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Alternative Sigma Factors and Their Roles in Bacterial Virulence

Department of Food Science, Cornell University, Stocking Hall, Ithaca, New York 14853

SUMMARY

INTRODUCTION

STRESS RESPONSE ALTERNATIVE SIGMA FACTORS

Sigma B

Pathogenic Bacillus species.

Staphylococcus species.

Listeria monocytogenes.

Mycobacterium tuberculosis {sigma}B and {sigma}F.

Sigma S (RpoS)

Escherichia coli.

Salmonella species.

Pseudomonas aeruginosa.

SIGMA 28 SUBFAMILY

FliA

Salmonella enterica serovar Typhimurium.

Other species.

ECF SIGMA FACTORS

Sigma E (RpoE)

PvdS and FpvI

Mycobacterial ECF Sigma Factors

HrpL

SIGMA 54

Sigma N

Pseudomonas aeruginosa.

Pseudomonas syringae.

Vibrio species.

Other species.

CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Sigma factors are a class of proteins constituting essential dissociable subunits of prokaryotic RNA polymerase. The association of appropriate alternative sigma factors with core RNA polymerase provides a mechanism for cellular responses mediated through redirection of transcription initiation. Sigma factors provide promoter recognition specificity to the polymerase and contribute to DNA strand separation; they then dissociate from RNA polymerase core enzyme following transcription initiation (16). As the regulon of a single sigma factor can be comprised of hundreds of genes, sigma factors provide effective mechanisms for simultaneously regulating large numbers of prokaryotic genes. In some cases, the genes comprising a sigma factor regulon have a clearly defined primary function (e.g., genes regulated by the sporulation sigma factors in Bacillus subtilis [171]); in others, the genes comprising a regulon contribute to multiple functions (e.g., the stationary-phase and general stress response genes regulated by ^B in Listeria monocytogenes [100]). One newly emerging field is identification of the specific roles of alternative sigma factors in regulating expression of virulence genes and virulence-associated genes in bacterial pathogens.

Virulence and virulence-associated genes are those that contribute to at least one aspect of bacterial disease transmission and infection processes. Specifically, virulence genes encode proteins whose functions are essential for the bacterium to effectively establish an infection in a host organism. Examples of virulence genes are L. monocytogenes inlA, which encodes the internalin-A protein important for invasion of nonprofessional phagocytes (129), and the spv gene cluster of Salmonella enterica, which allows for bacterial growth inside macrophages (128). In contrast, virulence-associated genes can contribute to bacterial survival in the environment (e.g., the ica operon of Staphylococcus aureus, which produces an adhesin important for biofilm formation on plastic surfaces such as those on indwelling medical devices [141]) or to survival in the host (such as bsh of L. monocytogenes, encoding bile salt hydrolase, which enhances bacterial survival in the intestinal environment prior to intracellular infection [48]). Therefore, activation of virulence-associated genes may enhance the capacity of the bacterium to spread to new individuals or to survive passage through a host organism. As alternative sigma factors have been shown to regulate expression of both virulence and virulence-associated genes, these sigma factors can contribute both directly and indirectly to bacterial virulence.

Virulence factor expression appears to be tightly regulated in bacterial pathogens. In some cases, pathogens have a "master regulator" of virulence gene expression, such as the positive regulatory factor A (PrfA) in L. monocytogenes. PrfA, a transcriptional activator, is required for expression of the majority of recognized L. monocytogenes virulence genes. Alternative sigma factors often function to regulate expression of virulence and virulence-associated genes in response to particular stimuli. Alternative sigma factors may regulate a small number of genes, each of which may be critical to infection (e.g., PvdS of Pseudomonas aeruginosa [discussed below] [147]), or they may regulate functions that contribute to virulence but also have additional physiological roles in the cell. For example, Salmonella enterica serovar Typhimurium ^E regulates genes that provide resistance to oxidative stress, which also aids bacterial survival in macrophages (82). This review focuses on both direct and indirect roles of selected alternative sigma factors in regulating virulence of bacterial pathogens of plants and animals.

Sigma factors can be classified into two structurally unrelated families: the ⁷⁰ and the ⁵⁴ families. Table 1 lists sigma factors in both the ⁷⁰ and the ⁵⁴ families that are currently recognized as contributing, either directly or indirectly, to bacterial virulence. For several alternative sigma factors, nomenclature in the literature has been inconsistent. In this document, in general, we refer to sigma factor families by number (e.g., the ⁵⁴ family) and to specific sigma factors by letter (e.g., P. aeruginosa ^N). For certain sigma factors, we use the predominant designation from the literature instead (e.g., FliA).

Although no sequence conservation exists between ⁵⁴ and ⁷⁰-like family members, both types bind to core RNA polymerase. However, the holoenzyme formed with ⁵⁴ sigma factors has different properties than the ⁷⁰ holoenzyme. While the C terminus (region III) of ⁵⁴ enables DNA binding, all ⁵⁴ species require a separate activator protein along with the core RNA polymerase (RNAP) to form an open promoter complex. The ⁵⁴ N terminus, which inhibits isomerization in the absence of the appropriate activator, stimulates initiation upon activation (19). Further, promoter structures recognized by ⁵⁴-RNAP differ from those recognized by ⁷⁰-RNAP. ⁵⁴ promoters are highly conserved, short sequences that are located at positions –24 and –12 upstream of the transcription initiation site, whereas ⁷⁰ promoter sites are typically located at –35 and –10 upstream. ⁵⁴ promoters, which are called –24/–12 promoters, are almost completely invariant at the –24/–12 positions (GG and GC, respectively) and in their spacing in both gram-negative and gram-positive bacteria. For reviews on the structure-function relationships of ⁵⁴, see references 19 and 142.

We present several examples of alternative sigma factors that have been shown to contribute to virulence in at least one organism. The text is organized by sigma factor to include the three subfamilies (stress response, ²⁸, and ECF) within the ⁷⁰ family, as well as those within the ⁵⁴ family. For each sigma factor, when applicable, examples will be drawn from multiple bacterial species.

STRESS RESPONSE ALTERNATIVE SIGMA FACTORS

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The ability to reproduce, or simply survive, under a wide variety of environmental conditions contributes to a microbial pathogen's potential for transmission by various routes. For example, to establish a food-borne infection in a human host, a bacterium first must survive transit in a contaminated food. Following ingestion, the bacterium then must survive exposure to rapid and dramatic changes in environmental conditions, including the acidic pH within the stomach, followed by vastly differing conditions during intestinal passage and/or infection (e.g., exposure to bile, vacuolar stresses, etc.) Survival under these extreme and rapidly changing conditions requires timely and appropriate alterations in gene expression and protein activity that occur in a bacterial cell in response to stimuli signaling these new environmental conditions. At the transcriptional level, these alterations are often controlled by changes in associations between different alternative sigma factors and core RNA polymerase, which essentially reprogram promoter recognition specificities of the enzyme to allow expression of new sets of target genes.

The general stress-responsive alternative sigma factors ^S (RpoS) and ^B transcribe genes contributing to bacterial survival under conditions of environmental stress in gram-negative and in gram-positive bacteria, respectively (Table 2). ^S was identified in both Escherichia coli and S. enterica serovar Typhimurium as an alternative sigma factor that activates the expression of numerous genes required to maintain cell viability during stationary phase (51, 119). ^S also plays a key role in protecting E. coli and S. enterica serovar Typhimurium from different environmental stress conditions, including starvation, hyperosmolarity, oxidative damage, and reduced pH (51, 119). Since its initial discovery, the presence of ^S and its role in the stress response has been confirmed in many gram-negative bacterial species, including P. aeruginosa, Borrelia burgdorferi, and Vibrio cholerae (49, 93, 227). Through enhancing environmental survival, as well as by directly activating virulence genes, ^B and ^S have both direct and indirect roles in bacterial pathogenesis.

View this table: [in this window] [in a new window]   TABLE 2. Virulence genes and virulence-associated genes regulated by stress response sigma factors B and S and phenotypes of sigma factor null mutants in selected bacterial species

View larger version (19K): [in this window] [in a new window]   FIG. 1. (A) sigB operon structures in various gram-positive bacteria. Promoter sites are marked by arrows. PA promoters are transcribed with RNAP-A, and PB promoters are transcribed with RNAP-B. (B) Posttranslational regulation of B activity via a partner-switching mechanism. Proteins shown are encoded by the B. subtilis and L. monocytogenes sigB operons depicted in panel A. Arrows indicate activation of protein activity, and T-bars indicate repression of protein activity. "P" represents a phosphate group. The proteins indicated by dark gray are encoded in all bacteria listed in panel A, whereas RsbU (light gray) is absent in the pathogenic Bacillus spp. and the proteins indicated by white are encoded only in L. monocytogenes and B. subtilis.

Although all seven Rsb proteins identified in B. subtilis and L. monocytogenes are not conserved among all bacterial species bearing ^B, two key proteins (RsbV and RsbW) are conserved among all species examined to date and thus appear to be minimally essential for regulating ^B activity (54). Specifically, in log phase, nonstressed B. subtilis cells, ^B is inactivated by its association with the anti-^B protein, RsbW (i.e., the "anti-sigma factor"). In stressed cells, however, the unphosphorylated form of the anti-^B antagonist protein, RsbV, (i.e., the "anti-anti-sigma factor") competes for binding to RsbW. As the relative concentration of the RsbW-RsbV complex increases, the concentration of free ^B also increases, thus allowing ^B to bind to core RNA polymerase (47). In B. subtilis, both environmental and energy stress signals induce dephosphorylation of RsbV. Environmental stress signals specifically activate the B. subtilis RsbU serine phosphatase through involvement of RsbR, RsbS, RsbT, and RsbX (211, 212, 223). In addition to its role in partner-switching regulation under environmental stress, B. subtilis RsbX also functions as a feedback regulator for ^B activity (Fig. 1B) (12). While both energy and environmental stresses have been shown to activate L. monocytogenes ^B (25), specific interactions among the Rsb proteins have not yet been investigated. To date, specific activation mechanisms have been most extensively reported for B. subtilis ^B.

Pathogenic Bacillus species. At least two pathogenic species of Bacillus encode ^B (55, 208). In Bacillus anthracis, only ^B, RsbV, and RsbW are encoded in the sigB operon (Fig. 1A). A third rsb gene, rsbY, encodes a protein with low similarity to B. subtilis RsbP. rsbY is located in close proximity to, but not within, the B. anthracis sigB operon (55). As in B. subtilis and L. monocytogenes, the sigB operon is autoregulated by ^B and is induced by heat shock and entry into stationary phase (55). A B. anthracis sigB mutant strain is virulence attenuated, producing less than half the mortality of the parent strain in the mouse model of anthrax (55). More-detailed studies are needed to determine if virulence attenuation is due to direct or indirect effects.

The organization of the Bacillus cereus sigB operon is identical to that of B. anthracis sigB, and likewise, the sigB operon is autoregulated by ^B and is induced by heat shock and entry into stationary phase (208). Through use of a combination of two-dimensional gels and Northern hybridizations, 15 B. cereus genes and proteins were determined to be ^B dependent, including RsbV and the KatE catalase (209). The activity of currently recognized B. cereus virulence factors, including protease, lecithinase, and hemolytic activity, as well as production of nonhemolytic enterotoxin, was not affected by disruption of sigB (208), suggesting that ^B does not directly contribute to B. cereus virulence.

Staphylococcus species. Staphylococcus aureus was the first pathogenic bacterium in which sigB was identified. (115, 224) (Fig. 1A). In S. aureus, the sigB operon is comprised of four genes, which are homologous to B. subtilis rsbU, rsbV, rsbW, and sigB. As in B. subtilis, all genes in the operon are expressed during exponential growth, presumably from the ^A-dependent promoter. The internal P_B promoter was confirmed as ^B dependent through in vitro transcription analyses (44). Transcriptional regulation of the S. aureus sigB operon is complex, generating multiple transcripts that appear to include a bicistronic sigB-rsbW transcript as well as a sigB monocistronic transcript. In support of an autoregulatory role for S. aureus ^B under conditions of environmental stress, an rsbV-W-sigB transcript was induced following exposure of cells to either 4% ethanol or a 48°C heat shock (115).

S. aureus ^B activity is regulated posttranslationally by Rsb proteins. The open reading frame immediately upstream of S. aureus sigB encodes the anti-sigma factor, RsbW (146). As in B. subtilis, S. aureus ^B also is activated via an RsbU pathway (62). An 11-bp deletion in rsbU in the NCTC8325 strain generated some phenotypic characteristics similar to those of a sigB strain, (e.g., decreased H₂O₂ resistance [114]). Giachino et al. (62) confirmed that NCTC8325 does not produce a functional RsbU and that complementation of this strain with a complete rsbU allele restored phenotypes to those of the rsbU⁺ Newman wild-type strain. However, some NCTC8325 phenotypes were identical to those of other rsbU⁺ strains (e.g., lipase production [see below]), suggesting the existence of multiple S.aureus ^B activation pathways, including at least one that is RsbU independent (114). As with RsbU, loss of RsbV results in a dramatic decrease, although not complete loss, of S. aureus ^B activity (165).

Through application of full-genome microarray screens for ^B-dependent genes in three S. aureus strains, as many as 251 genes have been identified as being ^B regulated (14), including several genes encoding proteins involved in synthesis of capsular polysaccharides. A number of adhesins, which are involved in Staphylococcus virulence, are upregulated by ^B. Multiple genes encoding exoenzymes and toxins (e.g., hla and nuc) are downregulated as ^B is activated (14), which may reflect ^B's role in controlling expression of S. aureus virulence gene regulators. For example, a number of the exoenzymes and toxins that are downregulated by ^B depend on an effector RNA produced from the agr locus (RNAIII) for heightened expression (204). RNAIII levels are reduced when ^B activity increases. The mechanism responsible for this phenomenon remains unclear (13, 14) but may involve the regulator SarA (8, 44, 79).

Multiple groups have described S. aureus sigB mutants as having pigment loss and decreased peroxide resistance, but higher -hemolysin, coagulase, clumping factor, and lipase activity, compared to the wild type (27, 62, 79, 114, 152). These characteristics have all been associated with S. aureus virulence (61, 67, 98, 131, 150, 184). It is likely that optimal levels of virulence factor expression and activity are required for efficient S. aureus infection and that too much or too little activity or expression at the wrong time is detrimental for the infection process. These hypotheses remain to be rigorously tested. In various animal models, wild-type S. aureus and an otherwise isogenic sigB strain showed no difference in virulence (22, 152). In additional, conflicting studies, Horsburgh et al. (79) found no difference in virulence between rsbU⁺ and rsbU mutant strains in a murine skin abscess model, while Jonsson et al. (92) showed that both rsbU and sigB mutant strains displayed decreased virulence phenotypes compared to the wild-type strain in murine septic arthritis, including reduced mutant persistence in kidneys and reduced mouse mortality, weight loss, arthritis, and interleukin-6 production.

The contradictory evidence surrounding the role of ^B in S. aureus virulence suggests that ^B contributions to virulence may be indirect or not detectable in some model systems. For example, ^B may contribute indirectly to S. aureus virulence through regulation of biofilm formation. Biofilm formation can be a prerequisite for establishing infection by staphylococci, and ^B has been shown to enhance microcolony and biofilm formation in Staphylococcus species (5, 177). Two studies have shown induction of S. aureus biofilm formation in a ^B-dependent fashion (5, 177), although another showed that a sigB strain formed biofilms and produced PIA, the polysaccharide adhesin encoded by the ica operon, equally as well as the wild type (207). S. aureus ^B contributions to biofilm formation likely occur through ^B-dependent transcription of the ica operon, which encodes essential elements of biofilm biosynthesis (177).

Staphylococcus epidermidis also encodes ^B. The sigB operon of S. epidermidis is similar to that of S. aureus (Fig. 1A); however, ^B serves different functions in the two species. Processing of lipase, a virulence factor, is dependent on ^B in S. epidermidis (102), while in S. aureus, lipase production is higher in a sigB mutant than in the wild-type strain (114). Multiple studies of ^B and S. epidermidis virulence suggest that ^B's effects are mediated primarily through its influence on biofilm formation in this organism (33, 106, 107). Stress induction of ^B in S. epidermidis increases biofilm formation and synthesis of the adhesin PIA, but an rsbU mutant does not form biofilms or produce PIA (106). As in S. aureus, S. epidermidis ^B contributes to biofilm formation via regulating expression of ica genes. By downregulating the icaR repressor, active ^B causes an increase in icaA expression and a biofilm-positive phenotype (107).

Listeria monocytogenes. ^B has also been extensively studied in the gram-positive pathogen Listeria monocytogenes. While the sigB operon structures are identical in L. monocytogenes and in B. subtilis (11, 219) (Fig. 1A), signal transduction pathways differ in the two organisms. In B. subtilis, environmental and energy stresses are conveyed to ^B through two interconnected but separate pathways. The environmental stimulus pathway is transmitted by regulatory proteins encoded in the sigB operon (RsbT, RsbU, RsbV, and RsbW). In addition to requiring RsbV and RsbW, the B. subtilis energy stress pathway also requires proteins encoded in a two-gene operon (rsbQ-rsbP) that is physically separate from the sigB operon (18). This operon is not present in L. monocytogenes. Instead, both energy stress and environmental stress activation of ^B in L. monocytogenes occurs through a single pathway, which includes RsbT, RsbU, RsbV, and RsbW (25).

A genome-wide search for predicted ^B-dependent promoters by using a hidden Markov model followed by application of a specialized, partial microarray identified 54 genes under positive control of ^B in L. monocytogenes, although the full regulon is likely to be as large as that of B. subtilis (100). ^B regulates expression of virulence and virulence-associated genes in L. monocytogenes (Fig. 2A; Table 2). bsh encodes a bile salt hydrolase that is important for virulence of L. monocytogenes (48) and is directly regulated by ^B (100, 199). Another recently identified ^B-dependent virulence-associated gene is hfq, which encodes an RNA-binding regulatory protein (29). Deletion of the ^B-dependent opuC (57), which encodes an osmotransporter, also negatively affects L. monocytogenes virulence (194, 217).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Examples of regulatory networks involving sigma factors and other transcriptional regulators or multiple sigma factors. (A) The B-PrfA network of L. monocytogenes. Some genes are activated solely by B (e.g., hfq and opuCA), some solely by PrfA (e.g., actA and hly), and some by both factors (e.g., inlA and bsh). (B) The short sigma factor cascade regulating type III secretion in P. syringae. N mediates transcription of hrpL, which encodes a sigma factor responsible for transcription of the hrp and avr genes of the type II secretion system, as well as other virulence genes. All transcription depicted in panels A and B is the result of direct activity by the sigma factor or regulator at the promoter sites. (C) The complex interaction of several sigma factors that affect virulence in M. tuberculosis. Multiple sigma factors activate expression of other sigma factors and of virulence-associated genes (white). The interactions depicted here were deduced from global expression profiles and may be the result of either direct or indirect regulation by the sigma factor(s).

Several PrfA-regulated genes are also ^B dependent, suggesting interplay between the two regulators (143) (Fig. 2A). For example, expression of the PrfA-regulated inlA gene, which encodes the cell surface protein internalin-A, is also at least partially ^B dependent (100, 200). Internalins are cell wall-anchored proteins with important roles in the intracellular pathogenesis of L. monocytogenes, and several members of the internalin gene family show reduced expression in a sigB mutant compared to in the wild type (100). Internalin-A, specifically, is responsible for invasion of nonprofessional phagocytes (129). Loss of ^B reduced invasiveness of the mutant strain compared to that of wild-type L. monocytogenes in two intestinal epithelial cell lines, Henle-407 and Caco-2 (103). In addition, inlA transcription was greatly reduced in the sigB strain, and internalin-A was undetectable by Western blotting (103). None of the effects of the sigB deletion on inlA were mediated through loss of ^B-dependent transcription of PrfA, however, as a P2_prfA strain had the same levels of invasiveness, inlA transcription, and internalin-A concentration as the wild-type strain.

Wiedmann et al. (219) tested the effect of a sigB deletion on virulence in a mouse model and found a small, but significant, decrease in spread of the mutant strain to the liver compared with that of the wild-type strain. Mouse infection experiments have been widely used to evaluate virulence characteristics of L. monocytogenes, including the preliminary evaluations of the sigB mutant (151, 219). In recent years, however, increasing evidence suggests that the murine model does not appropriately represent human L. monocytogenes infection by the oral route (121, 122). The gastric pH of mice is higher than the pH of the human stomach (97); thus, the role of L. monocytogenes acid tolerance is likely to be less important in mouse infection than in human infection. More importantly, in the human, L. monocytogenes has the ability to cross the intestinal barrier, the blood-brain barrier, and the fetoplacental barrier. Human E-cadherin acts as an L. monocytogenes internalin-A receptor, and the interaction between the receptor and the internalin-A surface protein contributes to the ability of L. monocytogenes to target and cross human intestinal and placental barriers (123). Murine E-cadherin, which differs in amino acid sequence from human E-cadherin, does not interact effectively with L. monocytogenes internalin-A, and hence mice show limited susceptibility to intragastric L. monocytogenes infection (121). In fact, in the mouse, translocation of L. monocytogenes across the intestinal barrier is typically no greater than that of the nonpathogenic Listeria innocua. Further, L. monocytogenes also does not appear to target the murine brain stem or the fetoplacental unit, even following intravenous injection (121, 122). L. monocytogenes strains do vary in their ability to cause systemic infection in intragastrically infected mice (38), and some strains of mice (A/J) are also more susceptible than others (C57BL/6) to intragastric infection (39). However, as a consequence of the biological differences in murine and human L. monocytogenes translocation across the intestinal barrier, data from mouse infection experiments may underestimate a given strain's human virulence following oral infection.

The guinea pig has emerged as a more appropriate model than the mouse for studying oral L. monocytogenes infection (121, 122). Like humans, guinea pigs exhibit gastroenteritis following L. monocytogenes infection by the oral route (122). Cultured guinea pig epithelial cells allow internalin-A-dependent L. monocytogenes entry, and both guinea pig and human E-cadherins bear a proline at critical amino acid position 16. When guinea pigs were inoculated orally with L. monocytogenes strain EGD or an otherwise isogenic inlA strain, significantly higher numbers of the wild type than of the inlA strain were recovered from guinea pig liver and spleen. In contrast, in the mouse model, low, statistically indistinguishable wild-type and inlA numbers were recovered from mouse organs (121). Lecuit et al. (121) also demonstrated that transgenic mice expressing human E-cadherin enable bacterial invasion of host cells. Taken together, these results illustrate the importance of appropriate internalin-A/E-cadherin interactions in the development of systemic listeriosis following oral infection with L. monocytogenes.

Mycobacterium tuberculosis ^B and ^F. Mycobacterium tuberculosis, a high-GC-content bacterium, has 13 sigma factors (for a review, see reference 137). Two of these 13, ^B and ^F, appear to share an evolutionary origin (54). M. tuberculosis ^F appears more similar to ^B of the low-GC gram-positive bacteria than to ^F of B. subtilis, which is a sporulation factor. Specifically, M. tuberculosis ^F is antigenically closer to B. subtilis ^B (43) and has the same consensus promoter recognition sequence (10, 60), and expression patterns for its encoding gene are similar to those of B. subtilis sigB (42, 133). As with B. subtilis ^B, M. tuberculosis ^F is regulated posttranslationally by an anti-sigma factor and anti-anti-sigma factor partner-switching mechanism (10). The gene encoding M. tuberculosis ^F is immediately downstream of the gene encoding its anti-sigma factor, UsfX, as is the case with B. subtilis ^B and its anti-sigma factor, RsbW. M. tuberculosis sigB, on the other hand, is located 3 kb downstream of the gene for the primary sigma factor, ^A, and is not flanked by genes encoding sigma factor regulatory proteins (45). The sigB genes in M. tuberculosis and in L. monocytogenes also share some characteristics. For example, expression of M. tuberculosis sigB is growth phase dependent, as is expression of sigB in other species (42, 80). The same studies also showed that sigB transcription is induced under a variety of stresses, including peroxide stress, heat shock, and cold shock. In spite of these observations on ^B stress induction, no studies on contributions of this protein to either M. tuberculosis stress resistance or virulence have been reported.

Microarray analysis of the M. tuberculosis ^F regulon identified ahpC, a gene implicated in virulence, as greatly reduced in expression in a sigF mutant (60). In addition, another sigma factor, sigC, which is required for M. tuberculosis lethality in mice (202), is also ^F dependent (Table 3). Several studies have linked M. tuberculosis ^F with virulence. Mice infected with a sigF strain displayed a longer time to death than mice infected with the wild-type strain, and the weight loss caused by wild-type M. tuberculosis did not occur in mice infected with the mutant strain (26). In a separate study, CFU counts recovered from the lungs and spleens of infected mice were approximately 40 times higher for the wild type than for the sigF strain. Histopathological analyses showed that the sigF mutant caused fewer, smaller granulomas and less inflammation than the wild type (60) after 12 weeks. In summary, multiple lines of evidence support direct and indirect roles for ^F in M. tuberculosis virulence.

Escherichia coli. A few reports have examined associations between ^S and E. coli virulence, but little direct evidence of a link exists. Wang and Kim (214) demonstrated that E. coli K1 invasion of brain microvascular endothelial cells was higher for stationary-phase cells than for exponentially growing cells, possibly due to stationary-phase activity of ^S. Indeed, complementation of rpoS into an rpoS mutant significantly increased invasion for one E. coli isolate but not for another (214). ^S is not essential for murine urinary tract colonization (37) and actually appeared to be detrimental during competitive colonization experiments in the mouse intestine (113). It is also possible that the lack of an appropriate animal model for investigating all aspects of E. coli pathogenesis (e.g., the absence of an appropriate model for studying hemolytic uremic syndrome infections caused by enterohemorrhagic E. coli [193]) has impeded identification of a direct role for ^S in E. coli pathogenesis.

It is likely that ^S contributes indirectly to E. coli pathogenesis. E. coli O161:H7 strains tend to be acid resistant, and rpoS mutants show decreased acid resistance and fecal shedding in mice and cattle (175). Several studies have shown that rpoS transcription and ^S activity are induced under stress conditions such as osmotic shock, heat, and low pH and that survival of rpoS mutants is reduced under these same conditions (3, 37, 59, 74, 215). Thus, in addition to enabling survival in high-acid and high-salt foods, ^S may enhance E. coli host survival and transmission.

Salmonella species. S. enterica serovar Typhimurium ^S is highly similar to E. coli ^S, in both function and regulation. In contrast with E. coli, however, numerous studies have shown the unequivocal dependence on ^S for full virulence of S. enterica serovar Typhimurium. For example, the plasmid-borne spv gene cluster is required for S. enterica serovar Typhimurium virulence, and several studies have demonstrated that transcription of this gene cluster is ^S dependent (51, 68, 112, 157) (Table 2). In fact, an rpoS mutant is up to 10-fold less virulent than an rpoS⁺, plasmid cured (spv-negative) strain, and the levels of plasmid-cured rpoS⁺ bacteria in the intestine were significantly higher than those of plasmid-cured rpoS mutants (51, 153), indicating that the effect of ^S on virulence is likely due to its role in regulating expression of chromosomal genes in addition to its effects on the plasmid-borne spv locus. In addition, mouse-based virulence assays show that in comparison to the wild-type strain, the rpoS mutant has a 3- to 4.5-log-unit higher 50% lethal dose (LD₅₀) (35, 51, 153). Similarly, an rpoS aroA strain was more virulence attenuated than an aroA strain, which has been used in vaccine candidate trials, as determined by spleen bacterial counts and time-to-death analyses (35). ^S does not contribute to levels of S. enterica serovar Typhimurium adherence, invasion, or intracellular survival, however (153).

Further evidence for the role of ^S in Salmonella virulence was obtained through analysis of rpoS alleles from recognized avirulent or virulence-attenuated strains. For example, the Salmonella enterica serovar Typhi vaccine strain Ty21a contains an rpoS sequence that generates a nonfunctional ^S (181). Virulence attenuation in the Salmonella enterica serovar Typhimurium LT2 strain may be a consequence of low levels of rpoS mRNA translation due to the presence of a rare UUG start codon on the transcript (124, 203). As in laboratory-generated rpoS mutants, the LT2 strain is greatly decreased in its ability to reach the spleen and liver of mice (221).

Pseudomonas aeruginosa. P. aeruginosa produces many exotoxins that contribute to its pathogenesis. ^S appears to have multiple regulatory roles in P. aeruginosa. In some cases, ^S positively regulates P. aeruginosa toxin expression; in others, it negatively regulates expression; and in still others, it appears to have no effect at all. For example, in an rpoS mutant, both exotoxin A and alginate production are approximately 50% of that of the wild type (196, 201) (Table 2). However, both reduced rpoS expression (109) and loss of ^S (196, 201) resulted in increased expression of pyocyanin, an antibiotic that also inhibits lymphocyte proliferation. In two studies, loss of ^S was shown to have little to no effect on production of elastase or LasA protease (196, 201). Some of the phenotypic effects on P. aeruginosa virulence factor production that are associated with loss of ^S may be indirect, for example, resulting from reduced expression of quorum-sensing systems (Table 2). ^S contributes to expression of members of the P. aeruginosa rhl and las quorum-sensing systems (168, 218). These quorum-sensing gene products are responsible for regulating production of several virulence factors, including lectins (190, 222); aminopeptidase, endoproteinase, and lipase (158); and rhamnolipid (166, 232). Several studies have shown quorum-sensing mutants to be avirulent or less virulent than the wild-type strain in mouse (167, 185, 195, 232), amoeba (34), and rat models (126). Finally, the role of ^S in P. aeruginosa virulence is highly dependent on the model system in which it is assessed. For example, while an rpoS mutant was as virulent as the wild type in a rat chronic lung model (201), it was approximately half as virulent as the wild-type strain in a Galleria mellonella (silk moth) larva model (196).

SIGMA 28 SUBFAMILY

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²⁸ is a subfamily of the ⁷⁰-like sigma factors. Members of this subfamily are structurally and functionally related and span many genera of both gram-positive and gram-negative bacteria. While the primary regulatory role of ²⁸ in many bacterial species is to transcribe genes required for flagellar synthesis and bacterial motility (69, 144), it also contributes to other functions. For example, in the nonmotile Streptomyces coelicolor, ²⁸ contributes to expression of a diverse set of genes, including those responsible for sporulation and agarase production (96). Examples of ²⁸ factors are FliA of enteric bacteria and ^D of B. subtilis.

Other species. Regulation of flagellar gene expression in Yersinia enterocolitica is similar to that in S. enterica serovar Typhimurium. fliA encodes a ²⁸ factor responsible for motility of the bacterium, and the master regulators FlhC and FlhD are required for expression of all genes encoding proteins active in subsequent flagellar synthesis (85). Motility is also required for full Y. enterocolitica invasion efficiency (228). However, another group of enteric bacteria has a different strategy for regulating flagellar gene expression. Helicobacter pylori, Campylobacter jejuni, and Vibrio cholerae do not have the flhDC master operon. Instead, early flagellar gene expression is carried out via a ⁵⁴ factor, while later genes are transcribed by FliA (89, 105, 154). These species also encode one or more ⁵⁴ activator proteins, such as FlgR. Virulence in these species is linked to production of flagella. In C. jejuni, virulence proteins are secreted through the flagella, and full virulence requires a complete flagellar export apparatus (110). Multiple studies have shown H. pylori virulence to be dependent both on expression of flagellin proteins and on flagellar motility (94, 140).

ECF SIGMA FACTORS

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Members of the extracytoplasmic function subfamily of ⁷⁰ sigma factors regulate functions related to sensing and responding to changes in the bacterial periplasm and extracellular environment. These sigma factors are conserved in both gram-positive and gram-negative species. The first ECF sigma factor identified was E. coli ^E, which was recognized as a second heat shock sigma factor in this organism (213). Although ^E does not appear to affect virulence in E. coli, other ECF sigma factors contribute to regulation of virulence genes and virulence-associated genes in a number of bacteria, including S. enterica serovar Typhimurium, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. A recent review covers some aspects of ECF sigma factors and their involvement in pathogenesis (4).

^E also appears to contribute to oxidative stress resistance in other gram-negative pathogens. In Haemophilus influenzae, rpoE expression was discovered to increase 102-fold inside macrophages, and survival of an rpoE mutant was reduced relative to that of the wild type in the macrophage (36). Vibrio cholerae rpoE mutants are virulence attenuated, exhibiting a reduced ability to colonize the mouse intestine and an LD₅₀ that is 3 log units higher than that of the wild type (111). Although ^E is not essential for growth in H. influenzae or V. cholerae, interestingly, it is essential for growth in Yersinia enterocolitica (76). ^E in Y. enterocolitica also appears to regulate htrA, which is important for virulence in this bacterium as well (77, 127).

AlgU of P. aeruginosa (also called AlgT) is homologous and functionally equivalent to ^E of E. coli (139, 229). As in the pathogens mentioned above, P. aeruginosa algU mutants have increased sensitivity to oxidative stress (139) and reduced survival in macrophages and neutrophils (230). Additionally, AlgU regulates biosynthesis of alginate, a major virulence factor in P. aeruginosa infections of cystic fibrosis patients. Expression of the major alginate biosynthesis gene algD and production of alginate are dependent on algU (138, 189). Thus, while regulation of oxidative stress resistance is functionally conserved between P. aeruginosa AlgU and ^E in multiple bacterial species, AlgU also has an additional role in P. aeruginosa virulence through the regulation of alginate production.

The genes involved in pyoverdine synthesis are located in three clusters on the P. aeruginosa chromosome, with the major genes comprising the pvd locus. Among these genes is pvdS, which encodes an alternative sigma factor. PvdS appears to be predominantly responsible for regulating genes in the pvd locus as well as other pyoverdine synthesis genes (147, 160, 198). The binding of iron by pyoverdine, which occurs outside of the cell, initiates a signaling cascade that leads to enhanced expression of pvd genes and additional secretion of pyoverdine and other virulence factors. Upon forming a complex with iron, pyoverdine binds to the FpvA cell surface receptor protein. FpvA is responsible for transporting the pyoverdine into the cell, but it also triggers a signal cascade to the membrane-bound anti-sigma factor, FpvR, which releases PvdS and allows it to transcribe the pvd genes. FpvR also controls the activity of another sigma factor, FpvI (9). The signal from bound pyoverdine also results in release (and hence activation) of this factor, which is responsible for expression of fpvA.

In addition to increasing pyoverdine synthesis and secretion, free PvdS also activates transcription of genes encoding two more virulence factors, those encoding exotoxin A and PrpL endoprotease. Expression of genes responsible for pyoverdine, exotoxin A, and PrpL production is also controlled by the regulator PtxR; expression of ptxR is also controlled by PvdS. A pvdS deletion mutant generates less PrpL (220) and only 5% of the exotoxin A produced by a wild-type strain (159).

Loss of PvdS results in decreased P. aeruginosa virulence in a rabbit aortic endocarditis model (226). The PrpL endoprotease contributes to the ability of P. aeruginosa to persist in a rat chronic pulmonary infection model (220). PvdS is required for virulence and appears to regulate only virulence-related genes.

Recently, deletion of sigC was shown to render M. tuberculosis unable to cause death in infected mice (202). Deletion of another sigma factor gene, sigH, also produced a nonlethal strain (99). Interestingly, despite the inability to cause fatalities, both sigC and sigH mutants grew to wild-type numbers in macrophages and murine tissues (99, 135, 202). Although the reasons for the similar phenotypes in the two different mutant strains are unknown, it is possible that a subset of virulence-associated genes are regulated by both factors. Alternatively, ^C and ^H may provide similar contributions to M. tuberculosis, but through different mechanisms. ^E appears to affect M. tuberculosis virulence differently than ^C and ^H. As with sigH, sigE expression is induced inside macrophages (64, 90). Loss of ^E, however, does result in decreased strain survival in macrophages and a greater susceptibility to killing by activated macrophages (134). In mouse infection models, the sigE mutant is delayed in its ability to cause lethality but is not completely compromised, as with the sigC and sigH mutant strains (2, 136). Manganelli et al. (136) reported a lower number of sigE mutants in the lungs compared to the wild type, while Ando et al. (2) reported no difference. This discrepancy may be due to differences in mouse strains used in the two studies.

Multiple studies suggest that ^D also contributes to M. tuberculosis virulence. Deletion studies of sigD show the mutant to be less virulent than the wild type in BALB/c and C3H:HeJ mouse infections, allowing substantially longer mouse survival (20, 179). The sigD strain did not show a difference in time to death in SCID mice, which lack T and B cells (20), suggesting that ^D regulates pathogenicity in a manner that is dependent on cell-mediated immunity. In addition, loss of ^D resulted in much milder tissue damage and granuloma formation in lung tissue histopathology in BALB/c mice (179).

Several alternative sigma factors present in M. tuberculosis affect virulence, whether through direct, indirect, or both types of strategies. In addition, some alternative sigma factors of M. tuberculosis autoregulate transcription of their own genes. Many sigma factors also activate transcription of other alternative sigma factors (Fig. 2C). In all, M. tuberculosis appears to have control over expression of its virulence genes via a complex network of multiple alternative sigma factors.

Another important phytopathogen, Erwinia amylovora, also utilizes an hrp-encoded type III secretion system. As in P. syringae, E. amylovora HrpL is an alternative sigma factor that directs transcription of several hrp genes (104). Inactivation of HrpL prevents E. amylovora from causing disease in susceptible plant species or HR in resistant plants (216). E. amylovora also has a dsp, or "disease-specific," gene cluster which is homologous to the avr genes of P. syringae (15). dspA is dependent on HrpL for expression and is required for virulence (58). In addition to E. amylovora, several other members of the Erwinia genus carry hrpL and other hrp genes, including the tumorigenic pathogen Erwinia herbicola (149, 155, 156) and the soft-rot pathogens Erwinia carotovora (24, 125, 180) and Erwinia chrysanthemi (6). The hrp-encoded type III secretion system is thus a common virulence mechanism among plant pathogens and is widespread among several types of pathogens, including tumorigenic, macerating, and soft-rot-causing species.

SIGMA 54

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⁵⁴ forms a distinct subfamily of sigma factors, apart from the ⁷⁰-like family. In almost all species, the ⁵⁴ factor is called ^N. ^N has been identified in many species, spanning a diverse phylogeny, including Legionella pneumophila (88), Pseudomonas spp. (72, 86, 108), Enterococcus faecalis (40), Campylobacter jejuni (89), and Listeria monocytogenes (183). A physiological theme for ^N-dependent genes has not yet emerged, as the regulated genes described to date control a wide diversity of processes (Table 4). Often nitrogen metabolism is controlled by ^N, but other functions of ^N-dependent genes can be found in several organisms.

View this table: [in this window] [in a new window]   TABLE 4. Virulence genes regulated by N in multiple bacterial species

Flagellar motility and pilus-mediated attachment are established virulence factors in P. aeruginosa (148, 186). Pili are external structures that are responsible for adhesion to host cells and interactions such as internalization. P. aeruginosa rpoN mutants do not produce pilin or form pili (206), and they demonstrate drastic loss of adhesion to multiple cell types (28, 32, 172). Wild-type P. aeruginosa also is internalized by host cells more efficiently than an rpoN mutant (172), suggesting an enhanced capacity of the wild-type strain to invade host cells. Reduced virulence due to loss of flagellar motility is also possible in rpoN-disrupted strains, as mutants are decidedly nonmotile (73, 206). rpoN mutants also do not produce the proteinaceous flagellin subunit or form flagella (206). Several studies have shown that P. aeruginosa strains lacking flagella are severely virulence attenuated (46, 52, 148).

P. aeruginosa rpoN mutants are also less virulent than wild-type strains in multiple infection models. An rpoN mutant strain showed diminished cytotoxicity to Madin-Darby canine kidney (MDCK) cells (32) and reduced virulence in several mouse models specifically developed to study P. aeruginosa pathogenicity; compared to the wild type, rpoN mutants cause lower mortality rates in infected mice (32, 73) and reduced fecal carriage and recovery from gastrointestinal tissues (170). In addition, no pathology was observed following infection with an rpoN mutant in a murine corneal scratch model (173). Cohn et al. (30) reported that rpoN mutants did not readily colonize human tracheal epithelium xenografts implanted in mice, although the difference in bacterial numbers of the mutant and wild-type strains was not statistically significant. In general, the defects associated with the rpoN mutation were greater than with strains that were specifically pilin negative, indicating the existence of an additional, pilus-independent mechanism through which ^N also contributes to virulence (28, 32, 170, 172).

Pseudomonas syringae. ^N of P. syringae controls hrp gene expression and influences virulence. Regulation occurs via a short regulatory cascade, wherein ^N and its enhancer-binding proteins HrpR and HrpS direct transcription of hrpL, the product of which is the alternative sigma factor required for expression of the hrp and avr genes (83) (Fig. 2B). Xiao et al. (225) showed that while expression of hrpL and HrpL-dependent genes requires hrpR and hrpS, constitutive expression of hrpL can provide full expression of HrpL-dependent genes with or without hrpR and hrpS. In addition, avrD, which is transcribed from an HrpL-dependent promoter, requires rpoN, hrpL, and hrpS for its expression (192). Characterization of P. syringae pv. Maculicola rpoN mutants identified a more severe phenotype than in hrpL mutants, however (72). rpoN mutants were nonmotile, displayed nitrogen utilization defects, and were unable to produce the phytotoxin coronatine, cause disease or HR, or induce host defense mRNAs. Complementation of hrpL into this strain partially restored some phenotypes but did not restore coronatine production. Other studies have also shown that