulator of Rot promoter activity in post-exponential phase of growth (125). In addition, Rot upregulates sarS transcriptionally, resulting in affecting genes transcription through SarS (107). In this case, a down regulation of the rot expression would result in a reduced level of sarS expression. However, SigB has also been shown to upregulate sarS transcription in a microarray approach (9). Therefore, other regulators would also involved in the sarS expression. Other regulators involve in the transcription regulation of sarS are the Agr and SarA (24). In terms of SarA down-regulation of sarS transcription, it results from SarA down-regulation of sarT expression, which SarT is an upregulator of sarS 29

expression. The down-regulation of sarT expression leads to the down-regulation of SarU (78). SarU would up-regulate Agr, which forming a feedback loop between Agr, SarT, and SarU (78). On the other hand, there has little change in Agr RNAIII transcript levels in SarS mutants (2). A model has been proposed for the regulation of between the SigB, Agr, SarA, SarS, SarR, SarT, and SarU in relationship to the spa and hla (Figure 3) (27). However, the exact mechanism and the hierarchy of gene regulation among the Agr, SarA, SarS, and Rot are not well understood.

3. The enterotoxins produced by S. aureus The staphylococcal enterotoxins (SEs) are important virulence factors for staphylococcal food poisoning. A number of staphylococcal enterotoxins have been identified that are distinguished serologically, or by amino acid sequence differences. They are designated as staphylococcal enterotoxin A (SEA) through R (SER), excluding SEF which was previously the designation of toxic shock syndrome toxin (6, 69, 94). These enterotoxins are superantigens that bind to the T cell receptor Vβ region. Subsequently, the activation of the T cells results in the release of large quantities of inflammatory cytokines, thus damaging the host immune system. The structure of the enterotoxins revealed two conserved domains, a N-terminal oligosaccharide/oligonucleotide binding domain and a Cterminal α-helix with a four stranded β-sheet (119). The N-terminal domain

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contains hydrophobic residues that interact with the MHC-II molecule and the αhelix interacts with the T cell receptors (1, 56, 115). Different culture techniques and media compositions have been reported to affect enterotoxin gene expression (14, 34, 106). Generally speaking, brain heart infusion (BHI) broth is a good culture medium for the enterotoxin production. Other media, such as protein hydrolysate powder (PHP), defined amino acids medium, N-Z amine, and tryptic soy broth (TSB) have supported a good yield of enterotoxins (86, 106). In addition, the conditions of culturing were found to have impact on enterotoxin production, including the pH of the medium, oxygen availability, time, temperature of incubation, and carbohydrate composition of the medium, such as the presence of glucose, sucrose, or glycerol (99, 105). The ideal pH for enterotoxin production is from 5.5 to 7. Low pH (