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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Shigella flexneri is the causative agent of bacillary dysentery and is a facultative intracellular pathogen. Its virulence regulon is subject to tight control by several mechanisms involving the products of over 20 genes and an array of environmental signals. The regulon is carried on a plasmid that is prone to instability and to integration into the chromosome, with associated silencing of the virulence genes. Closely related regulons are found in other species of Shigella and in enteroinvasive Escherichia coli. A wealth of detailed information is now available on the Shigella virulence gene control circuits, and it is becoming clear that these share many features with regulatory systems found in other bacterial pathogens. All of this makes the S. flexneri virulence gene control system a very attractive topic for those interested in the nature of gene regulatory networks in bacteria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Shigella flexneri is a Gram-negative facultative intracellular pathogen and is the causative agent of bacillary dysentery. This is an invasive disease of the lower gut in which the bacteria enter and replicate within colonic epithelial cells and move between cells. The genetics and cell biology of the complex processes underlying M-cell entry, basolateral invasion of the epithelia and macrophage killing are now becoming clear. The structural genes required for invasion and intercellular spreading are encoded within a 31 kb region of a 230 kb virulence plasmid (Fig. 1). The ipa genes encode secreted proteins needed for host cell entry, and the products of about 20 genes (mxi and spa) are required for Ipa protein secretion (Parsot et al., 1995). The plasmid also harbours key regulatory genes, and further regulatory genes are located on the chromosome. A very similar virulence system has been described in enteroinvasive Escherichia coli (EIEC), and much of what is written below applies equally to EIEC. Virulence gene expression is subject to strict control and is activated by growth at a temperature of 37°C in a medium of moderate osmolarity (with an osmotic pressure similar to that of physiological saline) and a pH of 7.4. It is thought that tight control by environmental factors prevents inappropriate expression of the virulence genes in or outside the host.

image

Figure 1. . Summary of the key features of the S. flexneri virulence gene regulatory cascade. The upper portion of the figure summarizes the chief components of the virulence gene regulon located within a 31 kb region on the 230 kb plasmid. The sites at which positive transcriptional regulatory inputs are made by the VirF and VirB proteins are shown by vertical arrows. The regulatory regions of the virB and virF genes are shown in the enlargements at lower left and right respectively. At the virB promoter, the overlapping binding sites of the positively acting VirF protein and the negatively acting H-NS protein are shown. The positive inputs of IHF and DNA supercoiling are indicated by arrows, with supercoiling being promoted by DNA gyrase (probably under environmental control) and antagonized by the DNA-relaxing enzymes topoisomerases I and IV. At the virF promoter, IHF is a positive regulator, while H-NS has a repressive influence. At the level of virF mRNA translation, the MiaA and VacC proteins have a modulatory role, and the same may be true of certain tRNA species. The diagrams are not drawn to scale.

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In recent years, a large number of virulence gene regulatory mechanisms have been described in a range of pathogens, together with other genetic features that contribute to the modulation of virulence gene expression. The S. flexneri system is particularly interesting because it possesses so many of these control elements and illustrates how they can be integrated to optimize the expression of a single phenotype.

The plasmid-linked virulence genes

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

The invasion genes are located in large, divergently transcribed operons on the 230 kb plasmid. The ipa genes coding for the invasion proteins that are secreted into the extracellular medium are located within one operon, and upstream and transcribed on the other DNA strand are the mxi and spa genes that specify the type III secretion system for invasion protein deployment. Both groups of genes are regulated collectively at the level of transcription, with regulation being exerted by the products of the plasmid-located virF and virB genes (Fig. 1; Adler et al., 1989). Regulation involves a cascade, with the virB gene being activated by the virF gene product. The plasmid-linked icsA (virG ) gene is located at a distance from the main virulence gene operons and encodes a product required for intercellular spreading. It represents a branch point in the regulatory circuit because it is activated directly by the virF gene product (Fig. 1). This gene is regulated post-transcriptionally by the product of the plasmid-located virK gene (Table 1). The virA gene, located upstream and transcribed divergently from icsA (virG ), is involved in invasion and spreading. It is the only gene outside the main virulence gene operons to be regulated by the VirB protein (Fig. 1).

VirF, an AraC-like regulatory protein

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

The VirF protein is a member of the AraC family of transcription regulators (Table 1). With one exception (the repressor protein CelD), these proteins are transcription activators, and they may be divided into two groups on the basis of the signals to which they respond (Gallegos et al., 1997). One group is made up of proteins that respond to chemical signals. The prototypic member of the family, AraC, is a member of this group. It regulates transcription of the ara operon in several Gram-negative enterobacteria in response to the carbohydrate arabinose. It is composed of an amino-terminal domain to which arabinose binds, a carboxy-terminal domain that contains a DNA-binding motif and a linker region that connects the functionally distinct domains. AraC forms dimers in solution, and its interactions with DNA are modulated by arabinose.

VirF belongs to the second group of proteins that regulate gene expression in response to a physical signal, usually temperature. Amino acid sequence similarity between the sugar-binding proteins and the thermally regulated ones is confined to the carboxy-terminus, a fact that is thought to reflect conservation of a DNA-binding motif. Indeed, VirF does appear to possess two helix–turn–helix (H–T–H) motifs within its carboxy-terminus, just as AraC does. In AraC, the first H–T–H is believed to be critical for DNA binding, while the biological significance of the second is unknown. VirF is much more closely related to other thermally regulated AraC-like proteins than to the carbohydrate binders. It shows a particularly close relationship with Rns (36% amino acid sequence identity), a protein that activates transcription of the cso genes encoding CS1 fimbriae in enterotoxigenic E. coli (ETEC). Interestingly, Rns can substitute for VirF in the activation of S. flexneri virulence gene expression, although VirF cannot substitute for Rns in activating cso gene expression (Porter et al., 1998). Evidence from dominant-negative mutant studies suggests that VirF can oligomerize, but direct biochemical evidence to support this is lacking (M. E. Porter and C. J. Dorman, unpublished data).

VirB, a vassal regulator

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

The VirB protein (called InvE in S. sonnei ) is believed to bind to DNA, although there is no direct evidence for this (Table 1). This 35.4 kDa polypeptide shows amino acid sequence similarities to proteins known to be involved in plasmid maintenance, such as the ParB protein of plasmid P1 and the SopB protein of plasmid F (Watanabe et al., 1990). The region of maximum amino acid sequence homology to ParB involves the central portion of the protein. This part of ParB is involved in DNA binding and includes a H–T–H motif. Nothing is known of the domain structure of VirB, and it is not known if VirB must oligomerize to become active. There are no footprinting data for VirB interaction with the structural gene promoters (such as icsB and ipgD ; Fig. 1). Nevertheless, the simplest interpretation of the available data is that VirB is a DNA binding protein that binds to operator sites at the promoters of these genes and activates them.

Environmental signalling

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

The primary environmental stimulus that activates S. flexneri virulence gene expression is a temperature of 37°C. Optimum induction also requires a moderate level of osmotic stress and a pH of 7.4. The key event in virulence gene activation is the conversion of the virB promoter to an open transcription complex. This event absolutely requires VirF protein. A binding site for VirF in the promoter region of the virB promoter has been identified by in vitro footprinting with a VirF–MalE hybrid protein. The footprint extends from position −17 to −117 with respect to the transcription start site (+1). Potentially, VirF could contact RNA polymerase at this promoter, improving its ability to form an open complex, or it could act by recruiting RNA polymerase to the promoter. It is not known if VirF binds to the operator site as a dimer or higher order oligomer or whether oligomerization occurs before or after binding.

Tobe et al. (1995) have shown that the VirF protein is present in vivo at both permissive and non-permissive temperatures and that it can bind in vitro to the virB promoter at both temperatures. Therefore, the thermal activation signal does not act simply by inducing the production of VirF. Instead, evidence has been presented to show that an increase in temperature produces a reduction in the linking number of virB promoter DNA, and the resulting change in DNA supercoiling is responsible for the VirF-dependent activation of the virB promoter. Interestingly, increases in osmolarity also result in elevated levels of negative DNA supercoiling, and supercoiling is also responsive to variations in pH (Porter and Dorman, 1997a). When the degree of supercoiling at the virB promoter is increased by a temperature-independent mechanism, VirF can activate virB gene transcription even at 30°C, a temperature normally too low to permit this to happen. The simplest interpretation of these results is that the alteration in DNA topology facilitates a productive interaction between DNA-bound VirF protein and RNA polymerase. It is also possible that the change in DNA supercoiling may promote VirF oligomerization on the DNA, and this may represent an activation step analogous to arabinose binding by the AraC protein.

Other evidence supporting a role for DNA supercoiling comes from work with DNA gyrase inhibitors (Dorman et al., 1990; Tobe et al., 1994), with mutations in topA, the gene encoding DNA topoisomerase I, and with plasmids overexpressing the parC and parE genes, encoding DNA topoisomerase IV (McNairn et al., 1995; Table 1). A mutation in the rho gene, encoding the transcription terminator protein Rho, results in a loss of thermal regulation at the virB promoter. In the rho mutant, DNA supercoiling levels respond poorly to temperature and are set at a value intermediate between that seen at 30°C and 37°C. It appears that the DNA supercoiling sensitivity of the virB promoter causes it to be expressed constitutively in the rho mutant (Tobe et al., 1994).

Transcription of the virulence genes is maintained as the cell enters the stationary phase of the growth curve. This seems paradoxical as DNA becomes relaxed under these conditions. However, proteins such as IHF are thought to counteract this effect through their influence at the virF and virB promoters (see below). Interestingly, no role has been found for the stationary phase sigma factor, RpoS, in controlling the plasmid-linked virulence genes in S. flexneri (Porter and Dorman, 1997b), although this protein is involved in acid resistance, acting via chromosomally located genes (Waterman and Small, 1996).

H-NS, an antagonistic regulatory factor

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Early work by Maurelli and Sansonetti (1988) identified a chromosomal locus (virR ) that appeared to encode a repressor of virulence gene transcription. This gene was subsequently shown to be allelic with the osmZ (hns) locus of E. coli (Dorman et al., 1990), demonstrating that the S. flexneri virulence genes belong to the large family of H-NS-repressed genes. While minor inputs are detectable at virF (Porter and Dorman, 1997a), H-NS makes its primary input at the virB gene promoter. A footprint extending from position −20 to + 20 has been detected in vitro at the virB promoter, suggesting that H-NS may exclude RNA polymerase from binding there (Tobe et al., 1993).

Inactivation of the hns gene results in virulence gene expression at low temperatures and at low osmolarity, although not to the full level seen in the wild type at 37°C and high osmolarity (Hromockyj et al., 1992; Porter and Dorman, 1994). This is because thermally and osmotically activated expression of the virB promoter is still required even in the absence of H-NS. This activation appears to involve increases in DNA supercoiling (see above), and the presence of the AraC-like VirF protein is absolutely required. Observations of antagonistic relationships involving H-NS and AraC-like transcription activators are becoming common. For example, the regulation of Cfa/I fimbrial expression in enterotoxigenic E. coli involves cfa gene activation by the AraC-like protein CfaD, with this activator being required chiefly to overcome the negative influence of H-NS at the structural gene promoter (Jordi et al., 1992). In the case of the S. flexneri virulence gene regulatory cascade, it is clear that VirF is not there simply to overcome the repressive effects of H-NS on the virB promoter but to play a direct role in promoter activation.

Like E. coli, S. flexneri possesses a copy of the stpA gene, which encodes a protein showing 58% amino acid sequence identity to H-NS. The relationship between H-NS and StpA is still the subject of intensive research, but it is becoming clear that the proteins have both distinct and overlapping roles in the cell and that they can form heteromeric complexes with one another (Williams et al., 1996). Preliminary work has shown that overexpression of StpA protein approximately mimics the negative effect of overexpressing H-NS on the expression of the S. flexneri virulence genes. However, an stpA null mutation has no significant effect, suggesting that the StpA may act simply as a molecular backup for H-NS in the case of this virulence gene regulon (M.E. Porter, A. Free and C.J. Dorman, unpublished; Table 1).

The integration host factor, a positive regulator of the virulence genes

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

The integration host factor (IHF) is emerging as a broad domain regulator of bacterial gene expression. This heterodimeric sequence-specific DNA binding protein is both a structural element in the cell and a conventional regulator of gene expression. Its subunits are encoded by the chromosomally located ihfA and ihfB genes. In its architectural role, its ability to bend DNA by angles of up to 180° facilitates the formation of nucleoprotein complexes for such diverse functions as site-specific recombination, DNA replication, genome organization and transcription control (Rice et al., 1996). In its guise as a transcription factor, it can bind upstream of promoters and contact the alpha subunit of RNA polymerase in a manner reminiscent of regulators such as CRP (Goosen and van de Putte, 1995).

IHF makes a positive contribution to the expression of the virulence genes of S. flexneri. It is required for the full activation of virF in both exponential and stationary phase cultures and for the activation of virB in stationary phase. IHF makes independent and direct inputs at both the virF and the virB promoters and, in the case of the latter, it appears to have an antagonistic relationship with H-NS (Porter and Dorman, 1997c). IHF levels vary with growth phase, increasing by a factor of five- to 10-fold as the culture enters stationary phase (Ditto et al., 1994). Under stationary phase conditions, DNA relaxes (Dorman et al., 1988) and the function of IHF may be to offset the inhibitory effect of relaxation on virulence gene expression (Porter and Dorman, 1997c).

OmpR/EnvZ, a two-component regulator

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Genetic studies have revealed a role for the OmpR/EnvZ two-component regulatory system in the expression of the S. flexneri virulence phenotype (Bernardini et al., 1990). These proteins are encoded by the ompB locus on the chromosome and function as a cytoplasmic membrane-located sensor of osmotic stress (EnvZ) and a cytosol-located DNA binding protein (OmpR). EnvZ communicates with and modulates the function of OmpR by phosphotransfer, and OmpR binds to specific operator sites located in the promoter regions of genes belonging to the OmpR regulon. While loss of ompB expression has been shown to result in a decrease in the expression of the plasmid-located virulence genes, these have not been shown to be under the direct control of OmpR. Furthermore, loss of expression of the OmpC outer membrane porin, encoded by a known member of the OmpR regulon, ompC, has been found to be a significant cause of the loss of virulence seen in ompB mutants of S. flexneri (Bernardini et al., 1993). It is possible that the ompB mutation and its associated loss of OmpC porin expression bring about indirectly the reported downregulation in plasmid-linked virulence gene expression.

pH regulation and CpxA/CpxR, a second two-component system

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Transcription of the virF gene is under pH control, although thermal regulation can override this effect at genes downstream in the regulatory cascade (Porter and Dorman, 1997a). In S. sonnei (and almost certainly also in S. flexneri and EIEC), this pH control is dependent on the chromosomally located cpxRA genes, whose products show homology to the sensor (CpxA) and response regulator (CpxR) members of the two-component family of regulatory proteins (Nakayama and Watanabe, 1995). Direct binding of the CpxR protein to the virF promoter has not been demonstrated, leaving open the possibility that the effect is indirect.

Virulence plasmid integration and stability

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Data from experiments with EIEC and S. flexneri show that the virulence plasmid can integrate into the bacterial chromosome and that this results in silencing of the virulence genes. Integration results in methionine auxotrophy, is RecA dependent and is site specific. The silencing effect is dependent on the H-NS protein and is caused by a failure in the expression of the virB gene. It has been postulated that this may result from the virB promoter being placed in a DNA structural context that impedes its activation and/or results in more efficient inactivation by H-NS (Colonna et al., 1995).

The virulence plasmid can be lost from the cell when the virulence gene regulon is induced. This instability depends on activation of the virB and virF genes, either in cis or in trans (Schuch and Maurelli, 1997), and may indicate that virulence structural gene transcription and/or post-transcriptional events make the plasmid difficult to maintain. The plasmid is also prone to rearrangements that inactivate the regulatory genes, virF and virB, as well as the structural genes (Porter and Dorman, 1997b; Schuch and Maurelli, 1997). These rearrangements can be mediated by insertion sequences or by other means, involving both RecA-dependent and RecA-independent pathways. Inactivation of the virulence genes provides a mechanism for stabilizing the plasmid and, in the case of some insertion sequence mutations in virF, the lesion can revert to wild type, allowing the virulence genes to be expressed again (Schuch and Maurelli, 1997).

Post-transcriptional regulation of virulence gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Expression of the S. flexneri tyrT gene in multicopy can partially complement the effect of an hns deletion on virulence gene expression. The mechanism by which this occurs remains unknown, but it may involve an effect on the translation machinery of the cell that is not necessarily specific to this tRNA gene (Hromockyj et al., 1992). This is supported by the finding that the mia gene, coding for the tRNA N 6-isopentyladenosine (i6A37) synthetase, required for the production of the modified nucleoside 2-methyl-N 6-isopentyladenosine, is necessary for the expression of the genes of the virulence regulon. This mechanism operates post-transcriptionally on the virF regulatory gene (Durand et al., 1997). Further evidence that tRNA modification is important for virulence gene expression comes from the observation that transcription of the ipa operon is decreased in strains mutated in vacC, an allele of the E. coli tgt gene. This is because the product of vacC, tRNA-guanine transglycosylase, is required for normal translation of the VirF protein, without which the other genes in the regulatory cascade cannot be transcribed (Durand et al., 1994). The vacB gene is located close to purA on the chromosome and is required for normal expression of the ipa virulence genes. This control is also exerted at the post-transcriptional level (Tobe et al., 1992). The mechanism is not understood.

Other regulators

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

A number of other chromosomal loci and at least one further plasmid-linked locus have been identified as being involved in the virulence gene regulon (Table 1). On the plasmid, a gene called mxiE has been found by sequence analysis to encode yet another member of the AraC family of transcription regulators. The location of the gene in the mxi operon suggests that it will emerge as a regulator of the virulence genes, but information about its input is lacking at present (Allaoui et al., 1993; Gallegos et al., 1997).

It appears that the expression of the virulence genes may be coupled to the bacterial cell cycle. Evidence for this comes from the identification on the chromosome of the cell division gene ispA as a determinant of the ability of the bacteria to spread within host cells and to polymerize actin. Other chromosomal loci encode factors critical for the pathogenesis of S. flexneri, but these remain poorly characterized in terms of the mechanisms (if any) by which they affect virulence plasmid gene expression. These include the vacJ gene (encoding a surface-expressed lipoprotein necessary for cell-to-cell spreading of S. flexneri ; Suzuki et al., 1994) and vacM (required for normal expression of the ipa operon at the level of transcription; Mac Síomóin et al., 1996; Table 1).

Integration of the regulatory mechanisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References

Clearly, the S. flexneri virulence regulon is subject to multiple regulatory inputs, and much more information is required before an accurate blueprint of the regulatory circuitry can be drawn up. At present, much of the information available comes from genetic studies, making it hard to determine which controls act directly and which are indirect. The best characterized component of the regulon is the virB promoter. Here, footprint analysis has revealed the probable binding sites of the VirF and H-NS promoters, and band shift assays have shown a direct role for IHF in binding there. In fact, H-NS and IHF remain the only chromosomally encoded regulators known to influence the plasmid virulence genes directly by interacting with their DNA.

The plasmid location of the virulence regulon is suggestive of genetic mobility. This impression is strengthened by the discovery that the type III secretion system encoded by the mxi spa operons of S. flexneri has counterparts in many other pathogens and that these are frequently found on their chromosomes (Groisman and Ochman, 1993). The ability of the virulence plasmid to integrate and excise from the bacterial chromosome is reminiscent of the behaviour of temperate bacteriophage and, indeed, the IHF protein was discovered originally as a regulator of the life cycle of phage lambda. These observations, the presence of very closely related plasmids in other organisms such as EIEC and the abnormally low G + C content of the virulence genes suggests that the S. flexneri virulence system may have been imported at some time in the past. This creates an interesting problem in terms of gene regulation. Specific regulators of transcription are usually site specific in their activity, preferring to bind to particular operator sequences. It would be surprising if a newly arrived regulon could find such regulatory proteins in its new host cell. Instead, it would have to bring its specific regulatory proteins with it and possibly exploit general regulatory features of the host cell, such as those influencing DNA topology or the process of mRNA translation. This description is consistent with current knowledge of the S. flexneri virulence regulon. The virF and virB regulatory genes are on the same plasmid as the structural virulence genes. Most of the chromosomal genes that influence virulence gene transcription have not been shown to interact directly with promoters on the plasmid. Of those that have, H-NS is a general DNA binding protein, and IHF has a long track record of interacting with mobile genetic elements. The virB promoter is sensitive to changes in DNA supercoiling, and this seems to be the basis of its environmental regulation by VirF. The virF gene is emerging as one subject to tight control at the level of translation. Issues that require immediate attention concern the mechanism by which VirF activates virB transcription in response to environmental signals and the mechanisms by which the many chromosomal loci, several of which seem to act indirectly, influence plasmid-linked virulence gene expression. New information about these processes is eagerly anticipated. Recent and imminent advances in bacterial genomics (Hinton, 1997) may help to identify the origin of the S. flexneri virulence genes and assist our understanding of the evolution of their regulatory apparatus.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The plasmid-linked virulence genes
  5. VirF, an AraC-like regulatory protein
  6. VirB, a vassal regulator
  7. Environmental signalling
  8. H-NS, an antagonistic regulatory factor
  9. The integration host factor, a positive regulator of the virulence genes
  10. OmpR/EnvZ, a two-component regulator
  11. pH regulation and CpxA/CpxR, a second two-component system
  12. Virulence plasmid integration and stability
  13. Post-transcriptional regulation of virulence gene expression
  14. Other regulators
  15. Integration of the regulatory mechanisms
  16. Acknowledgements
  17. References
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