Regulation of virulence gene expression in Shigella flexneri, a facultative intracellular pathogen

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Int. J. Med. Microbiol. 290, 89-96 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm

Regulation of virulence gene expression in Shigella flexneri, a facultative intracellular pathogen Charles J. Dorman, Sorcha McKenna, Christophe Beloin Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Republic of Ireland

Abstract Shigella flexneri and its close relatives are facultative intracellular pathogens of humans and are the etiological agents of bacillary dysentery. These bacteria secrete proteins that enable them to enter human epithelial cells via an elaborate and fascinating cell biology. This behaviour depends on a complicated regulon of virulence genes, whose expression is controlled in response to a multiplicity of environmental signals. This review describes and attempts to interpret these gene control mechanisms. Key words: Shigella, gene regulation – virulence plasmid – regulatory cascade

Shigellosis Shigella flexneri, together with S. sonnei, is a prominent cause of shigellosis, or bacterial dysentery, in humans. It is a particular problem in the developing world and children are especially at risk. Transmission is via the oral route, the organisms are extremely infectious, and fewer than 100 bacterial cells will suffice to establish the disease (DuPont et al., 1989). The disease affects the lower gut, where the bacteria enter and replicate within colonic epithelial cells, and move between cells. Shigella is unable to enter epithelia via their apical surfaces so it uses the antigen-sampling M cells as a means of gaining access to the baso-lateral surfaces (Wassef et al., 1989). The macrophages in the region beneath the epithelial layer engulf the bacteria, but are then propelled into apoptosis by their prokaryotic prey (Zychlinsky et al., 1992). The activation of caspase 1 in the dying macrophage causes the release of pro-inflammatory IL-1β cytokines (Zychlinsky et al., 1994). These in turn recruit polymorphonuclear (PMN) cells which proceed

to destabilize the epithelium, permitting more bacteria to gain access to the baso-lateral membrane of the epithelial cells (Perdomo et al., 1994a, b). Cytokines released by the infected epithelial cells recruit more host immune cells, enhancing the inflammation. The host displays symptoms such as intestinal cramps, fever, and the bloody diarrhoea that are characteristic of the disease (Philpott et al., 2000).

Plasmid-encoded system for host cell invasion The genes required for Shigella virulence reside on a 230-kbp plasmid where they are grouped within a 31 kbp portion (Fig. 1) (Maurelli et al., 1985; Sasakawa et al., 1988). Here, two divergently transcribed regions code for the secreted Ipa invasion proteins and the Mxi-Spa type III secretion apparatus. This Secindependent secretion system has counterparts in many other pathogens, including Salmonella where one also

Corresponding author: Charles J. Dorman, Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Republic of Ireland, Phone: +353 1 608 2013, Fax: +353 1 679 9294, E-mail: [email protected] 1438-4221/01/291/2-089 $ 15.00/0

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epithelial cell becomes phagocytic and engulfs the bacterium are brought about by the activities of IpaA and IpaC. Once internalized, the bacterium escapes from its vacuole through lysis of the vacuole membrane by IpaB (High et al., 1992). The liberated bacteria acquire mobility based on the recruitment and polymerization of host actin. This process requires the outer membrane protein IcsA, another factor that is encoded by the virulence plasmid (Bernardini et al., 1989). The motion resulting from the association of the bacterium with polymerized actin not only allows it to traverse the cell it has entered, but also to penetrate into neighboring cells, spreading the infection and associated tissue damage across the epithelial layer.

Environmental influences on virulence gene expression

Fig. 1. Schematic representation of the 230-kbp virulence plasmid of S. flexneri. The relative locations of the virulence genes are shown. The major regulatory genes, virF and virB are highlighted by hatching.An expanded view of the 31-kbp invasion locus is shown below the circular map. The angled arrows indicate directions of transcription, and the functions of the genes are shown, where known. Rep, origin of replication. Not to scale.

participates in host cell entry and Yersinia where another secretes anti-host factors (Cornelis, 2000; Hueck, 1998). Such systems are responsible for the secretion of signal transducing proteins in enteropathogenic Escherichia coli, toxins in Pseudomonas aeruginosa, flagellum assembly in Salmonella and Bacillus subtilis, and virulence factor secretion by plant pathogens (Alfano and Collmer, 1997; Mecsas and Strauss, 1996; van Gijsegem et al., 1993). The four Ipa proteins, IpaA, B, C, and D, are required for host cell entry. They are produced and stored within the bacterium and then released through the Mxi-Spa secretion system upon contact with host cells (Ménard et al., 1994). The rate at which this happens is thought to be regulated by a complex formed by IpaB and IpaD (Ménard et al., 1996). Once outside the bacterium, IpaB and IpaC form a complex that interacts with the epithelial cell membrane to form a pore through which other Ipa proteins are thought to enter the cytoplasm (Blocker et al., 1999). The cytoskeletal rearrangements through which the

Expression of the Shigella virulence phenotype is regulated at least at three levels: transcription, translation, and protein secretion. Transcription of the structural genes on the 230-kbp plasmid is controlled in response to temperature, osmolarity and pH, with optimal expression in vitro being achieved at 37 °C (Maurelli et al., 1984), an osmolarity equivalent to that of physiological saline (Porter and Dorman, 1994), and a pH of 7.4 (Nakayama and Watanabe, 1995). It is likely that this profile of environmental signals approximates to that existing at the site in the host where the virulence genes are to be expressed if cellular invasion is to result, i. e. the lower gut. The temperature and osmotic dependence will preclude expression outside the host while the requirement for a moderate pH will avoid expression, for example, in the acid environment of the stomach.

A transcriptional regulatory cascade Genetic analysis has revealed that transcription of the virulence structural genes on the large plasmid requires two positively-acting regulatory genes, virF and virB (Fig. 2) (Adler et al., 1989). Expression of the virB gene depends on virF, and expression of the structural genes in the ipa and mxi-spa regions depends in turn on virB. Analysis of the amino acid sequence of the VirF protein shows this to be a member of the AraC family of bacterial transcription factors (Dorman, 1992). Genetic and biochemical evidence demonstrates that VirF activates the promoter of the virB gene directly, but the mechanism by which it does so remains unclear (Tobe et al., 1993). The VirB protein (called InvE in some of the S. sonnei References) is re-

Regulation of virulence gene expression in S. flexneri

lated to a family of DNA binding proteins concerned principally with plasmid segregation (Watanabe et al., 1990). Thus far, evidence that VirB activates structural gene transcription directly is lacking, as is evidence that it is a DNA-binding protein. This leaves open the possibility that another factor or factors may act below VirB or in parallel with it in the regulatory cascade. The promoters that lie below VirB in the cascade are those required for expression of the ipa operon of eight genes, four of which code for the Ipa invasion proteins, the promoter of the mxi-spa operons, and the promoter of the virA gene. The product of virA, the 46-kDa VirA polypeptide, is a secreted protein that is not required for invasion (Demers et al., 1998). The icsA structural gene is activated by VirF directly, without a requirement for VirB, and thus represents a branch-point in the cascade (Fig. 2). Activation of the genes in the cascade in response to thermal signals in vitro reveals a gearing effect as one descends to each level in turn. The virF gene responds over a narrow range (about two-fold) as measured by Northern blotting, while the virB gene shows a 10-fold range of response, and the mxi operon of structural

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genes a 100-fold range of response (Porter and Dorman, 1997a). In addition, the strictness of the regulation increases as one descends the cascade, with virF transcription being most loosely regulated, the structural genes most tightly, and virB showing an intermediate degree of regulatory strictness. In addition, each promoter in the cascade shows individual characteristics of its response which may permit fine-tuning during activation. It has been reasoned that this regulatory pattern has evolved to prevent wasteful expression of the secreted virulence factors while ensuring strong and appropriate activation when this is required (Porter and Dorman, 1997a).

Global regulators and the Shigella virulence regulon In addition to the regulators encoded by the virulence plasmid, the virulence genes are controlled at the level of transcription by pleiotropic factors encoded by genes located on the chromosome. One of these is H-NS, the histone-like nucleoid structuring protein, a 15.6-kDa

Fig. 2. Summary of the main regulatory inputs in the Shigella virulence gene regulon. The plasmid-linked virulence genes are shown as thick horizontal arrows, with the arrowheads indicating directions of transcription. The divergently arranged virA and icsA genes, and the virF gene, are at a distance from the major structural gene operons and virB, and this is indicated by the breaks in the horizontal line (the drawing is not to scale). Regulatory inputs are shown by arrows. Question marks associated with VirB indicate that the activation may be direct or indirect. Plasmid-encoded regulators are written within ovals whereas those encoded on the chromosome are written within rectangles. Positive inputs are denoted by a (+) sign, and negative ones by (–). PT indicates that an input is posttranscriptional. Optimal conditions for activation of the cascade are indicated at the top of the figure.

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DNA-binding protein which exerts widespread effects on gene expression in many Gram-negative bacteria, including Shigella (Atlung and Ingmer, 1997; Bertin et al., 1999; Dorman et al., 1999). Identified originally as VirR, a repressor of virulence gene transcription (Maurelli and Sansonetti, 1988), this regulator was subsequently recognized as the global regulator H-NS (Dorman et al., 1990; Hromockyj et al., 1992). H-NS makes a negative contribution to the regulation of many of the promoters in the virulence gene regulon. A wellcharacterized instance concerns repression by H-NS of the virB promoter (Fig. 2). Here, footprinting experiments have identified an H-NS-binding site extending from –20 to +20 with respect to the transcription start site (+1) and shown that this overlaps with the putative binding site for RNA polymerase (Tobe et al., 1993). Removal of H-NS from the cell by mutation of the hns gene allows virB gene expression to increase at 30 °C, suggesting a role for this protein in the thermoregulation of the gene. However, the requirement for the positive input of VirF remains, and full activation at 37 °C is not seen in the absence of this AraC-like protein, even in an hns mutant (Tobe et al., 1993). Over-expression of H-NS has a negative effect on expression of the virulence genes, even at the permissive temperature. This effect is mimicked when the H-NS-like protein StpA is over-expressed; however, an stpA null mutation has no effect on virulence gene expression (Dorman and Porter, 1998). This is of interest in the light of the close similarity of StpA and H-NS (58 % identical amino acid sequences) and their ability to form heteromeric complexes and influence expression of many genes (Deighan et al., 2000; Williams et al., 1996). It seems likely that StpA acts simply as a less proficient backup for H-NS in the case of the S. flexneri virulence genes. The interaction of H-NS with the virF promoter has also been examined in detail. Here, two binding sites were identified centered at positions –250 and –1 and separated by a region of intrinsically-curved DNA. HNS bound cooperatively to both sites at 32 °C but not at 37 °C, and it was found that the DNA composed of these sites and the intervening sequence underwent a specific conformational change with variations in temperature (Falconi et al., 1998). This suggests that reductions in temperature below 37 °C alter DNA conformation and so facilitate cooperative binding of HNS, in turn leading to repression of the virF promoter. Genetic evidence shows that VirF represses its own promoter (Porter and Dorman, 1997a) but how this negative effect is integrated with that mediated by HNS is not known. The input of H-NS is not confined to the virB and virF regulatory genes; binding to structural gene promoters is also detectable, suggesting a wide-ranging role for this protein within the regulon (Beloin and Dorman, unpublished data).

The integration host factor (IHF) plays a role in many cellular processes, such as transcription control, transposition, and DNA recombination (Freundlich et al., 1992; Goosen and van de Putte, 1995). Its many contributions arise from its role as a DNA architectural element. This 22-kDa heterodimeric, sequencespecific DNA-binding protein can introduce bends in DNA of up to 180° and so exert influence at longrange over DNA transactions (Rice et al., 1996). In S. flexneri cells deficient in IHF, expression of the virulence genes is repressed, particularly on entry into stationary phase (Porter and Dorman, 1997b). IHF stimulates the virF promoter (Fig. 2) in both the logarithmic and early stationary phases of growth; in contrast it stimulates the virB promoter only in stationary phase; the icsA gene, like virB, is VirF-dependent, and it shows a virB-like profile of response to IHF. The inputs of IHF at the virF and virB promoters are direct, and at the virB promoter the primary role of IHF appears to be in overcoming the negative influence of H-NS (Porter and Dorman, 1997b).

DNA supercoiling The involvement of DNA supercoiling in the functioning of the S. flexneri virulence gene regulatory cascade has been recognized for some time (Dorman et al., 1990). The possibility of such involvement arose following the discovery that changes in temperature result in changes in the level of supercoiling in bacterial DNA (Goldstein and Drlica, 1984). Given the key role for temperature in controlling the S. flexneri virulence gene expression, it was logical to investigate the possibility of supercoiling involvement. Sensitivity to changes in DNA supercoiling has been described for the virF and the virB promoters (Fig. 2). At virF, supercoiling influences the formation of a repression complex involving H-NS and the promoter DNA (Falconi et al., 1998) (see above). At virB, an increase in temperature results in a decrease in the linking number of the DNA leading to VirF-dependent activation of the promoter (Tobe et al., 1995). If the reduction in DNA linking number is achieved by an artificial method, such as creation of local negative supercoiling by an upstream, divergently-oriented inducible promoter, VirF-dependent activation of virB transcription can occur even at 30 °C (Tobe et al., 1995). The observation that VirF is present in the cell at 30 °C as well as at 37 °C, and that it can bind to the virB promoter at either temperature, suggests that the thermal activation of virB most likely operates via changes in the superhelicity of its promoter and that these modify the interaction of VirF with itself, with RNA polymerase, or both.

Regulation of virulence gene expression in S. flexneri

Expression of the virulence gene cascade is strongly affected by mutations in genes coding for DNA topoisomerase I or topoisomerase IV (both of which relax DNA) or by antibiotic treatments that inhibit DNA gyrase, a topoisomerase that negatively supercoils DNA in bacteria (Dorman et al., 1990; McNairn et al., 1995; Ní Bhriain and Dorman, 1993; Tobe et al., 1995). These observations support the hypothesis that this environmentally regulated virulence gene regulon is supercoiling sensitive. Other supporting evidence comes from the observation that a mutation in rho, the gene coding for the Rho transcriptional terminator, results in loss of virB thermal regulation (Tobe et al., 1994). The significance of this effect is due to the influence of the rho mutation on global levels of DNA supercoiling in the cell. In the mutant, supercoiling responds poorly to changes in temperature and is set to levels intermediate between those normally seen at 30 °C and 37 °C (Tobe et al., 1994).

Two-component systems Regulatory systems of the classical two-component type play important roles in controlling the expression of virulence genes in many pathogens (Gross et al., 1989), and the same is true of Shigella. The pH responsiveness of the virF promoter has already been referred to. In S. sonnei, and very probably also in S. flexneri, this response is mediated via the CpxR-CpxA twocomponent regulatory system, encoded by the chromosomal genes cpxRA (Nakayama and Watanabe, 1995). Here, CpxA shows homology to the sensor family of bacterial histidine protein kinases while CpxR is a response regulating DNA-binding protein. The Cpx system is an activator of virF transcription, and mutations in either cpxA or cpxR abolish pH regulation. The CpxR protein binds directly to the upstream region of the virF promoter (Fig. 2) between position –103 and –37, and this binding activity is enhanced in vitro when the protein is phosphorylated. It has been suggested that CpxA may function as a phosphatase of the phosphorylated CpxR at low pH, inactivating the DNA-binding activity of the latter under acid conditions (Nakayama and Watanabe, 1998). Mutations in the ompB locus of S. flexneri cause a decrease in expression of members of the virulence gene regulon (Bernardini et al., 1990). This bi-cistronic operon encodes the EnvZ-OmpR two-component regulatory system in which the membrane-located EnvZ kinase senses, inter alia, changes in osmotic pressure, and transmits signals to the DNA-binding protein OmpR by phosphorylation (Egger et al,. 1997). Phosphorylation enhances the DNA-binding activity of OmpR and it regulates the expression of a regulon of genes that includes the outer membrane porin proteins OmpC and

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OmpF. Significantly, loss of expression of OmpC has been found to be largely responsible for the loss of virulence seen in ompB mutants of S. flexneri (Bernardini et al., 1993). How these factors influence expression of genes on the virulence plasmid is unknown.

Control at the level of translation The level of VirF protein in the cell is sensitive to tRNA modification via the tgt and miaA loci (Fig. 2). The tgt gene is responsible for the presence of quenosine at position 34 (the wobble position) while miaA is required for 2-methylthio-N6-isopentenyladenosine (ms2i6A37) at the 3’ side and adjacent to the anticodon (Curran, 1998). If VirF protein is overexpressed in a tgt or a miaA mutant, the effect is to restore full virulence (Durand et al., 2000). This indicates that the effect of these mutations is to reduce the level of translation of virF mRNA, and not that of any other mRNA in the cascade. An early report had suggested that the translation of VirFdependent icsA mRNA was influenced by a conserved virulence plasmid locus called virK (Nakata et al., 1992); how this fits with the more recent data on VirF translational control is unclear. It has been suggested that the sensitivity of virF mRNA translation to tRNA under-modification might explain previous reports in the References that over-expression of tRNA1Tyr and tRNA1Leu can suppress the deregulated expression of the virulence regulon seen in an hns mutant at 30 °C (Hromockyj et al., 1992). The proposal is that the drain on tRNA modification resources occasioned by the overexpression of these transfer RNAs could result in under-translation of VirF and thus the observed downregulation of virulence gene expression in the hns mutant (Durand et al., 2000).

Overview The Shigella virulence gene regulon is an example of a complex gene control system operating over three levels: transcription, translation, and protein secretion. This review has focused on the first two (Fig. 2), although it is becoming clear that all three are integrated to some degree (Demers et al., 1998). At the level of transcription, the regulon possesses specific control elements in the form of VirF and VirB, and sequence analysis of the virulence plasmid has shown the presence of other, as yet uncharacterized candidates for other transcription regulators, such as the AraC-like MxiE protein (Gallegos et al., 1997). The established regulatory link is that between VirF and virB; the mechanism by which the VirB protein influences the promoters of the structural genes downstream in the regulon remains

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unclear and may yet be shown to involve additional control factors. VirF is controlled at the translational as well as the transcriptional level, and this may open it to environmental influences additional to temperature, osmolarity and pH, such as iron starvation, amino acid availability, and oxygen limitation (Durand et al., 2000). If true, these influences would be expected to be fed through the regulon to affect structural gene expression; as yet, no hard data on this are available. The chromosomal loci tgt and miaA act on the regulon at virF indirectly through tRNA modification. However, other chromosomal loci, hns and ihfA and ihfB, exert a direct effect. The H-NS repression of virB transcription was characterized first, but the importance of its negative role at virF has now been recognized too. The mechanisms by which it acts at these two promoters are different, and the finding that it influences directly at least some structural gene promoters (Beloin and Dorman, unpublished) shows that this nucleoid-associated protein has a major function in controlling the regulon. IHF seems to be involved in relating virulence gene expression to growth phase and its influence is probably subtle, as befits a fine-tuning element. More overt transmitters of growth phase signals, such as the sigma factor RpoS, do not seem to have a role in the system (Dorman and Porter, 1998). Also exerting a direct effect is cpxR, the gene encoding the response regulator that binds at virF and regulates it in response to pH. The regulon is certainly sensitive to a multiplicity of environmental signals, but temperature retains a preeminent place among them. How the thermal signal activates virulence gene transcription is still not understood fully. Clearly, there is an important role for DNA supercoiling at both the virF and virB promoters. At the former it is concerned with assisting H-NS to repress virF transcription at low temperature, at the latter it enables bound VirF to activate virB transcription at the permissive temperature. However, the detail of how virB is activated by VirF is still lacking. This will continue to be just one of many important research topics associated with this fascinating gene regulatory cascade for the immediate future. Acknowledgements. This work was supported by grants ERBFMRXCT98-0164 from the European Union and SC/99/432 from Enterprise Ireland.

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