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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

The accessory genes of Staphylococcus aureus, in-cluding those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two-component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time- and population density-dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full-scale test.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

Staphylococcus aureus is a remarkably versatile organism. It is adaptable, flexible and multifaceted in its interactions with its surroundings. It can exist comfortably in inanimate sites as well as in various niches in the animal host. It can exist harmlessly as a commensal, inhabiting the skin or mucous membranes, and it can survive in the blood or in a variety of tissue sites where it is responsible for disease states ranging from minor skin infections to toxinoses and systemic, life-threatening illnesses.

This versatility depends on a tremendous range of adaptive or accessory gene systems. A subset of these is involved in pathogenesis, which is best viewed as an adaptation to the hostile environment of the host and its formidable antibacterial defences. Most of the 50 or more accessory genes involved in pathogenesis encode proteins that are either displayed on the bacterial surface or released into the surroundings. These enable the organism to evade host defences, to adhere to cells and the tissue matrix, to spread within the host and to degrade cells and tissues, for both nutrition and protection. These accessory genes are collectively known as the virulon, even though they are not all exclusively devoted to pathogenesis – for example, the exoenzymes are presumably of major importance for existence in the inanimate environment. Note that the virulon is very likely to overlap significantly with the set of genes involved in maintaining the state of colonization. It does not represent the same set of genes as that identified by in vivo genetic manipulation [IVET (Morfeldt et al., 1996a), STM (Mei et al., 1997), etc.], as the latter set includes housekeeping genes, which are ipso facto required for pathogenesis, because the organism must be able to multiply in order to cause disease. However, it has been difficult to define the entire virulon, one gene at a time, because with the exception of the toxinoses, staphylococcal disease is dependent on the combined action of many exoproteins. A key finding therefore was the identification of a global regulator, agr, that controls the expression of most of the exoprotein genes (Recsei et al., 1986; Morfeldt et al., 1988; Peng et al., 1988). In Table 1 are listed the most important of these genes and their products. The demonstration that agr mutants are attenuated for virulence in several animal models (Foster et al., 1990; Abdelinour et al., 1993; Gillaspy et al., 1995) has established agr as a global regulator of staphylococcal virulence. Further support for the concept of global regulation of virulence was obtained by the subsequent isolation of other pleiotropic mutants, including sarA (Cheung et al., 1992), sae (Giraudo et al., 1994), sarH1 (also known as sarS; Tegmark et al., 2000) and rot (McNamara et al., 2000). In Table 2 is a list of the most important regulatory and transcription factors identified thus far. These regulators also control the synthesis of a large class of cytoplasmic proteins (Recsei et al., 1986) including many catabolic enzymes (Dunman et al., 2001), such as amino acid deaminases and dehydratases, phosphotransferases, nitrate reductase, etc. (Table 3). Their possible role could be the utilization of amino acids released by the secreted proteases, particularly with respect to the acquisition of organic nitrogen. Whether or not any are involved in pathogenesis remains to be determined.

Table 1. . Staphylococcal extracellular accessory proteins.
GeneLocationProductActivity/functionTimingAction of regulatory genesReferences
agr saeRS rot sarA sarS sarT tst
Superantigens
 seaPhageEnterotoxin AFood poisoning, TSSxpa0     01, 2
 sebSaPI3Enterotoxin BFood poisoning, TSSpxp+   b   2, 3
 secSaPI4Enterotoxin CFood poisoning, TSSpxp+      4
 sedPlasmidEnterotoxin DFood poisoning, TSSpxp+      5
 etaETA phageExfoliatin AScalded skin syndromepxp+      6
 etbPlasmidExfoliatin BScalded skin syndromepxp+       
 tstSaPI1,2,bov1Toxic shock toxin-1Toxic shock syndromepxp+   b   2, 7
Cytotoxins
 hlaChromα-HaemolysinHaemolysin, cytotoxinpxp++ b 2, 7, 8, 9, 10, 23
 hlbChromβ-HaemolysinHaemolysin, cytotoxinpxp++ b    7, 10, 23
 hldChromδ-haemolysinHaemolysin, cytotoxinxp+0 ++07, 8, 9, 10
 hlgChromγ-HaemolysinHaemolysin, cytotoxinpxp+  b    18, 23
 lukS/FPVL phageP-V leucocidinLeucolysinpxp+    2, 23
Enzymes
 SplA–FChromSerine protease-likePutative protease +     23
 sspChromV8 proteaseSpreading factorpxp+0 0 2, 16, 19
 aur Metalloprotease  (aureolysin)Processing enzyme?pxp+     16, 19
 sspB Cysteine proteaseProcessing enzyme? +     19, 23
 scp Staphopain  (protease II)Spreading, nutritionpxp+     19
 gehChromGlycerol ester  hydrolaseSpreading, nutritionpxp+0 b   15, 23
 lip Lipase (butyryl  esterase)Spreading, nutritionpxp+0  b    20
 fmeChromFAMEFatty acid esterificationpxp+   b    20
 plc PI-phospholipase C pxp+      19
 nucChromNucleaseNutritionpxp++     15
 hysChromHyaluronidaseSpreading factorxp b        
 coaChromCoagulaseClotting, clot digestionexp +++   10, 11, 14, 23
 sakPhageStaphylokinasePlasminogen activatorpxp+0     7
Surface proteins
 spaChromProtein AAnti-immune, anti-PMNexp  b + +  7, 10, 17, 23
 cnaPT isletCollagen BPCollagen bindingpxp0      12
 fnbAChromFibronectin BPAFibronectin bindingexp   +   13
 fnbBChromFibronectin BPBFibronectin bindingexp   +   13
 clfAChromClumping factor AFibrinogen bindingexp0      14
 clfBChromClumping factor BFibrinogen bindingexp0 +0   22, 23
Lactoferrin BPLactoferrin binding        19
Capsular polysaccharides
 cap5ChromPolysacch.  cap. type 5Antiphagocytosis?pxp+  +   21
 cap8ChromPolysacch.  cap. type 8Antiphagocytosis?pxp+      21
Table 2. . Known accessory gene regulation and transcription units in S. aureus.
Regulatory unitDescriptionRoleReference
AgrACDB/rna IIITCS, autoinduced by peptideRegulates many extracellular and cytoplasmic protein accessory genes Novick et al. (1993)
SaePQRS TCS, autoinducedRegulates many extracellular protein genes Giraudo et al. (1999)
ArlRS TCSRegulates autolysis and certain accessory genes Fournier et al. (2001)
SvrA Membrane proteinRequired for the expression of agr Garvis et al. (2002)
SrrAB TCSRegulates certain accessory genes at low PO2 Yarwood et al. (2001)
σ B Rpo sigma factorActive in late exponential phase; regulates many accessory genes Kullik et al. (1998)
SarA Transcription factorAssists in agr autoinduction under certain conditions; pleiotropic repressor Cheung et al. (1996)
SarS Transcription factorActivates transcription of spa and possibly other surface protein genes Tegmark et al. (2000)
SarT Transcription factorRepresses transcription of hla and possibly other exoprotein genes Schmidt et al. (2001)
SarR Transcription factorMinor transcription factor for sarA and possibly sarS Manna and Cheung (2001)
RotTranscription factorMajor transcription factor for hla and other exoprotein genes McNamara et al. (2000)
Table 3. . Post-exponentially regulated cytoplasmic genes.
GeneProteinActivation ratioa
  • a

    . Transcript level during post-exponential phase divided by level during early exponential phase (Dunman et al., 2001).

arcA Arginine deaminase5.7
arcB Aspartate/ornithine carbamoyltransferase8.7
arcC Carbamate kinase7.9
arcD Arginine/ornithine antiporter6.4
arcR Transcriptional regulator Crp/Fnr family18.9
Transcriptional regulator GntR family5.2
hutI Imidazolone-5-propionate hydrolase41.5
hutU Uroconate hydratase23.3
sdhA l-serine deaminase6.5
sdhB l-serine dehydratase beta subunit7.6
bsaA Glutathione peroxidase5.8
ald Alanine dehydrogenase11.9
bmfBAB Thiamine pyrophosphate-dependent dehydrogenase26.8
narG Anaerobic dehydrogenase93.4
tdcB Threonine dehydratase33.8
folD 5,10-Methylene tetrahydrofolate dehydrogenase6.5
heml Glutamate-1-semialdehyde aminotransferase10.9
gyrA DNA gyrase6.2
mnhA Multisubunit Na+/H+ antiporter11.5
nasE Assimilatory nitrate reductase116.0
dLtD DttD protein58.3
Phenol-soluble modulin beta 26.2
treP Phosphotransferase system IIC component13.1
treA Alpha-glucosidase10.8
ssaA Secretory antigen precursor17.0
hla α-Haemolysin38.8

Production of virulence factors is carefully controlled in response to cell density, energy availability, environmental signals and SAgs so that these factors are produced when required. In vitro, the production of different exoproteins follows a specific temporal programme in which adhesins are made before haemolysins or proteases and other degradative enzymes. Recent studies suggest that this is also the case during infection (J. S. Wright and R. P. Novick, unpubl. data). Transcript profiling has shown that genes encoding surface proteins are downregulated early in growth, whereas those encoding secreted proteins are upregulated post-exponentially (Dunman et al., 2001; Y. Fang and R. P. Novick, unpubl. data). This shift in expression pattern is correlated with the population density-sensing agr two-component system, which is activated in mid-exponential phase and is known to upregulate a number of secreted proteins and to downregulate several surface proteins (Novick et al., 1993; Dunman et al., 2001). However, when energy or biosynthetic metabolites are insufficient – as with respiratory chain (McNamara and Proctor, 2000) or citric acid cycle mutants (Somerville et al., 2002a), many of these proteins are not synthesized, enabling the organism to maintain its housekeeping functions. Several environmental signals affect the production of extracellular proteins, in some cases perhaps in relation to the need to conserve resources under detrimental conditions. Thus, 1 M NaCl, subinhibitory ethanol and subinhibitory concentrations of antibiotics (Herbert and Novick, 2001) that inhibit ribosome function (Gemmel and Shibl, 1976; Chan and Foster, 1998a) have a general inhibitory effect on the synthesis of the exoproteins listed in Table 1. In other cases, the response may be related to specific environmental exigencies – thus, acid pH (≈ 5.8) downregulates tst* (see Table 1 for gene abbreviations) and hla, but upregulates ssp and spa (B. Weinrick and R. P. Novick, unpubl. data). Added to this elaborate set of regulatory patterns is the genetic variability of SAg genes; as the SAgs in and of themselves cause toxinoses, a strain expressing one has no need of most other exoproteins. Because these only complicate matters by evoking host defences, the toxin shuts down their genes (Vojtov et al., 2002).

In this review, we outline what is known about the regulatory strategies underlying this complex set of behaviours and begin to model the organization of the overall regulatory network. As virtually all the important information on the regulation of staphylococcal virulence and other accessory genes has been obtained by studies in vitro, this review is based primarily on in vitro data. Most of these studies have involved a single strain, NCTC8325, and its derivatives. This focus has caused at least two problems, one specific and one general. 8325 and its derivatives have an 11-base deletion in rsbU, which encodes a phosphatase that activates σB (Kullik and Giachino, 1997), an alternative sigma factor that is involved in the regulation of the virulon (see below). The general problem is that there are important differences in regulatory patterns among S. aureus strains. Smeltzer and coworkers have begun to address strain differences (Blevins et al., 2002), as have we (unpubl. data). Ultimately, it may be possible to understand these variations within an overall regulatory framework.

Two-component systems (TCSs)

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

Signal receptors are a major source of information on the external environment and, for S. aureus, appear to be the primary regulatory modality for expression of the virulon. Four distinct TCSs are presently known to be involved, agr (Recsei et al., 1986), sae (Giraudo et al., 1994; 1999), srr (Yarwood et al., 2001) [independently analysed by Throup et al. (2001) and referred to as srh] and arl (Fournier and Hooper, 2000) (see Fig. 1.B–D). These four represent one-quarter of the putative TCS identified by examination of the S. aureus genome (Brunskill and Bayles, 1996; Martin et al., 1999; D. McDevitt, pers. comm.), and there is every reason to believe that some of the others are also involved.

image

Figure 1. The two-component systems known to affect the virulon. A. The agr system. The pro-AIP peptide is processed and secreted by AgrB, binds to an extracellular loop in the receptor-HPK, AgrC, activating autophosphorylation (or dephosphorylation), followed by phosphorylation or dephosphorylation of the response regulator, AgrA, which, in conjunction with SarA, activates the two agr promoters, P2 and P3, leading to the production of RNA III, which controls transcription of the target genes via one or more intracellular regulatory mediators, including a second two-component module, saeRS. B. Sae. The sae locus, about 3.5 kb, contains four ORFs, P, Q, R and S. R and S form a classical two-component signalling module. The functions of P and Q are unknown. Sae is transcribed from two or three promoters, one of which is active in an agr-null strain and the other(s) is activated by RNA III. All three major transcripts, A, B and C, end at ter. D may be independently transcribed or derived from C by processing. PCR probes used to map the transcripts are shown. C. arlRS (adapted from Fournier et al., 2001). The arlRS locus encodes a receptor-HPK (arlS) and a response regulator (arlR), driven by a single promoter and followed by a terminator stem–loop. D. SrrAB (adapted from Yarwood et al., 2001). The srrAB locus encodes a receptor-HPK (srrB) and a response regulator (srrA), driven by a single promoter that generates two transcripts whose relative significance is unknown.

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The agr system

We begin with a summary of the agr system, which consists of an ≈ 3 kb locus (Fig. 1A) containing divergent transcription units, driven by promoters P2 and P3. The P2 operon encodes a two-component system and its autoinducing ligand (Novick et al., 1995). The primary function of the operon is to activate the two agr promoters. The P3 transcript, RNA III, rather than the response regulator, AgrA, is the intracellular effector of target gene regulation (Janzon and Arvidson, 1990; Novick et al., 1993). As agr is autoinduced by an extracellular ligand, it represents a sensor of population density. Because the activating ligand is encoded within the operon, the circuit is doubly autocatalytic, resulting in a very rapid burst of activity once the autoinduction threshold has been reached. The expression of this system entails a tremendous metabolic burden, resulting in frequent spontaneous agr mutants in the laboratory (Bjorklind and Arvidson, 1980; Somerville et al., 2002b), especially in strains that lack σB, which modulates the agr regulon (see below). The agr-activating ligand is a post-translationally modified peptide (AIP), seven to nine aminoacyl residues in length, which is processed from a propeptide encoded by agrD (Ji et al., 1995). The AIP binds to the N-terminal transmembrane domain of the agr signal receptor, agrC (Ji et al., 1995; Lina et al., 1998; Lyon et al., 2000), activating the agr TCS, of which AgrA is the response regulator. Activated AgrA then upregulates promoters P2 and P3.

RAP.

 It has been proposed that ribosomal protein L2 (or ‘RAP’) is an alternative agr activator that is supposed to act early in growth. The protein is reported to be present in (post-exponential) staphylococcal culture supernatants, although it lacks a signal peptide. Although it was originally claimed that this agr-activating protein could be prepared from agr-null supernatants (Balaban et al., 1998), more recent reports have described its preparation, by ultrafiltration, only from agr+ culture supernatants, which contain the AIP. We and others have been unable to demonstrate any extracellular signalling protein in supernatants (Novick et al., 2000). Agr-null supernatants do not contain agr-activating activity (Novick et al., 2000), and all such activity in agr+ supernatants is dialysable, is recovered quantitatively in the dialysate and thus represents the AIP (Novick et al., 2000). Although the AIP can be removed by dialysis, we suspect that it cannot be quantitatively removed by ultrafiltration and is therefore responsible for the reported activity of ultrafilter retentates. We also suspect that the presence of L2 in post-exponential supernatants is the result of autolysis; this possibility has not been addressed.

Agr specificity groups. 

Agr is conserved throughout the staphylococci with interesting variations in the B–D–C region (see Fig. 1). These variations have resulted in at least four agr specificity groups in S. aureus and probably one or more in each of 15 other staphylococcal species examined (Ji et al., 1997; Otto et al., 1999; Jarraud et al., 2000; Dufour et al., 2002). The groups are defined by the mutual inhibition by their peptides of the agr response in heterologous pairings, resulting in a novel type of bacterial interference in which the agr regulon, rather than growth, is blocked (Ji et al., 1997). The ability of an AIP to activate its cognate receptor is highly sequence specific; a single amino acid substitution can change group specificity (see AIPs I and IV in Fig. 2). The N-terminal one-third of agrB and the C-terminal (cytoplasmic) histidine protein kinase domain of AgrC are highly conserved, whereas the intervening sequences are highly divergent, constituting the hypervariable region indicated in Fig. 1. The divergent regions determine group specificity and must therefore have evolved in concert. Functional variants within the agr locus are designed for cross-group and, presumably, cross-species interference rather than co-operative communication, so that they serve to isolate populations and may represent a major determinant of strain and species divergence. In keeping with this idea, it is predicted that the agr groups, at least in S. aureus, will be correlated with specific biotypes. Indeed, agr groupings are broadly correlated with strain genotypes as defined by multilocus sequence typing and by amplicon fragment length polymorphisms (Jarraud et al., 2002), resulting in general congruence between phylogenetic trees and agr groups (Jarraud et al., 2002). Evidence in support of agr group-specific biotypes has begun to emerge with respect to clinical features. Thus, most menstrual TSS strains belong to agr group III (Ji et al., 1997), as do all the 16 strains recently found to cause leucocidin-induced necrotizing pneumonia (Gillet et al., 2002); most of the recently encountered VISA strains belong to agr group II (Sakoulas et al., 2002), and most exfoliatin-producing strains belong to agr group IV (Jarraud et al., 2000; McDowell et al., 2001). A similar species-specific divergence has been described for the identically organized comAP locus, responsible for transformation competence in bacilli (Tortosa et al., 2001). Intergroup interference in S. aureus has enabled a test of the effects of blocking agr on an experimental staphylococcal infection, the skin abscess model of Barg et al. (1992), in which co-administration of the synthetic group II AIP along with or immediately after the bacteria sharply attenuated an infection caused by a group I strain (Mayville et al., 1999; J. Wright et al., in preparation). The growing realization that agr groups are biologically and clinically significant has prompted the development of agr typing methods. Several of these use the polymerase chain reaction (PCR) to generate agr group-specific products that are identified by restriction site polymorphisms (Papakyriacou et al., 2000) or hybridization tests (Dufour et al., 2002). In the author's laboratory, activation or inhibition of a luciferase or green fluorescent protein (GFP)-tagged RNA III promoter during adjacent co-culture on an agar surface is used (J. Wright et al. in preparation).

image

Figure 2. Structure and activity of the AIP. At the right is a computer-predicted structure for AIP-II determined by an energy-minimizing algorithm, kindly provided by G. Lyon (personal communication). Residues belonging to the macrocycle are shown in green, those to the linear tail portion in magenta. At the left are diagrammatic representations of the four known S. aureus AIP structures. For AIP-II, the two critical residues, N3 and S6, are indicated with larger green circles; the replacement of either with alanine generates a universal agr inhibitor for S. aureus. One of these, position 6, is the site of the only difference between AIPs I and IV and, therefore, the determinant of groups I and IV specificity, as shown. Cross-hatched circles represent tail residues, dotted circles ring residues. The conserved essential cysteine is enclosed by a circle with small grey bubbles. The S-C =  O of the thiolactone bond are indicated in red.

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AIP synthesis, structure and activity.

The agrD-encoded propeptide is both N- and C-terminally processed to form a unique thiolactone ring between the conserved central cysteine and the peptide's C-terminal carboxyl (Ji et al., 1997) (see Fig. 2). With one known exception, this cyclic thiolactone is essential for AIP activity and is the hallmark of these peptides. The only exception is a serine in some strains of Staphylococcus intermedius (Dufour et al., 2002). These strains produce an active AIP (G. Lina and F. Vandenesch, pers. comm.), which has been shown by mass spectrometry to be a nonapeptide containing a cyclic lactone (Kalkum et al., 2003). There is considerable evidence suggesting that the propeptide is processed and secreted by AgrB, a polytopic transmembrane protein (Ji et al., 1997; Saenz et al., 2000; Zhang et al., 2002). However, the possibility that one or more other proteins is involved has not been ruled out, and a possible candidate is SvrA, a recently described polytopic transmembrane protein required for agr activation (Garvis et al., 2002). The group specificity of AgrB is less stringent than that of the AIP–receptor interaction. Thus, AgrB-I* and AgrB-III will each process AgrD-I and AgrD-III with equal efficiency, but neither will process AgrD-II or AgrD-sl and vice versa (Ji et al., 1997). [Nomenclature: agr group is indicated by a roman numeral appended to a gene or protein abbreviation; thus, agrD-I, AIP-II and AgrC-III refer to the group I agrD gene, the group II AIP and the group III AgrC protein respectively. AIPs of non-aureus strains are referred to by species abbreviations – thus, Si-I and Se-II refer to AIPs produced by S. intermedius specificity group I and S. epidermidis specificity group II respectively (see Fig. 4 for a list of species abbreviations).] The agrD sequence has been determined for nearly 30 different strains, including representatives of some 16 different staphylococcal species (Ji et al., 1997; Van Wamel et al., 1998; Otto et al., 1999; Jarraud et al., 2000; Dufour et al., 2002) (see Fig. 3). The AIPs from strains of all four S. aureus agr specificity groups and from representative strains of S. lugdunensis, S. warneri and S. epidermidis have     been     sequenced     and/or     synthesized    in     vitro. (Jarraud et al., 2000; Lyon et al., 2002a). AIPs I and IV are octapeptides, AIPs-II and Si-I are nonapeptides (Ji et al., 1997; Kalkum et al., 2002) and AIPs-III and Sl-I and II are heptapeptides, suggesting that most staphylococcal AIPs are seven to nine amino acids in length. The AIPs form a coherent group with generally conserved structural features, including a strong gradient of increasing hydrophobicity from N- to C-termini, culminating in two bulky hydrophobic residues, limited to FLVY, plus an occasional M.

image

Figure 4. RNA III secondary structure (adapted from Benito et al., 2000). Computer-predicted structure was confirmed by enzymatic and chemical analyses. Numbers 1–14 refer to hairpins; A, B and C indicate long-distance interactions; the S–D sequence (70–4) and start (85–7) and stop (163–5) codons for hld are in bold. See Benito et al. (2000) for details. Regions of complementarity with the hla mRNA leader are highlighted in red (Novick et al., 1993; Morfeldt et al., 1995) with the spa mRNA translation initiation region in green (unpublished). The hld coding sequence and potentially translatable regions upstream are outlined in blue, solid and dashed respectively.

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image

Figure 3. Comparison of agrD sequences. Sequences were aligned visually. Predicted AIPs are in bold and are set between spaces. Those for which sequence has been confirmed by in vitro synthesis or by mass spectroscopy are highlighted in grey. Saur, S. aureus; Sarc, S. auricularis; Sarl, S. arletta; Scap, S. capitis; Scapr, S. capri; Scarn, S. carnosus; Sconc, S. cohneii cohneii; Sconu, S. cohneii urealyticum; Sepi, S. epidermidis; Sgal, S. gallinarum; Sint, S. intermedius; Slug, S. lugdunensis; Ssim, S. simulans; Swar, S. warneri; Sxyl, S. xylosus.

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Detailed structure–function analyses have been performed for AIPs II and I. The general structure of the AIPs and the structure–function results for these two are summarized in Fig. 2 (details of the AIP structure–function analysis are included in Supplementary material). The cyclical structure is generally required, whether the peptide is acting as an agr activator or as an inhibitor (Mayville et al., 1999; Lyon et al., 2000; McDowell et al., 2001). Replacement of the thiolactone by a lactone or lactam bond virtually eliminates the activation but not the cross-group inhibition function of the peptide, although these variant peptides are not self-inhibitors. Removal of the tail region of AIPs I or II converted either peptide into a universal inhibitor of S. aureus agr function (Lyon et al., 2002a,b). The interaction between activating and inhibiting peptides at the receptor is strictly competitive (Lyon et al., 2002b). A second cyclic peptide autoinducer has recently been identified as a ligand for the fsr receptor, an AgrC homologue that regulates gelatinase synthesis in Enterococcus faecalis (Nakayama et al., 2001; Qin et al., 2001). In this case, the AIP, an 11-residue peptide with an 8-member lactone ring, was more stable to protease degradation in culture supernatants than a linear peptide of the same primary sequence (J. Nakayama, personal communication). Thus, the cyclic structure may provide stability and may also be well suited to the dual activation–inhibition role required for these peptides. Nevertheless, a variety of linear peptides serve as signal receptor ligands in other Gram-positive bacteria, including Streptococcus pneumoniae, Bacillus subtilis and lactobacilli.

Mutations affecting agr have been tested in a variety of animal models, including skin abscesses (Mayville et al., 1999), endocarditis (Cheung et al., 1994), septic arthritis (Abdelinour et al., 1993) and peritoneal sepsis. Interestingly, agr is not expressed in the peritoneal sepsis model (Bellinger-Kawahara et al., 2002; R. Jin and R. P. Novick, unpublished data), and does not appear to affect virulence (J. Wei and R. P. Novick, unpublished data), probably because the agr autoinducer does not accumulate locally.

A linear peptide YSPWTNF (‘RIP’), alleged to be the agr inhibitor produced by strain RN833, was originally proposed to have been derived from the putative agr activator protein, RAP (Balaban et al., 1998) (i.e. ribosomal protein L2; Novick et al., 2000). Balaban et al. (1998; 2001) assumed that YSPWTNF arose by mutation from YKPITN, an internal sequence in the N-terminal region of L2. Subsequently, after our realization that RN833 is not a mutagenized derivative of an S. aureus strain, as originally thought, but actually a native strain of S. warneri (Novick et al., 2000), producing a typical AgrD-derived AIP, YSPCTNFF (Novick et al., 2000), Balaban et al. (1998; 2001) now claim that YSPWTNF (RIP) is encoded in the RN833 genome, in addition to the true agrD peptide (Novick et al., 2000). In fact, YSPXTNF, the actual sequence determined by Balaban et al. (1998) for the RN833 agr inhibitor, with ‘X’ being indeterminate, corresponds very well to the true RN833 AIP, YSPCTNFF, except that it lacks the second phenylalanine and contains a tryptophan arbitrarily inserted in place of the genetically determined cysteine (see Fig. 3). Several laboratories have independently synthesized YSPWTNF and have been unable to demonstrate agr inhibition by this peptide at concentrations up to 50 µM (about 2000-fold higher than the IC50s of native AIPs; T. Muir, personal communication, M. Booth, personal communication, J. Larrick, personal communication). Nevertheless, YSPWTNF (synthetic ‘RIP’) and related linear peptides have been reported to interfere with the ability of S. aureus to cause lethal subcutaneous abscesses in mice (Balaban et al., 2000; Gov et al., 2001); however, the reported therapeutic protocol involved preincubating the bacteria with the peptide at a concentration of 2–10 mM (representing a concentration in the mouse ≈ 10 000- to 100 000-fold greater than the effective concentration of a native inhibitory AIP in the same model; Mayville et al., 1999). These peptides have not been injected separately from the bacteria, nor have there been any tests of the peptides at these concentrations on the overall health of the bacteria or on organisms other than S. aureus. Their reported interference with staphylococcal infections is therefore of questionable significance.

AgrC, the agr signal receptor.

AgrC was inferred by sequence analysis to be a histidine protein kinase, was shown by phoA fusions to have a polytopic transmembrane N-terminal domain (Lina et al., 1998) and by pull-down studies to be the only cellular protein capable of binding the AIP (Ji et al., 1995), thus confirming that AgrC is the receptor of the agr TCS. It is unclear, however, whether agr activation follows the classical TCS paradigm. Thus, our original report that AgrC is phosphorylated in response to the AIP (Lina et al., 1998) has recently been re-examined by G. Ji (personal communication) who found AgrC to be at least partially phosphorylated in the absence of any AIP. Comparative genomic analysis (performed by G. Lyon) has suggested that the cytoplasmic domain of AgrC is dimeric, containing the typical four-helix bundle, so that autophosphorylation would be a trans process.

Predictably, group specificity resides in the N-terminal transmembrane domain of AgrC, as demonstrated by switching the two domains between AgrCs of different groups (Lyon et al., 2002a). Additionally, switching the proximal and distal halves of the N-terminal (receptor) domain of the AgrCs has localized the specific recognition of AIPs I and IV to the distal subdomain (Wright et al., 2003), suggesting that the single amino acid that differs between these two AIPs (aspartate versus tyrosine at position 5) makes a specific contact in this region of the receptor. Other chimeras of this type, however, have given results that are very difficult to explain on the basis of the classical ‘lock-and-key’ model for intermolecular interactions. In particular, the I–III (chimeras of this type are represented by the AgrC group of the promoter-proximal subdomain followed by that of the promoter-distal subdomain) and IV–III chimeras could not be inhibited but rather were activated by a variety of AIPs, including some that are strong inhibitors of other AgrCs. These results have given rise to a model in which interaction with the receptor involves two distinct events – first, the peptide enters a hydrophobic pocket of the receptor in a non-sequence-specific manner, requiring the two bulky C-terminal residues of the AIP. Next, it makes one or more specific hydrophilic contacts with specific sites in the receptor, leading to activation or inhibition. The broadening of specificity in the I–III and IV–III chimeras could then represent a situation in which the receptor is misfolded in such a way as to be poised for activation by the binding of a hydrophobic AIP without the need for any specific activating contacts.

AgrA.

AgrA has the sequence features of a response regulator (Nixon et al., 1986) and is required for activation of the two agr promoters, P2 and P3, completing the autoinduction circuit (Novick et al., 1993; 1995). However, binding of AgrA to the agr promoters or any other DNA has not been demonstrable (Morfeldt et al., 1996a). Nevertheless, it seems likely that either AgrA or AgrA∼P binds in this region. AgrA homologues, PlnC, PlnD and SppR, in Lactobacillus plantarum and Lactobacillus sake (Diep et al., 2003), respond to autoinducing peptides and bind to heptanucleotide repeats that are similar to those in the agr intergenic region (Morfeldt et al., 1996a). Ji and coworkers have observed that AgrA is constitutively phosphorylated (G. Ji, personal communication), so that its activation may involve dephosphorylation. This provides a possible explanation for experiments in which phosphorylation of AgrA could not be demonstrated in vitro (Morfeldt et al., 1996b). Thus, the agr-specific phosphotransfer pathway and its precise role in activation of the two agr promoters remains unclear at present. Although no role has been demonstrated for AgrA outside of activation of the two major agr promoters, agrA has its own promoter, very weak in vitro (Novick et al., 1995), but highly active in vivo, as revealed by signature-tagged mutagenesis (Mei et al., 1997), implying a possible additional role for the protein.

Intergenic region.

The intergenic 120 bp region between agr promoters P2 and P3 must contain the site(s) of activation of the two promoters. There is a strong 17 bp inverted repeat (IR) in this region that has been proposed to be a bidirectional regulatory protein binding site (Bayer et al., 1996); there are also four heptanucleotide repeats, centred at −45 with respect to P2 and at −66 with respect to P3, of which the two middle repeats overlap with the IR and are conserved in S. epidermidis, S. simulans and S. warneri, in which activation of the P3 promoter by the agr TCS is also conserved (Benito et al., 1998; Tegmark et al., 1998). Only the two P3-proximal repeats are required for P3 activation, suggesting that the two agr promoters may be differentially regulated. Further, the mechanism of P3 activation is related to the 19 nucleotide (nt) spacing between the −10 and −35 elements of the promoter, as elimination of three nucleotides in this region causes constitutive expression of the promoter (Morfeldt et al., 1996a).

Agr-RNA III: the agr effector. 

The immediate consequence of agr autoinduction is the production of RNA III, the P3 transcript, which is the intracellular effector of the agr regulon (Novick et al., 1993). RNA III is highly abundant and has a long half-life, ≈ 15 min (Janzon and Arvidson, 1990). It has a complex secondary structure (Benito et al., 2000; see Fig. 4), which is well conserved (although the sequence is not) among several staphylococcal species (Benito et al., 1998; Tegmark et al., 1998), resulting in interspecific cross-reactivity of the molecule. RNA III acts reciprocally, upregulating transcription of most of the extracellular protein genes and downregulating that of many surface protein genes (Novick et al., 1993; Saravia-Otten et al., 1997). Extracellular protein genes that are not regulated by agr include entA, cna and clfA and B, of which the last two are strongly upregulated post-exponentially (Foster, 1994) by an unknown signalling element. The mechanism(s) by which RNA III regulates transcription is(are) unknown. Structure–function analysis of the molecule has revealed that: (i) the transcription termination loop is necessary and sufficient for repression of spa transcription (Benito et al., 2000); and (ii) there is functional redundancy within the molecule with respect to the activation of target gene transcription, in that non-overlapping 5′ and 3′ subregions of the molecule are independently active in stimulating hla transcription (Novick et al., 1993). This is most probably related to the presence of nearly identical sequences in the unpaired regions of stem–loops 7 and 14 (see Fig. 4), which are complementary to the canonical Shine–Dalgarno (S–D) sequence. Thus, RNA III could act by interfering with the translation of other regulatory proteins by virtue of these anti-S–D sequences. Another possibility is that these C-rich loops could serve as ‘decoy’ binding sites for regulatory proteins, as is the case with the regulatory RNA, CsrB in E. coli, which titrates CsrA and thus regulates gluconeogenesis (Liu et al., 1997; Romeo, 1998). RNA III could bind to individual transcription factors, causing allosteric modifications that affect their ability to bind target sequences (Arvidson and Tegmark, 2001), and it is possible that RNA III affects the stability of the target gene transcripts. We have observed that RNA III binds a single staphylococcal protein, Hsa, an HU homologue (Y. Fang and R. P. Novick, unpublished data). If Hsa, like HU, is a general transcriptional silencer, its titration by RNA III could increase transcriptional activity of a wide variety of genes. RNA III and its mechanism of action are thus areas seriously in need of study.

RNA III also acts secondarily at the level of translation for at least two of the products, α-haemolysin (Novick et al., 1993; Morfeldt et al., 1995) and protein A (unpublished data). The 5′ region of RNA III is complementary to the hla leader (see Fig. 4; gene symbols are listed in Table 1), which folds into an untranslatable configuration unless prevented from doing so by RNA III; it is likely that translation of the hld reading frame is required for this interaction with the hla leader. The 3′ end of RNA III is complementary to the translation initiation site of spa mRNA and blocks its translation (unpublished observations; see Fig. 4). Whether translation of other agr-regulated proteins is affected by RNA III is presently unknown.

The sae TCS

SaeRS (Giraudo et al., 1994; 1999) (Fig. 1B) was identified as a transposon insertion mutation with a pleiotropic defect in exoprotein production distinct from that seen with agr mutants. It thus represents the second major TCS involved in global regulation of the staphylococcal virulon. In the sae mutant, in particular, nuclease and coagulase were profoundly reduced in culture supernatants owing to reduction in transcription. The mutation had no effect on the production of RNA III and so is either downstream or epistatic to agr. Neither an sae clone nor an agr clone restored exoprotein synthesis in an agr–sae double mutant. We have found that the sae locus is larger and more complex than that described by Nagel and coworkers (Giraudo et al., 1999). In both agr+ and agr-null strains, a 2 kb sae transcript appears at the beginning of the exponential phase and disappears post-exponentially. In the agr+, two larger sae transcripts appear immediately after the onset of RNA III synthesis. All three of these have a common 3′ end. The longest transcript includes two open reading frames (ORFs), of 140 and 150 codons, 5′ to saeR, neither of which is contained within the 2 kb transcript or in any other detectable transcript. The longer transcripts are not seen in the sae mutant and are greatly reduced in an agr-null strain and in a sarA mutant, but appear after the induction of RNA III cloned under Pbla control in the agr-null strain. These ORFs are likely to be important for sae function and perhaps for transducing environmental signals.

Several environmental stimuli, such as high salt, low pH, glucose and subinhibitory antibiotics, affect the sae transcription pattern (unpubl. data). For example, in the presence of 1 M NaCl or a subinhibitory concentration of clindamycin, hla and spa transcripts as well as the larger sae transcripts are profoundly decreased, whereas with subinhibitory concentrations of β-lactam antibiotics, all four are upregulated. These environmental cues act independently of agr but may act through SarA or one of its homologues. Thus, sae appears to lie at a convergence of cell density and environmental signals.

ArlRS

ArlRS (see Fig. 1C) is a TCS that was identified on the basis of its control of autolysis and of the norA polyvalent export pump (Fournier and Hooper, 2000), and a single study of its properties has been published (Fournier et al., 2001). Agr mutants are reported to be defective in arlRS expression, whereas an arlS mutant apparently overexpresses agr, especially the agr P2 transcript, consistent with independent regulation of P2 and P3 (Fournier et al., 2001). Agr and arlRS thus formally represent an autorepression circuit such that arlRS counters agr autoinduction. Consistent with this is the reported downregulation by arlRS of overall exoprotein synthesis, presumably consequent to downregulation of agr. However, arlRS appears to act independently of agr with respect to protein A production. Unfortunately, the paper by Fournier et al. (2001) contains a number of substantive uncertainties (documented in the Supplementary material), and the results must be regarded as tentative until confirmed.

SrrAB / srhSR

The fourth TCS evidently involved in expression of the staphylococcal virulon, especially under microaerobic conditions, is srrAB (Yarwood et al., 2001) (Fig. 1D; also known as srhSR; Throup et al., 2001), a homologue of the O2-responsive resDE system of B. subtilis (Birkey et al., 1998). The srrAB/srhSR mutants are profoundly growth defective in the absence of oxygen, although they grow normally under aerobic conditions. This TCS appears to inhibit agr activation (Yarwood et al., 2001) and is itself downregulated by agr (unpublished data; P. Schlievert, personal communication). Agr and srrAB/srhRS thus represent a mutual cross-inhibition circuit that would necessarily have to respond to extrinsic regulatory inputs. SrrAB/srhRS also regulates many genes involved in energy metabolism and evidently regulates energy transduction under anaerobic conditions (Throup et al., 2001). It may be activated by menaquinone or a derivative, one of the intermediates in the oxidative respiratory pathway. Thus, this TCS may connect the agr signalling pathway with the overall energy metabolism of the cell. The report by Yarwood et al. (2001) is problematic as strain RN4220 and several of its derivatives were used exclusively. RN4220, derived from RN450 by nitrosoguanidine mutagenesis, contains an indeterminate number of uncharacterized mutations, including one that affects agr function, and is unsuitable for studies of regulation. The Throup et al. (2001) report used a clinical strain, WCUH29, but no data on virulence factors were presented. It appears that each of these three TCSs exerts its effects on the virulon and other accessory genes largely, although not entirely, through its interaction with agr. Thus, saeRS and agr appear to be mutually upregulatory; srrAB and agr are reported to be mutually downregulatory, and arlRS and agr seem to constitute an autorepression circuit. These different interactions, probably indirect, presumably represent a central regulatory logic, but one whose biology remains to be determined.

Alternative sigma factors

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

A second major mechanism of response to environmental stimuli is via alternative sigma factors, which are generally activated directly within the cell rather than through signal transduction. S. aureus possesses only one of these, a homologue of B. subtilisσB. σB is activated by environmental stress and energy depletion (reduced ATP/ADP ratio), as well as by environmental stimuli such as ethanol (Chan and Foster, 1998a) and salicylic acid (Bayer et al., 2000), and its activity is regulated by a complex post-translational pathway consisting of rsbU, V and W (Scott et al., 1999). σB is usually bound by RsbW, an antisigma factor, that phosphorylates RsbV, an anti-antisigma factor. Under conditions of environmental stress, RsbV∼P is dephosphorylated by either of two phosphatases, RsbU or RsbP, and then binds RsbW, releasing and activating σB. σB recognizes a unique promoter (GTTT(N14−17)GGGTAT), which has been identified for 23 different S. aureus genes (Gertz et al., 2000), including one of the three sarA and one of the three sarS promoters, plus genes encoding transport functions and others involved in generating NADH2. σB is also required for certain genes that lack a σB promoter; these are presumably regulated by σB-dependent transcription factors.

In S. aureus, σB feeds into the global regulatory network governing the expression of accessory genes, acting mostly through other regulatory genes and transcription factors, but also acting directly on those few that have σB-dependent promoters. Thus, it has reciprocal activities, upregulating some exoprotein genes at a very early stage of growth, such as coa and fnbB, of which the former has a σB-dependent promoter (Nicholas et al., 1999), and downregulating others at the end of the exponential phase in vitro. As many of the latter are involved in virulence, σB seems to be antagonistic to agr. One might therefore predict that inactivation of σB would increase virulence. However, virulence cannot be regarded as a simple function of the amounts of putative virulence factors that are produced by the organism in vitro and, in fact, mutation of sigB had little or no effect on the virulence of one clinical strain (Nicholas et al., 1999), although it greatly de-creased the biofilm-forming ability of another (Rachid et al., 2000).

Strains of the 8325 lineage do not produce the characteristic S. aureus golden pigment, a σB-dependent function, owing to their rsbU deletion. However, these strains are not totally deficient in σB function, as pigment synthesis can be induced by subinhibitory clindamycin (S. Herbert and R. P. Novick, unpublished data). We suggest that this could involve σB activation via RsbP. A small fraction of S. aureus clinical isolates are non-pigmented and overproduce various exoproteins. These are likely to be σB defective, supporting the idea that σB may not be required for pathogenesis. Derivatives of 8325 strains in which rsbU has been repaired (Bischoff et al., 2001; Horsburgh et al., 2002) show important differences in the overall biology of the organism, including a reduction in the lag phase of growth, an increase in overall growth yield and in starvation survival (Horsburgh et al., 2002), as well as in the expression of both regulatory and exoprotein genes (see below).

At this point, it is safe to say that σB is involved in the signaling pathways that regulate virulence and other accessory gene functions in staphylococci. However, its precise role in the overall regulatory network remains to be determined, as does the pathway leading to σB activation, especially by small molecule stimuli. Additionally, it is hypothesized that σB is present during the stationary-to-exponential phase transition (see below).

Transcription factors

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

In general, transmission of environmental signals recognized by transmembrane and intracellular receptors to effector (or target) genes involves intracellular transcription factors. Indeed, the response regulator component of a signalling module is typically a pleiotropic transcription factor; however, this is certainly not the case for the staphylococcal agr system, which uses a regulatory RNA as its intracellular effector (there is presently no information for the other staphylococcal TCSs). We note that there is no consensus sequence that could represent the regulatory target for agr among the target genes; consistent with this idea, it has long been our view that RNA III would have to act via a multiplicity of intermediary proteins (Novick et al., 1993). This possibility was tested by studying the effects of blocking protein synthesis on target gene transcription. An agr-null strain was incubated with erythromycin at an inhibitory concentration, and hla and spa transcripts were analysed (Novick et al., 1995). Inhibition by erythromycin was accompanied by a dramatic increase in hla transcription and an equally dramatic decrease in spa, mimicking the effects of RNA III and leading to the suggestion that hla expression is controlled by a labile repressor and spa by a labile activator. The hypothesis of intermediary proteins was subsequently confirmed by the isolation of mutations with phenotypes similar to the effects of erythromycin. In the absence of RNA III, hla is upregulated by mutation in rot or sarT (McNamara et al., 2000; Schmidt et al., 2001), and spa is downregulated by mutations in sarS (Tegmark et al., 2000) or rot (Said-Salim et al., 2003).

SarS, T and Rot are SarA homologues that are members of a vast and growing family of winged helix–turn–helix transcription factors. This family includes bacterial regulators of efflux pumps (Alekshun et al., 2001;Godsey et al., 2001), phage maturation proteins (de Beer et al., 2002), the arginine repressor family (Holtham et al., 1999), response-regulator proteins (Martinez-Hackert and Stock, 1997), etc.

SarA and its homologues

SarA and its homologues (Tegmark et al., 2000; Manna and Cheung, 2001) affect a wide variety of genes that may or may not encode virulence or other accessory functions (Dunman et al., 2001). The DNA-binding segments of these proteins are closely conserved, all containing the motif KXRXXXDER, whereas other parts of the proteins are less so. The first of these factors to be discovered was SarA (Cheung et al., 1996), a 14.7 kDa DNA-binding protein, distantly related to VirF of Shigella flexneri. SarA binds as a dimer, whereas at least three of its homologues, SarS, SarU and SarY, appear to be the result of duplications and therefore intrinsically dimeric. Given the degree of structural and sequence similarity among the members of this family (Cheung and Zhang, 2002), the possibility of heterodimeric combinations has been suggested (Tegmark et al., 2000). The Sar family has been recently and ably reviewed by Arvidson and Tegmark (2001) and Cheung and Zhang (2002).

The sarA locus is transcribed from three promoters, sarP1, sarP2 and sarP3 (see Fig. 5A), of which the last is a σB promoter. The three promoters are active at different times during the in vitro growth cycle, and there are inconsistent reports on whether there is significant temporal variation in the resulting level of SarA (Manna et al., 1998; Blevins et al., 1999; 2002). There are two short reading frames upstream of the sarA coding sequence that appear to have a stimulatory effect on sarA function, either as translation products or, more likely, through their effects on sarA transcription or SarA translation; these are not transcribed by promoter, sarP1 (Cheung et al., 1997). SarA transcription from the sarP2 promoter is inhibited by another homologue, SarR (Manna and Cheung, 2001), the role of which in the overall pattern of accessory gene expression has yet to be defined.

image

Figure 5. SarA and sarS maps. Adapted from the reports by Manna et al. (1998) and Tegmark et al. (2000) [note that sarH1 (Tegmark et al., 2000) has been redesignated sarS (Arvidson and Tegmark, 2001)]. Transcripts are indicated by wavy lines, terminators by stem–loops. P, promoter; T, terminator; LP, leader peptide.

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SarA binds as a dimer to AT-rich sequences distributed widely in the AT-rich staphylococcal genome, including the 5′ regions of several genes. Rechtin et al. (1999) have described multiple SarA binding sites in the intergenic region between agr promoters P2 and P3, suggesting the possibility of co-operativity, whereas Cheung and coworkers have identified only one site (Morfeldt et al., 1996a; Chien and Cheung, 1998). Indeed, there is no consensus on the SarA binding site. According to Wisell (2000), ‘A comparison of all sequences reported to bind SarA revealed no consensus binding sequence. However a high AT:GC ratio seemed to correlate with high affinity binding of SarA. Thus in some respects, SarA appears to belong to the group of regulatory proteins that bind DNA with limited sequence specificity (e.g. H-NS and HU)’.

In a few cases, SarA binding has been shown to affect the expression of the respective genes. SarA has been reported by Cheung et al. (1997) to upregulate the transcription of several genes, including the two agr operons and fnbA, and to downregulate several others, especially including ssp (Chan and Foster, 1998b; Chien et al., 1999) and cna (Blevins et al., 1999). The reported stimulatory effect of SarA on agr expression has been confirmed for organisms grown on an agar surface or in broth under suboptimal aeration, but not in optimally aerated broth cultures (Lindsay and Foster, 1999; Blevins et al., 2002). SarA is required for saeRS and arlRS activation, possibly via its effects on agr transcription. SarA downregulates the transcription of several of the exoprotein genes, independently of its effects on RNA III transcription, including ssp, aur, scp, cna and spa. This effect is consistent with SarA binding in the 5′ regions of these genes, deletion of which results in an increase in expression. Activation by SarA of fnbA, tst and hla has also been reported (Cheung et al., 1999; Wolz et al., 2000). There is, however, less than universal agreement on the upregulatory effects of SarA. Thus, the reduced fibronectin-binding activity observed with sarA mutants may not be at the level of transcription and may, instead, be a consequence of the increased proteolytic activity seen with these mutants (Blevins et al., 2002). Additionally, in our hands, SarA had no agr-independent effect on upregulation of tst (N. Vojtov and R. P. Novick, unpublished data); further, deletion of the SarA binding sites directly upstream of the hla, fnbA and agr P2 promoters completely eliminated the activity of each of these promoters, and it is likely that these deletions inactivated the respective promoters rather than removing regulatory targets. Moreover, in vitro analyses have revealed only inhibition by SarA (Chakrabarti and Misra, 2000), consistent with the suggestion by Arvidson and Tegmark (2001) that SarA is not ordinarily a transcriptional activator, and with the conclusion that SarA upregulates hla by downregulating the hla repressor, SarT (Schmidt et al., 2001).

Superantigens

Remarkably, at least two of the major staphylococcal SAg toxins, TSST-1 and SEB, are themselves transcription factors, acting as global repressors of most exoprotein genes at the level of transcription (see Table 1), and are also autorepressors (Vojtov et al., 2002). These two proteins, as well as the other staphylococcal and streptococcal SAgs, are structurally very closely related, as shown by X-ray crystallography (Ohlendorf et al., 1998). However, there is no striking sequence similarity corresponding to the inhibitory regions of the two proteins. The data establish clearly that the protein itself, rather than the mRNA or the DNA, is the inhibitor, and that a region ending about the middle of the C-terminal half of the protein is necessary (Vojtov et al., 2002). As the purified toxin has no effect when added to a culture, and a deletion derivative lacking the signal peptide retains its inhibitory activity (N. Vojtov, H. Ross and R. P. Novick, unpublished data), it is clear that an intracellular form, most likely the precursor, is the effector. Although the target of inhibition or autorepression is always the promoter, it appears that neither protein binds DNA directly, presumably acting through an intermediate transcription factor. As the inhibitory effects are manifest as early as one can detect the toxins, the putative intermediary must also be present at this early time. It remains unclear precisely how this regulatory paradigm fits into the overall regulatory network; however, it is evidently of major clinical importance: in post-surgical TSS, resulting from a contaminated wound, the infection is often very difficult to detect as, unlike the typical staphylococcal lesion, the wound is neither purulent nor inflamed. It is well-known that TSST-1 has major effects on the production of cytokines and very probably influences the inflammatory response by this means. It is additionally hypothesized that one or more of the exoproteins, possibly lipase, the synthesis of which is inhibited by the SAg, is responsible for attracting polymorphonuclear leucocytes and stimulating the inflammatory re-sponse. It is predicted that other variable genes encoding toxins that cause toxinoses are likely to act in this manner.

TRAP

TRAP (‘target of RAP’) is a putative cytoplasmic transcription factor that is stably histidine phosphorylated in a growth-dependent manner and is reported to be partially required for agr activation (Balaban et al., 2001). Its phosphorylation is reported to be stimulated by L2/RAP and inhibited by AIP-containing culture ultrafiltrates (Balaban et al., 2001). In a TRAP-defective mutant of a standard agr+ strain, RNA III production is delayed and decreased (Balaban et al., 2001), consistent with a role for this protein in the agr signalling pathway. TRAP was reported to be the only phosphorylatable protein in the staphylococcal cytoplasm – an implausible result which has, incidentally, been found to be incorrect (G. Ji et al., personal communication). According to Balaban et al. (2000; 2001), TRAP phosphorylation occurred in the agr-null mutant, apparently to the same extent as in the agr+ (Balaban et al., 2001) and was inhibited (in the agr+) by the cognate, agr-activating AIP (Balaban et al., 2001). It has been proposed that TRAP represents a regulatory intermediate between L2/RAP and agr-RNA III, leading to the suggestion by Garvis et al. (2002) that SvrA could be the signal receptor for L2/RAP. Aside from the irreproducibility of any role for L2/RAP in agr activation, this seems unlikely as agr is completely inactive in an svrA mutant but has considerable activity in a TRAP mutant. Moreover, if TRAP were a component of a second signalling pathway, it would be activatable by the signalling molecule in the agr-null background. This has not been tested. Given the above considerations, one possible interpretation of the Balaban et al. (2001) results is that TRAP∼P is a negative regulator in the agr pathway and that it is the non-phosphorylated form that has upregulatory activity. As the AIP has no effect on rna III transcription in the absence of the agr signalling pathway (Lyon et al., 2002a), TRAP would have to be acting within that pathway rather than by a second signalling module. Further studies of TRAP will be required to clarify its role in the agr activation pathway.

Regulatory organization

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

Temporal programme

Much of the facultative gene expression system, especially including the virulon, is temporally organized so that the component genes must contain regulatory sequences that are activated combinatorially in a time-dependent manner by different incoming signals acting through intracellular response elements. As suggested by the model in Fig. 6A, the entire accessory gene regulatory network must also be coupled to the overall energy metabolism of the cell, and it has been suggested that there must be a key coupling parameter, such as the levels of nucleotide polyphosphates or of other energy-transducing cofactors such as NADH2 (R. Proctor, personal communication). Key enzymes of intermediary metabolism, such as aconitase (Somerville et al., 2002a), could be involved in this coupling, possibly acting through one or more TCS, such as srr (srh). On the basis of results obtained with in vitro cultures, it appears that the surface proteins are probably required earlier in the course of an infection than the secreted enzymes, immunotoxins and cytotoxins and the above-mentioned intracellular metabolic enzymes. This sequential activation seems to be, at least in part, a function of population density. Starting with stationary phase, there would seem to be three key transition points in the in vitro growth cycle, possibly occurring in response to intracellular signals, such as GTP levels. First is the transition to exponential phase, which involves not only the revival of biosynthetic and other metabolic pathways required for growth and cell division, but also the synthesis of some surface proteins, coagulase and possibly other accessory proteins. The synthesis of these is probably initiated during the transition from stationary phase to exponential phase and may come under the general metabolic programme governing this transition. The nature of the signals (?) acting at this stage represents a key area for study. Other surface protein genes are switched on shortly after the onset of exponential growth and, as typified by spa, are switched off shortly thereafter, concomitantly with the appearance of agr-RNA III (Vandenesch et al., 1991). This clear reciprocity, however, is not seen with all strains and under all conditions (Tegmark et al., 2000), and may be related to σB activity (S. Herbert and R. P. Novick, unpublished data) or to growth conditions and media. The agr AIP reaches its threshold around mid-exponential phase, activating agr expression. In 8325 derivatives, however, certain exoprotein genes, such as coa, are sharply downregulated well before the appearance of RNA III, suggesting that some other inhibitory signal is responsible. The second transition, between the exponential and post-exponential phases (possibly a consequence of decreasing availability of oxygen owing to increasing population density), is, in most strains, accompanied by upregulation of the genes encoding secreted proteins. Agr, which, in 8325, is activated two or more hours earlier, sets the level of expression of most of these proteins, but not the timing (Vandenesch et al., 1991); in fact, upregulation of these genes occurs at the onset of the post-exponential phase, regardless of when, or even whether, RNA III transcription is activated (unpublished data). This is consistent with the results of temporal activation studies, in which activation of hla transcription may occur as much as 6 h after RNA III (cloned to the β-lactamase promoter and induced) reaches its maximum level (Vandenesch et al., 1991). In a sarA mutant, RNA III transcription is delayed by an hour and is closely co-ordinated with the onset of hla transcription. This effect can be attributed to SarS (also known as SarH1) because, in the double SarA/SarS mutant, RNA III is not delayed and there is again a timing differential (Tegmark et al., 2000). In some strains, such as those of agr group IV, hla and other exoprotein genes are upregulated earlier, concomitantly with RNA III synthesis (Jarraud et al., 2000), suggesting that the exponential to post-exponential phase transition may not be a critical regulatory point for hla and other exoprotein genes in these strains. A further complication is the apparent post-exponential upregulation of DNA gyrase by agr (≈ six-fold; Dunman et al., 2001), raising the possibility that agr regulation could involve changes in superhelix density, which are well known to occur during post-exponential growth and are well known to affect a variety of promoters (although there are very little data on this in staphylococci).

imageimage

Figure 6. A. Global regulation of the staphylococcal virulon – black-box model. See text for description.

B. Regulatory interactions involving SarA and its homologues. Arrows represent upregulation, bars represent downregulation. Blue lines represent translation; black lines represent interactions that are probably, but not always certainly, transcriptional. The interactions illustrated are based on reviews by Arvidson and Tegmark (2001) and Cheung and Zhang (2002) and on recent papers by Manna and Cheung (2003) and Said-Salim et al. (2003). Although the abbreviations are mostly in italics, on the assumption that the interactions are likely to be at the transcriptional level, there is actually very little evidence to indicate whether they are direct or indirect or at what level they occur. Question marks represent the most speculative. σB is shown entering the system via sarS and sarA, which have σB-dependent promoters and are likely to represent important intermediates in the pathways by which environmental signals are handled.

The third transition, from post-exponential to stationary phase, is accompanied by a major metabolic rearrangement that prepares the cell for long-term survival by shutting down most housekeeping and facultative genes and by activating genes required for long-term survival (Watson et al., 1998), by mechanisms that are not well understood in Gram-positive bacteria.

The environmental factors that affect expression of various components of the virulon would exert their effects whenever they are encountered. Certain of these (pH, O2 tension, CO2 concentration) would typically vary during growth in laboratory cultures and would be expected to have increased importance late in growth. Others would be encountered only under special circumstances and are not regarded as elements of the temporal programme.

Overall regulatory strategy – a black-box model (Fig. 6A)

Finally, we have attempted to conceptualize the overall strategy used for the regulation of accessory genes as a grand metabolic scheme, viewed as a condensation of the temporal programme just described. In this scheme, there are major unknown pathways, indicated by black boxes (BB) and black arrows, and much of the information implied by the coloured arrows is also rather sketchy at best. The largest segment of the regulatory system is the transcription factors, including the SarA homologues. These are viewed collectively here – a preliminary attempt to detail some of their individual activities is shown in Fig. 6B.

The overall metabolic machinery of the staphylococcal cell is impacted by energy resources dependent on oxygenation and nutrition. An unknown energy signal, possibly nucleotide polyphosphate (NPP) or NADH2 level, is transmitted to the agr locus through a black box (BB-1), which up- or downregulates agr according to energy resources, whereas housekeeping functions are fuelled preferentially by metabolic energy and nutritional resources. SvrA activates agr by an unknown mechanism. Agr autoactivates by means of the AIP and interacts with at least three other TCSs, sae, arl and srr(srh), which may up- or downregulate it, establishing one or more feedback loops. These interactions may or may not occur at the level of RNA III. RNA III, in turn, feeds into BB-2, generating signals that regulate transcription factors, especially the SarA homologues; the other known TCSs also transmit signals through BB-2 either to the transcription factors or directly to the target genes. Environmental inputs signal either through BB-5, which activates (or deactivates) σB, again by an unknown mechanism. σB, in turn, acts on those transcription factors and other genes that have σB promoters. Other environmental inputs signal through BB-3 to the transcription factors; these may or may not involve σB. The transcription factors, viewed as a pool, receive inputs from various sources, determining how they will interact with the target genes (represented here by the virulon and the responses to stresses such as heat, cold, etc.). Transcription factors also feed back to agr, establishing additional feedback loops and probably interact similarly with the other TCSs. Note that Rot is given a special role, possibly distinct from the other transcription factors, and may be the only one that interacts directly with RNA III. Finally, at least two of the SAgs, signalling through BB-4, transmit information through the transcription factors for downregulation of the various exoprotein genes.

Transcription factors

A blow-up of the transcription factor region in Fig. 6A is shown in Fig. 6B, which represents an example of the type of regulatory circuitry in which SarA and its homologues are involved. Intermediary transcription factors have been shown to affect the transcription of accessory genes, other SarA homologues and RNA III (Arvidson and Tegmark, 2001; Schmidt et al., 2001; Manna and Cheung, 2003), generating complex activation cascades and feedback loops and defining subsets of accessory genes. Presumably, RNA III interacts directly or indirectly with these factors, by regulating either their synthesis or their action. For example, a recent microarray analysis has revealed that Rot has broad regulatory effects on genes belonging to the agr regulon and generally acts counter to agr, downregulating genes encoding secreted proteins and upregulating surface protein genes (Said-Salim et al., 2003) (see Table 1). In order to account for the observed effects of agr, one must postulate that agr downregulates Rot. As rot is constitutively transcribed (McNamara et al., 2000), it is likely that any such downregulation would be post-transcriptional. In the case of SarS (see Fig. 6B), it is known that RNA III blocks expression (Tegmark et al., 2000), perhaps through Rot. It has also been observed that SarT downregulates agr (Schmidt et al., 2001), apparently acting via the newly described SarU (Manna and Cheung, 2003), an agr upregulator. The downregulation of sarT by agr thus generates a negative feedback loop as shown in Fig. 6B. Additional data such as those obtained by transcript profiling (Dunman et al., 2001; Said-Salim et al., 2003) will fill out this circuitry, perhaps defining regulatory subsets of target genes that could have biological/clinical relevance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Two-component systems (TCSs)
  5. Alternative sigma factors
  6. Transcription factors
  7. Regulatory organization
  8. Supplementary material
  9. References
  10. Supporting Information

Appendix A1. ONLINE SUPPLEMENT: 1. Lineups of AgrB and C sequences. 2. AIP structure-function analysis. 3. Uncertainties in Fournier, et al. (2000).

Fig. S1. AgrB. See Fig. 3 for abbreviations. Roman numerals refer to agr groups; for species no group is specified, only one has thus far been identified.

Fig. S2. N-terminal (receptor) domain of AgrC. See Fig. 3 for abbreviations. Roman numerals refer to agr groups; for species no group is specified, only one has thus far been identified.

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MMI_3526_sm_figS2.pdf3858KSupporting info item

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