Thesis
Reference
Molecular regulation of tst superantigen expression by global regulatory systems in Staphylococcus aureus
ANDREY, Diego Olivier
Abstract
Staphylocoque doré est un dangereux pathogène, capable de diverses formes d'infections, dont le Toxic Shock Syndrome. Ce travail étudie la régulation génique de tst, codant TSST-1, la toxine du Toxic Shock, au niveau moléculaire. Bien que situé sur un élément génétique mobile, les résultats montrent que tst est finement régulée et de manière complexe par les voies de régulation endogènes suivantes : SarA (S. aureus accessory regulator), le facteur de stress sigmaB, SarS, le senseur du quorum agr/Rot et le système deux composant SrrAB. A l'exception du senseur du quorum agr les autres voies semblent être répressives, soit directement comme le facteur de transcription SarA, qui se lie au promoteur de tst, soit indirectement comme sigmaB. La voie SrrAB (Staphylococcal respiratory response) active l'expression de TSST-1 en aérobiose et la réprime en anaérobiose, cela grâce à la phosphorylation de l'aspartate D53 de SrrA par l'histidine H369 de la kinase SrrB.
ANDREY, Diego Olivier. Molecular regulation of tst superantigen expression by global regulatory systems in Staphylococcus aureus. Thèse de doctorat : Univ. Genève, 2011, no. Sc. Méd. 2
URN : urn:nbn:ch:unige-181753
DOI : 10.13097/archive-ouverte/unige:18175
Available at:
http://archive-ouverte.unige.ch/unige:18175
Disclaimer: layout of this document may differ from the published version.
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Section de médecine clinique Département de médecine interne Service des maladies infectieuses
Thèse préparée sous la direction du Docteur William L. Kelley et du Professeur Daniel Lew
Molecular regulation of tst superantigen expression by global regulatory systems in Staphylococcus aureus
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le
Doctorat en Sciences médicales « MD-PhD » par
Diego Olivier ANDREY de
Genève (GE)
Thèse n° 2 Genève 2011
SUMMARY
Staphylocoque doré est un dangereux pathogène, capable de diverses formes d’infections, dont le Toxic Shock Syndrome. Ce travail étudie la régulation génique de tst, codant TSST-1, la toxine du Toxic Shock, au niveau moléculaire.
Bien que situé sur un élément génétique mobile, les résultats montrent que tst
est finement régulée et de manière complexe par les voies de régulation
endogènes suivantes : SarA (S. aureus accessory regulator), le facteur de stress
sigmaB, SarS, le senseur du quorum agr/Rot et le système deux composant
SrrAB. A l’exception du senseur du quorum agr les autres voies semblent être
répressives, soit directement comme le facteur de transcription SarA, qui se lie
au promoteur de tst, soit indirectement comme sigmaB. La voie SrrAB
(Staphylococcal respiratory response) active l’expression de TSST-1 en
aérobiose et la réprime en anaérobiose, cela grâce à la phosphorylation de
l’aspartate D53 de SrrA par l’histidine H369 de la kinase SrrB.
à Aline, mon épouse,
obrigado por ser tao perfecta e mais que tudo por seu amor
AKNOWLEDGMENTS
I want to give my great thanks to:
Bill Kelley, my thesis director, for these many hours of passionate discussion about science, travel and wine
Daniel Lew, my clinical mentor, for your guidance and trust
Adriana Renzoni, for your help in research world, your kindness and your always- present smile
Ambrose Cheung, for sharing your knowledge, expertise in S. aureus field and for these good moments spent with you in Geneva
Ambre Jousselin, for these laughs in the lab and beers after the lab
Régis Villet, for the hours spent after midnight purifying recombinant proteins, for your friendship and for letting me know “un vrai Parisien”
Antoinette Monod, for transmitting me your secrets of challenging cloning Christine Barras, for your help in the RNA world
Emmanuelle Lelong, for your good advices regarding how to finish a thesis David, my brother, for your help, as always and especially in digital graphics
The Andrey-Gomez family, Pilar, Christophe, David, Anabel as well as our beloved elderly Lelo and Yvonne, and my wife Aline for your presence and love
1
UNIVERSITE DE GENEVE FACULTE DE MEDECINE
Docteur William L. Kelley Professeur Daniel P. Lew
Molecular regulation of tst superantigen expression by global regulatory systems in Staphylococcus aureus
THESE
présentée à la Faculté de Médecine de l’Université de Genève pour obtenir le Doctorat en Sciences Médicales MD-PhD
par
Diego Olivier ANDREY de
Genève (Suisse)
Jury members:
Prof Dominique Belin - University of Geneva (President) Dr William L. Kelley - University of Geneva (Thesis Director)
Prof Daniel P. Lew - Geneva University Hospitals (Thesis Co-Director) Prof Ambrose L. Cheung - Dartmouth Medical School, USA (External Expert)
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TABLE OF CONTENTS
INTRODUCTION ... 4
Overview of Staphylococcus aureus clinical importance ... 4
Virulence of Staphylococcus aureus ... 6
Virulence factors ... 6
Regulation of virulence ... 10
Integration of virulence regulation ... 29
Staphylococcus aureus toxinoses ... 32
Toxic Shock Syndrome (TSS) ... 33
Physiopathology of TSS ... 35
Tst gene ... 37
Molecular epidemiology ... 37
Regulation of tst ... 38
ARTICLE 1 ... 41
ARTICLE 2 (in preparation) ... 42
ABSTRACT ... 43
MATERIALS AND METHODS ... 44
RESULTS ... 46
FIGURES AND TABLES ... 50
ARTICLE 3 (in preparation) ... 56
ABSTRACT ... 57
MATERIALS AND METHODS ... 58
RESULTS ... 63
FIGURES AND TABLES ... 68
DISCUSSION ... 78
Various models for tst study ... 78
sarA ... 81
sarS ... 82
sigB ... 83
rot/agr ... 84
mgrA ... 85
Circuitry and hierarchy over tst control ... 85
Role of the srrAB TCS ... 88
Architecture of the tst promoter... 91
REFERENCES ... 96
3
APPENDICES ... 111
I ... 111
II ... 112
III ... 113
IV ... 114
V ... 116
VI ... 117
VII ... 118
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INTRODUCTION
Overview of Staphylococcus aureus clinical importance
Staphylococcus aureus is a gram positive bacteria, belongs to the family of Micrococcacea, and is considered as a human commensal organism, that, in certain conditions can become a dangerous pathogen. Approximately 30% of healthy people are transiently colonized, asymptomatically, by S. aureus particularly in nostrils but also in axillae, pharynx, vagina, perianal region or damaged skin surfaces. The majority of these S. aureus strains are MSSA (Methicillin-Sensitive S. aureus) and only 1% approximately are MRSA (Methicillin-Resistant S. aureus), as shown by Tenover and colleagues in a study performed between 2001 and 2004 in the USA (Gorwitz, et al., 2008, Tenover, et al., 2008). In addition approximately 10- 20% of these people are persistent carriers (Casewell & Hill, 1986, Ruimy, et al., 2010).
In some cases, S. aureus turns into a virulent opportunistic pathogen that can cause a wide range of infections involving different organs. It can infect skin and soft tissues (folliculitis, impetigo, furuncles, abscesses, and fasciitis), the bloodstream (bacteriemia), heart valves (endocarditis), bones (osteomyelitis), and lower respiratory tract (abscessing pneumonia) among others (Que & Moreillon, 2010). An important determinant allowing this commensal organism to become a pathogen is the presence of a breach in skin or mucosal barriers. This allows spreading of the bacteria into surrounding tissues, or access to the bloodstream. A possible further step is blood-mediated metastatic dissemination followed by adherence to certain tissues – the prototypical situation being adherence to heart valves, or embolic abscess into lungs. Whether a simple breach in skin will allow such dramatic situations depends on a complex balance between virulence properties of the bacteria and efficiency of the immune response (Lowy, 1998).
This ability of S. aureus to efficiently infect organs where it encounters very different environmental conditions regarding oxygen supply, blood irrigation, pH, immune defenses mechanisms among others, is one of the most striking features of this organism, and
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explains the difficulty of the research approaches in order to reproduce faithfully the physiology of the bacteria in controlled experimental models.
An additional feature of the spectrum of diseases caused by S. aureus is its ability to produce a wide array of toxins. Sometimes toxins are involved in the infection process itself, acting in cooperation with many other virulence factors, and in other situations toxins can trigger a disease by themselves. (Lowy, 1998, Cosgrove, 2006, Que & Moreillon, 2010). This latter clinical condition, described as S. aureus toxinose, will be discussed in more detail below.
Another characteristic feature of this versatile pathogen is its ability to adhere and colonize synthetic materials forming so-called biofilms. The structured lifestyle of bacterial biofilm communities involves a coordinated sequence of events including primary surface attachment, microcolony formation, expansion, and dissemination (Hall-Stoodley, et al., 2004). S. aureus colonization of catheters, implanted pacemakers and joint prosthesis can lead to changes in drug sensitivities, thus complicating efforts of therapeutic eradication (Jefferson, et al., 2005). Generally, biofilm formation leads to surgical removal of the infected device, which, in the case of joint prosthesis infections as an example, is a very challenging surgical operation with high morbidity and mortality rates.
Efficiently treating S. aureus infections has become a challenge for clinicians due to the emergence of antibiotic resistant strains, and more particularly dissemination of MRSA strains, resistant to anti-staphylococci penicillins such as flucloxacillin (Chambers & Deleo, 2009, DeLeo & Chambers, 2009). MRSA strains have spread worldwide and are endemic in most hospitals, where they cause infection in patients with predisposing risk factors or illness such as diabetes, drug usage or surgery (Tuazon & Sheagren, 1974, Tuazon, et al., 1975). Whether HA-MRSA (Hospital-Acquired MRSA) strains are more virulent than MSSA is still debated. Nevertheless, HA-MRSA bacteremias have a high mortality rate (20%). Lately some MRSA strains, especially in the USA and some southern countries, have been shown to cause skin and soft tissue infections in healthy people with no traditional risk factor or illness, outside of hospital settings (in the community). In the USA one of these CA-MRSA (Community-acquired MRSA) strain, called USA300, has rapidly spread and is found in most of the cases of MRSA infection outside of hospitals, even generating epidemics typically in close contact situations such as schools, military barracks, sport teams or prison (Deleo, et
6
al., 2010). These infections cannot be treated with traditional anti-staphylococci penicillins and require the use of alternative anti-microbial combinations. In addition, CA-MRSA (and especially USA300) strains generally carry the lukS/lukF-PV genes, encoding the Panton- Valontine leucocidin (PVL), whose presence is highly correlated with serious skin infection and severe necrotizing pneumonias. However the precise role of this toxin in CA-MRSA pathogenicity remains to be defined and is the subject of intense debate (Deleo, et al., 2010).
Taken together, these characteristics of Staphylococcus aureus reveal a dangerous and versatile pathogenic organism; Staphylococcus aureus remains a great challenge for both the clinician and the researcher.
Virulence of Staphylococcus aureus
This ability to cause such a wide array of infections is due to S. aureus’ fantastic arsenal of virulence factors and virulence strategies. Actually S. aureus virulence (or any bacteria’s virulence) could be defined as its capacity to provoke damage to the host once it has been colonized. Its virulence level will determine its pathogenicity. To provoke this damage (to be virulent), S. aureus needs virulence factors; these are generally either membrane-anchored or secreted proteins that allow the bacteria to cause damage to the human cells and tissues.
In a even more general definition of virulence factors, all bacterial constituents that participate to the pathogenic process can be included, such as the gram positive cell-wall inserted teichoic acids (involved in adhesion) or polysaccharides (involved in biofilm formation). An overview of the major Staphylococcus aureus virulence factors follows.
Virulence factors
Except in the cases of the previously cited toxinoses, where a single toxin can provoke a disease upon simple host colonization, multiple virulence factors are generally necessary for an infection to occur. The following steps are generally described for the infection process by S. aureus: adherence, immune system evasion, and tissue invasion (Lowy, 1998, Fournier,
7
2008). Spreading to adjacent tissue or metastatic dissemination through the blood stream to other organs can happen with further repetition of the same cycle. Each of these steps has several virulence factors involved, typically cell wall bound virulence factors are involved in adherence and secreted factors in spreading and dissemination. Then, both membrane anchored factors (eg. Protein A) and secreted factors (eg. Superantigens, PVL) can contribute to evading the immune system. These virulence factors are also expressed in a defined temporal sequence with typically adherence factors highly present in the early infection stages (in vitro early exponential phase) and secreted factors produced late in infection (in vitro post-exponential phase)(Arvidson & Tegmark, 2001, Cheung, et al., 2004, Cheung, et al., 2008); this temporal organization is notably under control of the main regulator of virulence genes, the quorum sensing system agr, that will be discussed in more detail below (Novick, 2003).
The peptidoglycan cell wall as well as lipoteichoic acids are important for virulence determination. Peptidoglycan has a possible endotoxin-like effect by stimulating cytokine release by immune cells, probably via Toll receptors, and activation of complement, (Lowy, 1998), and teichoic acids are implicated in adherence to host tissues and escape from host defense mechanisms such as cationic antimicrobial peptides, especially when their surface charge is altered by D-alanylation (mediated by the dlt operon) (Peschel, et al., 1999, Neuhaus & Baddiley, 2003). In addition, S. aureus has a microcapsule; the microcapsular polysaccharides, of serotype 5 & 8 in most cases of infection, are important for evading the immune response, especially avoiding activation of the complement pathways (Nilsson, et al., 1997, Thakker, et al., 1998, Watts, et al., 2005). The polysaccharide intercellular adhesins (PIA), an extracellular polysaccharide encoded by the ica operon, are highly important in the process of biofilm formation since they form an intercellular network stabilizing cells after adhesion to the synthetic material (Cramton, et al., 1999).
Anchored to the peptidoglycan are so-called MSCRAMMS (Microbial Surface Components Recognizing Adhesive Matrix Molecules) proteins, produced in exponential growth phase in vitro, which help the bacteria to bind to extracellular matrices such as collagen or fibrinogen.
FnbP’s (fibronectin binding proteins) encoded by fnbA and fnbB, Clf’s (clumping factors) and Cna (collagen adhesin) are examples of such virulence factors (Fournier, 2008). In addition to
8
their adherence properties, rendering these factors essential for both colonization and infection, they also play a role in protection against phagocytosis (Palmqvist, et al., 2004) and mediate uptake into the cell (Sinha, et al., 1999, Massey, et al., 2001, Sinha & Herrmann, 2005). Strategies for development of vaccines against these MSCRAMMS are being pursued (Rivas, et al., 2004).
The cell wall anchored Protein A, encoded by spa, is a major player of virulence. It binds to the Fc part of IgG, produced in the context of the humoral immune response, thus positioning them in an incorrect manner for proper opsonization by neutrophils; probably no interaction between Fc domain of IgG and neutrophils Fc receptor can take place in these conditions (Foster & McDevitt, 1994).
In addition to the capsule, the MSCRAMMS and Protein A, there are also secreted virulence factors important for impeding proper complement activation and opsonization of S. aureus such as Staphylococcal complement inhibitor (SCIN), Staphylokinase (Sak), and Extracellular fibrinogen-binding protein (Efb). SCIN is a very efficient complement inhibitor that blocks action of C3 convertases (C4b2A and C3bBb), necessary for cleavage of C3 into C3a and C3b.
In this condition, the C3b opsonin, important for neutrophil phagocytosis when bound to S.
aureus surface, is no longer available (Rooijakkers, et al., 2005). Sak promotes activation of plasmin, which will subsequently cleave the following molecules: the surface-bound opsonin C3b and IgG at Lys222 removing the Fc fragment (necessary for C1q mediated action of complement classical pathway). Efb binds to C3 blocking further cleavage by convertases;
thus Efb also targets the central player downstream of all three complement activation pathways (Rooijakkers, et al., 2005, Foster, 2009). Other secreted proteins such as i) CHIPS (chemotaxis inhibitory protein of S. aureus), that block C5a action and thus subsequent neutrophil recruitment, and ii) staphylococcal superantigen-like protein 7 (SSL7) that bind C5, IgA 1 and 2, are also involved in controlling the immune response (Rooijakkers, et al., 2005, Fournier, 2008, Foster, 2009).
Exotoxins secreted by S. aureus, such as the hemolysins, leucocidins, proteases, lipases and coagulase, are involved in tissue lysis allowing dissemination of the bacteria in surrounding tissues. The -hemolysin ( -toxin), encoded on hla, can lyse erythrocytes and leucocytes,
9
meanwhile -hemolysin lyses mainly erythrocytes (Verdon, et al., 2009). Leucocidin acts as heterodimer to form pores in leucocytes. The Panton-Valontine leucocidin (PVL), whose two components are encoded on lukS-PV and lukF-PV genes on a mobile genetic element, lyses neutrophils and monocytes. PVL is generally associated with SCCmec cassette type IV of CA- MRSA strains. It has been involved in severe necrotizing pneumonias, primary skin abscesses and other dermatological diseases. However, although the presence of these genes in S.
aureus isolates is highly correlated with these severe infections, especially in southern countries, the exact physiopathologic role of this leucocidin is still debated (Deleo, et al., 2010). Several studies done on various animal models have shown different results regarding the role of the PVL itself. Some authors show that the presence of PVL does not contribute to the pathogenesis, in a murine skin abscess infection model (Voyich, et al., 2006, Bubeck Wardenburg, et al., 2007, Bubeck Wardenburg, et al., 2008). In contrast, other results suggest that PVL is involved in the pathogenicity of theses strains (in a murine acute pneumonia model or rabbit bacteremia) (Labandeira-Rey, et al., 2007, Diep, et al., 2008, Cremieux, et al., 2009, Diep, et al., 2010).
The S. aureus superantigens, such as TSST-1 (toxic shock syndrome toxin 1), exfoliatin toxins A & B and the numerous enterotoxins (that also have superantigenic properties) have precise molecular targets for exerting their virulence (see section below). Besides their direct pathogenic properties, they play an important role in preventing a normal immune response by triggering the unspecific activation of a large fraction of T-cells (as much as 30% of lymphocytes can be stimulated). Normal antigen-specific response cannot take place in this condition; thus immunosuppression and lack of immunological memory are the ensuing consequences (Schlievert, 1993).
Although S. aureus impressive arsenal of virulence factors has not been fully described here, we have presented some major virulence associated genes.
10 Regulation of virulence
It is clear that the range of diseases caused by Staphylococcus aureus is reflected in the expression of virulence factors. How S .aureus accomplishes this is only partially understood.
Research over the last decades has uncovered several global regulators that control virulence. Other studies have revealed the existence of regulatory proteins that control specific responses to certain nutrients or environmental conditions. In the following chapter will be reviewed some of the major regulatory pathways.
Quorum sensing system agrBDCA
The importance of the growth phase, in vitro, on the production of virulence factors has been discovered long ago. It has now been shown to be regulated by the quorum sensing (QS) system of S. aureus, which is mediated by the agr (accessory gene regulator). The QS allows the bacteria to assess the size of their own population, since they can sense the concentration of an AIP (auto-inducing peptide), a small thiolactone ring containing peptide, produced by the population and therefore proportionally concentrated in the medium. The agr system allows production of this AIP, its release into the environment, sensing of its concentration and finally regulation of downstream target genes in accordance (Novick &
Geisinger, 2008). The specificity of S. aureus QS system is that the final effector is not a transcription factor but a complex regulatory RNA, RNAIII (Novick, 2003). This agr/RNAIII system regulates a large number of genes; particularly it upregulates exotoxins and secreted virulence factors and down-regulates cell-wall associated virulence factors (Recsei, et al., 1986, Janzon & Arvidson, 1990, Dunman, et al., 2001). At the genetic level, the agr system is encoded by the agrBDCA operon, which is transcribed through a single transcript RNAII, under control of agrP2 promoter, and the effector RNAIII is divergently encoded, under control of the agrP3 promoter.
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Figure 1. The quorum sensing agr system (adapted from Novick and Geisinger, 2008)
The quorum sensing autoregulatory loop of the agr system functions in the following manner (see figure 1):
AgrD codes for a propeptide that will subsequently be processed by AgrB in order to produce the AIP. The propeptide is N-terminally membrane anchored and C-terminally interacting with AgrB, which is a transmembrane endopeptidase. The propeptide will finally be processed in both N-terminal and C-terminal extremities, probably by SpsB (Kavanaugh, et al., 2007) and by AgrB respectively, in order to generate AIP thiolactone ring; the AIP is finally secreted, even though the exact secretion mechanism remains unknown. After secretion in the environment, the AIP is detected by AgrC, the histidine kinase sensor of the agrCA two-component system. Once activated, AgrC promotes phosphorylation of AgrA, that will bind to the agr P2-P3 intergenic region and stimulate transcription of both RNAII and RNAIII. This autoinducing loop will amplify the production of the AIP on one hand and activate downstream gene regulation on another hand (Novick, 2003, Novick & Geisinger, 2008).
AgrA is known to bind in vitro to the agrP2-P3 intergenic region, and its phosphorylation (using acetylphosphate as a phosphate donor group) enhances its binding affinity (in an in
12
vitro binding assay). An AgrA recognition sequence ACAGTTAAG, present twice, as a tandem repeat with a 12bp interval, on both P2 and P3 promoters has been identified (Koenig, et al., 2004). AgrA only binds to these promoters; no other direct target of AgrA has been identified (with the possible exception of the phenol soluble PSM cytolysin genes (Queck, et al., 2008)). Thus apparently, only RNAIII is responsible for further downstream gene regulation mediated by the QS agr system.
In addition, sequence variations in agrBDC region (see figure 1) have allowed classification of S. aureus strains in so-called agr groups (I to IV); this typing system appears to be a useful marker for toxin secreting strains and correlates with certain diseases (Novick & Geisinger, 2008).
RNAIII
This 514 nucleotide regulatory RNAIII is extremely important in gene regulation, therefore extended efforts have been devoted to better characterize its structure and function (see figure 2). Although it has been known for a long time that RNAIII upregulates exotoxin production in post-exponential growth phase, the exact mechanism has only been described recently. Benito and co-workers first described RNAIII structure and proposed 14 hairpins (Benito, et al., 2000). RNAIII was shown to bind in an antisense manner to the rot mRNA, to abolish its translation, and to trigger endoribonuclase III-dependant degradation of the mRNA. Hairpins 7, 13 and 14 (see figure 2) have been involved in this interaction with rot mRNA (Boisset, et al., 2007). Rot (Repressor of toxins) is a transcriptional factor involved in repressing transcription of various exotoxins. RNAIII, by downregulating rot expression, indirectly promotes synthesis of these exotoxins. In addition RNAIII, through direct binding, diminishes translation of certain virulence factors transcripts such as as spa mRNA (Protein A) (Huntzinger, et al., 2005). The 3’end of RNAIII has partial complementary sequence with 5’end of spa mRNA, more precisely with its ribosome binding site (RBS) and adjacent nucleotides. Other mRNA targets, such as SA1000 (a fibrinogen-binding protein) mRNA, have been identified due to sequence complementarity with RNAIII, and subsequently proven to be under its control. Finally direct control of RNAIII over hla mRNA has been proposed in 1995 by Morfeldt and co-workers. In vitro a complex can be observed between RNAIII and
13
5’UTR (untranslated region) of hla transcript. The proposed mechanism for RNAIII-mediated activation of translation by an anti-sens mechanism is that RNAIII disrupts an intramolecular pairing of hla transcript at 5’ end, thus allowing access of ribosome to its RBS (Morfeldt, et al., 1995).
Figure 2. RNAIII structure (adapted from Novick and Geisinger 2008)
SarA family of transcriptional regulators
This large family of global regulators share similarities in their overall structure and DNA- binding properties. Here we will describe the main and first member of the family SarA.
Other members of the family will only be briefly presented. A detailed analysis of the whole family is beyond the scope of this work. For extended information refer to the following review (Cheung, et al., 2008).
14 SarA
SarA is a 14.5 kDa protein, has 124 residues, and binds DNA via its winged-helix-turn-helix domain. SarA is encoded by the sar locus which is transcribed by three transcripts of different sizes, under control of P1, P2, and P3 promoters. In vitro sarA transcript expression is maximal in post-exponential growth phase (Manna & Cheung, 2001), and their transcription is repressed by SarR (mostly P1 transcript), by SarA itself (P1 and P3) (Cheung, et al., 2008), and stimulated by sigmaB (P3) (Manna, et al., 1998)(these regulators are described below). However the total amounts of SarA remain elevated during growth cycle and the importance of SarA quantities on physiology is controversial (Manna & Cheung, 2001, Fujimoto, et al., 2009). There are hints that post-translational modifications such as phosphorylation can modulate SarA DNA binding (Didier, et al., 2010).
SarA has been shown to be an important transcription factor involved in virulence regulation. It was first identified in a study of a Tn917 transposon mutant that affected exoprotein synthesis (Cheung, et al., 1992). Since then, sarA has been extensively studied and shown to regulate a high number of virulence related genes, such as hla ( -hemolysin), spa (Protein A), tst (TSST-1), sec (S. aureus enterotoxin C), fnbA (fibronectin binding protein A), cna (collagen adhesin) among others (see Table 1) (Novick, 2003, Cheung, et al., 2004). It also modulates agrP2-P3 expression in conjunction with SarR (SarA Regulator) (see also appendix VI). SarA thus is a major regulator of virulence in S. aureus and can control expression of virulence factors both directly and indirectly by its modulating effect on agrP3- RNAIII expression. SarA has both stimulatory and repressing regulatory effects depending on its targets (See Table 1). In addition, inter-strain differences have been identified regarding the role of sarA on some virulence factors. For example exotoxins, and more specifically hla, is positively regulated by SarA in the widely used laboratory strain 8325-4 and derivatives, but repressed by SarA in several other strains such as UAMS-1 and Newman (Heinrichs, et al., 1996, Blevins, et al., 2002). These differences have been addressed in some studies and it has been proposed that this effect is due to the very strong expression of another regulator, a repressor of hla called SarS, in the sarA- mutant of 8325-4 strain compared to other strains
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where sarS is only lightly expressed. High levels of this repressor would be responsible for the low hemolytic pattern of 8325-4 sarA- mutant (Tegmark, et al., 2000, Oscarsson, et al., 2005, Oscarsson, et al., 2006). The respective role of the main global virulence regulators agr/RNAIII and sarA, their interactions with the other regulators, and their relevance in coordinating S. aureus virulence in vivo is still widely investigated.
A transcriptomic study done with a sarA defective strain showed approximately 120 genes affected by SarA absence, where 76 genes were upregulated and 44 downregulated (Dunman, et al., 2001). Interestingly, not only virulence genes appeared to be SarA-regulated in this study; actually, the majority of genes regulated by SarA were not related to virulence but rather linked to various cell metabolism pathways.
SarA is thus an important global regulator, involved in various physiologic processes. It has been proposed to be involved in oxidative stress resistance (Ballal & Manna, 2010), and to be a sensor of the redox state of the bacteria, with modification of its DNA binding properties as a function of the redox state and pH mediated by a highly reactive cysteine residue C9 present in the DNA-binding channel (Fujimoto, et al., 2009). On another hand, SarA is also important for biofilm formation, since it is known to regulate activity of the ica operon, in conjunction with IcaR and sigmaB (Beenken, et al., 2003, Jefferson, et al., 2003), and for resistance mechanisms against cell-wall acting antibiotics (Trotonda, et al., 2009).
Finally in a study by Roberts and colleagues, SarA was shown to be involved in post- transcriptional turnover of cna and spa mRNA (Roberts, et al., 2006).
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Table 1. Virulence factors under control of sarA and agr (adapted from Cheung et al. 2004)
The molecular structure of SarA has been extensively studied; it was crystalized by two different groups that obtained divergent results (Schumacher, et al., 2001, Liu, et al., 2006).
However it is clear that SarA binds DNA as a dimer (see figure 3). Arginine residues R84 and R90, situated on the winged region have been demonstrated to be critical for DNA-binding, since electrophoretic mobility experiments done with these mutant recombinant proteins
17
showed abolition of binding to the spa promoter (Liu, et al., 2006). A 26 bp DNA recognition sequence for SarA was obtained by footprint experiments and alignment analysis of the agr, hla, spa, fnbA, fnbB, sec and sarA promoters. The derived consensus is ATTTgTATtTAATATTTataTAATTg (Chien & Cheung, 1998, Chien, et al., 1999, Cheung, et al., 2008). Another approach, using SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure (a technique relying on iterative selection by SarA of specific DNA sequences from an initial random population) and alignment of almost the same set of promoters (agr, hla, spa, fnbA, sspA, cna), revealed a 7-bp motif ATTTTAT, generally present more than once on each promoter and included in the 26 bp binding site consensus (Sterba, et al., 2003). All authors agree on the affinity of SarA to AT-rich sequence, although the exact sequence might diverge substantially between promoters.
Since S. aureus genome is >60% AT-rich overall, defining SarA binding site by in silico analysis is problematic. A systematic study of high resolution contact site analysis has not been performed for SarA and its known sites. Further studies, including the one presented in this thesis, will contribute to refinement of the SarA binding site consensus.
Figure 3. Dimeric struture of SarA (adapted from Liu, et al. 2006)
SarR
SarR (SarA Regulator), a member of the Sar family, was first identified as a regulator of sarA transcription (Manna & Cheung, 2001). It possesses a helix-turn-helix domain and was shown to bind in vitro to an AT-rich sequence upstream all sarP1-P2-P3 promoters.
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Disruption of sarR increases the amount of sarA P1 transcript, arguing that it acts as a repressor of sarA P1 (Manna & Cheung, 2001). SarR also regulates agr expression and binds in vitro to the same DNA region as SarA, suggesting that both regulators might compete for similar binding sites (A. Cheung, personal communication). In fact either sarR or sarA disruption, alone, has only a moderate diminishing effect on a agrP3(RNAIII)-gfp reporter fusion, however, when both sarA and sarR are absent the activity of the reporter is strongly attenuated. These data suggest that SarA and SarR tighty co-regulate agr activity, and together are strong activators, at least in RN6390 strain where the study was performed (Manna & Cheung, 2006). In addition, a strong positive role for SarR in regulating other virulence genes, such as aur (aureolysin) and sspA (V8 protease), has been established (Gustafsson & Oscarsson, 2008). Recently a regulator of sarR transcription was identified and named rsr (repressor of sarR) (Tamber, et al., 2010).
Other SarA homolgs
Rot, (Repressor of Toxins), was first identified in a Tn917 transposon library screening for high hemolytic activity in an agr- background (McNamara, et al., 2000). A transcriptomic analysis comparing a rot- strain to its wild-type counterparts showed that a large number of virulence factors are regulated by rot; indeed Rot represses toxins and upregulates cell wall associated virulence factors (Said-Salim, et al., 2003). This inverse pattern of regulation compared to agr/RNAIII and the fact that in an agr-/rot- background production of toxins are rescued led to the assumption that part of RNAIII effect might be mediated through Rot repression, the latter being the direct regulator. Actually it was subsequently shown that RNAIII and rot mRNA interact in such a manner that translation of rot mRNA is blocked by RNAIII (Geisinger, et al., 2006, Boisset, et al., 2007). Other studies showed that Rot binds to spa promoter (Oscarsson, et al., 2005). In addition, in clinical strains, this inverse correlation between RNAIII and Rot seems to be preserved, suggesting that in vivo this regulator plays a particularly important role (Jelsbak, et al., 2010).
MgrA (Multiple global regulator, also called rat/norR) in addition to its role in autolysis and capsule production, has also been implicated in the regulation of virulence factors (Ingavale, et al., 2003, Ingavale, et al., 2005). Downregulation of spa and hla, and upregulation of
19
lukDSFM (Leucocidins), lip (lipase), sak (Staphylokinase) are mediated by this regulator (Luong, et al., 2006). The following cis-binding consensus for MgrA has been proposed:
TGTTGGN8ACAACG after footprint analysis of MgrA binding to one of its targets, the sarV promoter (Manna, et al., 2004). MgrA also regulates other regulators of the family, such as sarX and sarZ (Manna & Cheung, 2006, Ballal, et al., 2009). In addition, MgrA is involved in biofilm formation (Trotonda, et al., 2008), and has been involved in resistance to antiobiotic drugs (Truong-Bolduc, et al., 2003, Truong-Bolduc, et al., 2005, Truong-Bolduc & Hooper, 2007).
SarS (formerly named SarH1), as previously discussed, is a repressor of hla and activator of spa transcription (Cheung, et al., 2001). It was shown to bind to a similar recognition sequence as SarA. How exactly sarA and sarS co-regulate their common targets remains to be elucidated (Tegmark, et al., 2000, Oscarsson, et al., 2005).
Various other regulators named SarT, SarU, SarV, SarX, and SarZ have been identified and shown to be involved in the complex network of virulence regulators, and that is only beginning to be understood. (See Table 2) (Cheung, et al., 2004, Kaito, et al., 2006, Cheung, et al., 2008).
Table 2. Members of the SarA regulators family and their putative function (adapted from Cheung et al. 2008)
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Two-component histidine-kinase sensing systems (TCS)
Two-component histidine-kinase transductions systems are widely present among bacterial species and lower eukaryotes (Gao, et al., 2007, Gao & Stock, 2009). These TCS typically allow sensing of external stimulus with further transduction of the signal into the cell and subsequent regulation of downstream target genes. They have been implicated in almost all biological features of the bacteria, including regulation of virulence. An example is the previously described agr system, which contains in its operon a TCS encoded by agrCA genes. The prototypical TCS is composed of one histidine kinase (HK) sensor and a response regulator (RR), both being generally encoded on the same operon. The HK sensor classically senses an external signal, autophosphorylates on a histidine residue and transfer the phosphate group on the aspartate residue of the RR (see figure 4). The response regulator, generally a transcription factor, then modulates transcription of downstream target genes.
Figure 4. Scheme of a typical TCS. The conserved Histidine Kinase domain (HisK) mediates autophosphorylation and phosphoryl transfer to the Aspartate residue on the Receiver domain (REC) of the RR (adapted from Gao et al. 2007)
If the majority of RR do have DNA binding properties, some have other functional domains such as RNA-binding, protein binding, or enzymatic activities (see Figure 5). RR with DNA- binding domains have been sub-classified according to their DNA-binding domain type (Gao, et al., 2007).
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Figure 5. Classification of bacterial RR, upon the function of their effector domain (adapted from Gao et al. 2007)
In S. aureus 17 TCS have been identified by sequence analysis, and if some of them have been extensively studied, others remain largely uncharacterized. Generally, TCS are not essential genes; however in S. aureus one TCS, yycFG (walKR), appeared to be essential and could not be mutated (Martin, et al., 1999). Classical genetic approaches, coupled with genome-wide strategies have been helpful to analyze downstream regulation of some of these 17 TCS, however the exact nature of the upstream signal, their mechanism of binding and triggering of the sensor HK remain generally elusive and poorly described, with the notable exception of agr AIP (Gao, et al., 2007, Gao & Stock, 2009).
Finally, the importance of these regulatory systems involved in so many bacterial processes, and the fact that the histidine kinase activity is restricted to prokaryotes, have revealed histidine kinases as ideal targets for antimicrobial drugs, generating efforts for research in this direction (Barrett, et al., 1998, Stephenson, et al., 2000, Eguchi, et al., 2011).
SaeRS
SaeRS (staphylococcal accessory element) is an important positive regulator of many virulence factors, such as hla, hld, coa and a negative regulator of spa and sspA (Giraudo, et al., 1994, Giraudo, et al., 1997, Giraudo, et al., 1999). Novick and co-workers suggest that SaeRS helps coordinate environmental signaling with quorum sensing (Novick, 2003). The regulon of SaeR has been partially characterized and a DNA recognition site, GTTAAN6GTTAA proposed, based upon DNA footprint assay on the sae promoter region. Directed mutagenesis of each of these nucleotides separately validated this binding site in vitro (Sun,
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et al., 2010). Interestingly the strain Newman, widely used for virulence assays, carries a proline instead of a leucine (L18P substitution) in saeS responsible for the high level of phosphorylation of SaeR and subsequent high expression of some virulence factors. It has been proposed that some targets (class I) are directly under control of SaeS-phosphorylated SaeR, and others are activated by SaeR at low level of phosphorylation (probably not mediated via SaeS) (class II) (Mainiero, et al., 2010). Finally, in vivo studies have shown the importance of this TCS in animal infection models (Nygaard, et al., 2010). Interestingly hla expression in vivo was severly decreased in sae- mutant whereas in agr- and sarA- mutants it was not the case. This confirms that virulence regulatory pathways might differ notably between in vitro and in vivo model systems (Goerke, et al., 2001).
SrrAB
The srrAB (staphylococcus respiratory regulator) system, (also called srhSR), has first been identified by its similarity with the resDE (respiration regulator) of Bacillus subtilis. In this organism resDE is a major anaerobic gene regulator, regulating the fnr regulator, the nasDEF operon and hmp genes, involved in fermentation and nitrate respiration (Nakano, et al., 1996, Nakano, et al., 2000). Both ResD and SrrA belong to the OmpR family of RR and have a winged-HTH DNA binding domain. ResE and SrrB also share similarities, especially regarding their extracellular, transmembrane, and histidine-kinase domain positions. Nevertheless certain domains of ResE, such as the PAS domain (Taylor & Zhulin, 1999), are not identified in silico in SrrB, suggesting possible functional difference between both TCS (figure 6).
In S. aureus srrAB was first analyzed regarding its role in repression of virulence factors in anaerobiosis (especially TSST-1, which was known at the time to be strongly repressed in this condition). These studies showed that when srrAB was overexpressed, Protein A, TSST-1 and RNAIII were barely detectable, in both aerobiosis and anaerobiosis. This regulation was proposed to be mediated at the transcriptional level and to be direct, since SrrA binds in vitro to the promoter regions of these genes; however no binding recognition sequence has been described and almost no transcriptional data were provided in these studies (Yarwood, et al., 2001, Pragman, et al., 2004). Using alternative strategies employing an antisense srrA silencing another study showed that SrrA, in aerobiosis, was a positive regulator of tst, icaR
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and spa transcription (but surprisingly not RNAIII) (Pragman, et al., 2007). The authors proposed srrAB to be a dual regulator of virulence genes, both repressing and stimulating some targets in anaerobiosis and aerobiosis, respectively. The role of SrrB-phosphorylation of SrrA in this dual regulation remains unknown, as well as the exact nature of the signal triggering SrrB activation. Furthermore so far no global transcriptomic study has revealed the extent of genes regulated by the SrrAB TCS.
Figure 6: Scheme of ResE and SrrB structural domains. Alignement of SrrB and ResE homologs from Bacillus subtilis, Bacillus anthracis and Listeria monocytogenes. HisKA = Histidine kinase domain, HATPase = ATPase domain, HAMP, PAS TM = transmembrane.
In vivo data are also intriguing: on one hand a rabbit endocarditis model showed the strain overexpressing srrAB to be less virulent than its wild-type counterpart, suggesting that srrAB is a repressor of virulence (Pragman, et al., 2004); while on the other hand srrAB was necessary for full virulence in a murine sepsis model (Richardson, et al., 2006). Finally a srrA mutant turned up in a screen for attenuated virulence in C. elegans, suggesting again that the srrAB TCS is necessary for full virulence (Bae, et al., 2004).
Besides classical virulence factors regulation, SrrAB is an important direct modulator of the ica operon (intercellular adhesion locus), which codes for the enzymes necessary for the PIA (polysaccharide intercellular adhesin) biosynthesis and known to be important for biofilm formation. Indeed, a screen for mutants defective in biofilm formation in comparison to
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their wild-type counterpart (a highly biofilm-forming S. aureus clinical isolate) revealed srrA importance in this process (Tu Quoc, et al., 2007). In vitro phosphorylated-SrrA was shown to bind to the ica promoter (Ulrich, et al., 2007). In addition PIA is also involved in defense mechanisms against neutrophil phagocytosis and, in this context, the srrAB- mutant was shown to be very vulnerable to neutrophils, especially in anaerobiosis (Ulrich, et al., 2007).
In another study srrAB appeared to be part of a regulon induced by nitrosative stress and thus potentially required for full response against innate immunity. A srrAB mutant is particularly hypersensitive to NO. In this situation however, a role for srrAB remains unclear since the genes under control of SrrA and important for NO resistance have not been identified (Richardson, et al., 2006, Richardson, et al., 2008).
Figure 7. Overview of the interconnections between virulence regulation pathways and metabolic pathways and putative role of SrrAB (adapted from Somerville and Proctor 2009)
Finally, a metabolic role for srrAB, probably as a regulator for activation and repression of certain pathways when the bacteria switches from an aerobic to an anaerobic environment, was proposed in a proteomic study, comparing a wild-type strain and its srhSR- (another name for srrAB-) mutant derivative. This study showed that numerous proteins involved in energy metabolism were affected in the absence of srrAB. The authors showed that TCA
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cycle enzymes, such as aconitase, succinate dehydrogenase, fumarase and NADH dehydrogenase were repressed by srrAB; in addition other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology were affected (Throup, et al., 2001). It has thus been proposed that SrrAB might be responsible for repressing TCA cycle enzymes and favoring fermentative pathways, when nutrients are abundant or when oxygen is not available (Somerville & Proctor, 2009). Taken together these findings suggest that the srrAB TCS is an important link between energy metabolism and regulation of virulence, even though many elements remain unclear (see figure 7) (Somerville & Proctor, 2009).
ArlSR
The arlSR (autolysis-related locus) TCS has been shown to decrease expression of some virulence factors such as hla, sspA, spa, and regulators such as sarA and mgrA (Fournier, et al., 2001). It also down or upregulates RNAIII production, depending on strains. In vivo arlRS- mutants were less virulent in a murine pyelonephritis model (Liang, et al., 2005). Its role in virulence regulation remains to be defined as well as its triggering signal and subsequent mechanisms.
Other S. aureus TCS
Many other TCS have been identified in S. aureus. We will discuss shortly some of them hereafter, although they might not be directly involved in virulence regulation. Among them, one of the most studied is the vraSR system (vancomycin resistance associated regulator), which has been shown to be a capital sensor and activator of genes involved in resistance to cell-wall active antibiotics.
VraSR
VraR is strongly induced in the presence of these antibiotics, for example synthetic penicillins or glycopeptides, and data suggest that VraS somehow senses the cell wall “attack” (Kuroda, et al., 2003, Gardete, et al., 2006). The exact molecular trigger is unknown. Once activated, VraS phosphorylates its cognate RR VraR, which induces a regulon probably involved in
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resistance mechanisms, although precise details of this response remain to be elucidated.
Genetic disruption of the signal transduction, by impairing phosphorylation of VraR by VraS, has the effect of failing to induce VraR expression when the bacteria is challenged by cell wall active antibiotics. In addition, the emergence of first generation resistant colonies on plates containing low level glycopeptides (a phenomenon commonly observed with this class of antibiotics), is abolished when the TCS is not functional, as shown in recent work from our laboratory, - see appendix V and VII- (Galbusera, et al., 2010). A regulon of VraR has been proposed in a transcriptomic studies (Kuroda, et al., 2003, Utaida, et al., 2003), and the following DNA cis-recognition sequence for VraR proposed, ACG(Xn)AGT (Belcheva, et al., 2009). This TCS, though does not seem to be involved in virulence regulation per se, knowledge of its role in S. aureus is so important for clinical practice that deserves special attention (McCallum, et al., 2010).
WalKR
WalKR (formerly yycFG) is implicated in cell wall biosynthesis, biofilm formation and resistance to vancomycin. Part of its regulon has been described and is mainly composed of autolysins and genes related to cell wall degradation (Dubrac & Msadek, 2004). No virulence related genes have been identified in the WalKR regulon. A binding recognition site has been proposed in silico TGT(A/T)A(A/T/C)N5TGT(A/T)A(A/T/C) (Dubrac, et al., 2007). This TCS was strongly induced in a vancomycin resistant (less sensitive) derivative compared to its wild type parent, suggesting the importance of this TCS for cell-wall active antibiotic resistance and its potential interest as a target for new antimicrobial drugs (Jansen, et al., 2007, Gotoh, et al., 2010, Eguchi, et al., 2011).
The Staphylococcus aureus genome encompasses many other TCS such as NreBC (involved in nitrate and nitrite reduction), HssSR (involved in avoiding heme toxicity) and GraRS (involved in resistance to cationic peptides and cell-wall active antibiotics) (Meehl, et al., 2007, Stauff, et al., 2007, Schlag, et al., 2008); however these will not be discussed here, since they have not been demonstrated to be involved in virulence regulation.
27 SigmaB ( B)
Besides sigma factor A, the 2 ’ holoenzyme core of S. aureus RNAPolymerase can interact with sigmaB ( B), sigmaS and sigmaH to initiate transcription at specific promoters. Little is known about the two latest sigma factors, except that sigma H has been described to be necessary for transcription of the int (integrase) gene and subsequent prophage excision (Tao, et al., 2010), and sigmaS seems to be important for virulence (Shaw, et al., 2008). The alternative B factor, widely studied in B. subtilis and subsequently in S. aureus, is activated in stress conditions such as antibiotic attack, heat-shock, acid shock, and starvation of glucose (see figure 8). In both B. subtilis and S. aureus it has been established that in normal conditions B forms a complex with the anti-sigma factor RsbW and in consequence cannot interact with the RNA polymerase. In case of stress the RsbU phosphatase de- phosphorylates RsbV, which in this condition has a high affinity to RsbW, thus disrupting RsbW- B interaction. Once released, B can now interact with RNA polymerase and modulate transcription, as shown in figure 8 (Haldenwang, 1995).
Figure 8. Scheme of B activation by RsbU-V-W family in B. subtilis. Similar mechanisms exist in S.
aureus (adapted from Haldenwang, 1995)
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SigmaB is involved in the transcriptional regulation, directly or indirectly of hundreds of genes in S. aureus, as shown in a transcriptomic study (Bischoff, et al., 2004). The B cis- recognition sequence, originally described in B. subtilis (Haldenwang, 1995), could be identified upstream of many of these genes in an in silico analysis. Regarding virulence regulation, B is generally accepted to be a positive regulator of membrane associated factor genes, such as fnbA and spa, and a repressor of secreted toxins genes, such as -hemolysin and SEBs (Bischoff, et al., 2001, Entenza, et al., 2005); B shows an inverse pattern of regulation compared to the quorum sensing agr effector molecule RNAIII. Regarding its role as a regulator of regulators, the following findings are available. Several studies have shown that B influences sarA expression. Bischoff and co-workers using a gene-reporter strategy, as well as Gertz and colleagues in a proteomic study, find a positive regulatory role for B on sarA expression (Gertz, et al., 2000, Bischoff, et al., 2001). Genetic analysis and in vitro transcription run off assay showed that the sarA transcript under P3 promoter control is strongly dependent upon sigmaB (Deora, et al., 1997, Manna, et al., 1998). In contrast, however, Horsburgh and co-workers find no evidence of B influence on sarA transcription (Horsburgh, et al., 2002).
Regarding agrP3/RNAIII regulation, some studies find a repressive role of B whereas other studies reveal no influence. In one study, B was shown to downregulate RNAIII production in Newman strain and in 8325-4 derivatives; whereas a transcriptomic approach (in strains COL and Newman among others) did not confirm this observation (although the authors suggested that the high levels of RNAIII might have saturated the target oligonucleotides of the GeneChip) (Bischoff, et al., 2001, Bischoff, et al., 2004). Collectively, these findings concerning a B role in virulence were mostly obtained in vitro, but in vivo studies did not always confirm the importance of B in infection models. For example, Horsburgh reported no difference between 8325-4 (rsbU-) and SH1000 (rsbU+) in a murine skin abcess model (Horsburgh, et al., 2002), whereas Jonsson and co-wokers showed that B was necessary for full virulence in a murine arthritis model (Jonsson, et al., 2004). In another study, the wild- type sigmaB strain provoked bigger vegetations in a rabbit endocarditis model than the mutant counterpart, (and these vegetations could be attenuated by salicylic acid) (Kupferwasser, et al., 2003).
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Finally, regarding clinical isolates, some -hemolysin high producer clinical strains were shown to carry mutations in sigB (Wood46) or rsbU (KS26, V8) (Karlsson-Kanth, et al., 2006).
One could hypothesize that in infections where adhesion plays a central role, such as endocarditis, where bacteria adhere to the cardiac valves in spite of the blood stream, sigB is necessary for full virulence, whereas it is possible that sigB absence or inactivity could favor toxin-mediated diseases.
CcpA
Catabolite control protein A, when associated to phosphorylated Hpr binds to a CRE (catabolite repression element) on DNA and enhances or represses transcription of its target genes, depending of the position of the site. The CRE consensus sequence in B. subtilis is WWTGNAARCGWWWWCAWW (where W = A/T) (Miwa, et al., 2000). Regarding virulence regulation, this CRE element has been identified upstream of hla, spa and tst genes, these genes are repressed by CcpA in presence of glucose -almost no effect of the mutation can be detected in the absence of glucose- (Seidl, et al., 2006, Seidl, et al., 2008). The ccpA mutation has an impact on RNAIII production although no CRE site was identified suggesting that this effect could be indirect.
Integration of virulence regulation
In summary, we have presented some of the major transcriptional regulators involved in virulence control in S. aureus, although this is not an exhaustive list. Other recently discovered transcriptional regulators, such as Rex (involved in anaerobic repression of genes)(Pagels, et al., 2010), or post transcriptional modulators, such as the serine-threonine kinase PknB (also called Stk-1) and cognate phosphatase Stp-1 (Debarbouille, et al., 2009, Burnside, et al., 2010, Ohlsen & Donat, 2010, Tamber, et al., 2010), are apparently of critical importance for regulation of various metabolic pathways in general and virulence in particular, although their precise mode of action remains to be studied. Finally, of great interest is the potential role in S. aureus virulence of small regulatory RNAs, such as SprD (Chabelskaya, et al., 2010, Romby & Charpentier, 2010), and numerous other small RNAs so far uncharacterized (Beaume, et al., 2010).
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Integrating these last decades numerous findings in the field of virulence determinants regulation into a common general picture is not an easy task; nevertheless this ultimate goal is being pursued by several researchers. It is important to emphasize that not all information comes from the same strain background and that there are several notable inter-strain specificities and differences. More specifically, a large amount of information was obtained in strain 8325-4 (or derivatives such as RN4220, RN6390, ISP794) that does not possess a full sigmaB activity (rsbU-). Lately we have learned a lot from strains Newman or SH1000, that have a full sigmaB activity, although these strains themselves also carry mutations (in saeS, or tcaR genes respectively) that have physiologic consequences (Herbert, et al., 2010). There is no uniformly agreed upon reference strain used by all S. aureus research labs.
In addition, in vitro (in a flask) and in vivo (in animals or patients) findings do not always correspond. Nevertheless, the following general pictures have been proposed so far (see figures 9, 10 and 11) and several reviews try to propose a coherent model (Novick, 2003, Yarwood & Schlievert, 2003, Bronner, et al., 2004, Cheung, et al., 2004, Pragman &
Schlievert, 2004, Cheung, et al., 2008, Loughman, et al., 2009, Somerville & Proctor, 2009)
Figure 9. Integrated regulation model for hla expression, as a prototype of a secreted virulence factor (exoprotein) (adapted from Bronner et al. 2003)
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Figure 10. Integrated regulation model for spa expression, as a prototype of a membrane-anchored virulence factor (adapted from Bronner et al. 2003)
Figure 11. Regulation of virulence determinants by Sar family regulators, quorum sensing system agr and sigmaB in an idealized flask (adapted from Cheung et al. 2008)
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Staphylococcus aureus toxinoses
Besides the infections provoked by the presence and replication of the bacteria itself in a defined anatomical site, serious clinical situations can be triggered by exotoxins produced by S. aureus upon simple colonization. In these cases, an infection is not necessary for triggering the disease. Although an infection might be present, the toxin itself is generally sufficient to provoke the disease; this is typical of toxins with superantigenic properties which can, alone, be responsible of so-called toxinoses. This situation must be differentiated from classical infections, such as abscesses, where secreted exoproteins (typically proteases, coagulases, lipase, hemolysins) act collectively and whose main role is to allow the bacteria to spread into surrounding tissues.
S. aureus is responsible for three main toxic syndromes:
1) food poisoning caused by enterotoxins,
2) staphylococcal scaled skin syndrome (SSSS) caused by exfoliatins and,
3) toxic shock syndromes (TSS) caused by either TSS Toxin-1 (TSST-1) or enterotoxins with superantigenic properties
Food-borne intoxication is the typical situation where food, prior to ingestion, is contaminated by S. aureus and its secreted toxins. Once in the human digestive tract the toxin can provoke damage and, at that point, S. aureus itself is no longer involved; in this case neither infection nor colonization is necessary to provoke the disease (Que & Moreillon, 2010).
The SSSS is a clinical situation where young children, generally in the context of S. aureus cutaneous infection, suffer from blisters due to exotoxins exfoliatins A or B. Exfoliatins A and B cleave desmoglein-1, which mediates cell-cell adhesion of the epiderma, thus causing in mild cases bullous impetigo and in severe cases the whole-body generalized form SSSS (Amagai, et al., 2000). This disease does not generally occur in older children or adults due to protective antibodies (Patel & Finlay, 2003, Que & Moreillon, 2010). These exfoliatins are encoded by eta and etb genes, and are present only in a minority of clinical strains (as shown
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in a study performed in Geneva (Megevand, et al., 2010). Only recently has a study brought up some findings about their regulation characteristics (Kato, et al., 2011).
Staphylococcal TSS will be discussed hereafter in some detail, focusing on its physiopathological aspects. Clinical treatment approaches will not be discussed, but can be read in the following review (Lappin & Ferguson, 2009).
Toxic Shock Syndrome (TSS)
This life threatening disease, came to general attention in the early 1980’s when a series of approx. 900 menses’ related TSS cases where reported by the US Center for Diseases Control (CDC) with 73 deaths (1981, 1983). Young healthy women, generally menstruating, were critically ill and in some cases eventually died because of the production by S. aureus of a dangerous toxin with superantigenic properties in the vagina. This disease was suspected to be associated with the use of highly absorbent hygienic tampons (notably RELY from Procter&Gamble) (Reingold, et al., 1982, Schlech, et al., 1982). However these findings were strongly debated since the first described cases of TSS had happened before the introduction in the market of this product (although other brands of similar products existed before) (Todd, et al., 1978, Garrett, 1994).
At that time of the initial TSS outbreak the exact cause of the disease was unknown.
Intensive studies eventually showed an association of the disease with presence of S. aureus in the vaginal flora and then pointed out the role of an exotoxin (Enterotoxin F, since then renamed TSST-1) as the causative agent. The absolute confirmation, after a strong fight between the CDC scientists and Patrick Schlievert, was brought up by the latter when the toxin, injected into rabbits provoked the disease; in addition TSS’ causing strains (coded) were correctly identified from control strains in a blind study (Bergdoll, et al., 1981, Schlievert, et al., 1981, Schlievert & Kelly, 1982). TSST-1 encoding gene, tst, was finally identified and shown to be on a mobile genetic element, explaining why only some strains did provoke TSS (Kreiswirth, et al., 1983, Lindsay, et al., 1998).
