Virulence gene regulation in pathogenic Josée Harel, Christine Martin

To cite this version:

Josée Harel, Christine Martin. Virulence gene regulation in pathogenic Escherichia coli. Veterinary Research, BioMed Central, 1999, 30 (2-3), pp.131-155. ￿hal-00902563￿

HAL Id: hal-00902563 https://hal.archives-ouvertes.fr/hal-00902563 Submitted on 1 Jan 1999

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Virulence gene regulation in pathogenic Escherichia coli

Josée Harel Christine Martinb

’Groupe de recherche sur les maladies infectieuses du porc (GREMIP), département de pathologie et microbiologie, faculté de médecine vétérinaire, université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Quebec J2S 7C6, Canada b Laboratoire de microbiologie, Inra Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France

(Received 5 November 1998; accepted I January 1999)

Abstract - The ability to regulate gene expression throughout the course of an infection is important for the survival of a pathogen in the host. Thus, virulence gene expression responds to environmen- tal signals in many complex ways. Frequently, global regulatory factors associated with specific regulators co-ordinate expression of virulence genes. In this review, we present well-described reg- ulatory mechanisms used to co-ordinate the expression of virulence factors by pathogenic Escherichia coli with a relative emphasis on diseases caused by E. coli in animals. Many of the virulence-asso- ciated genes of pathogenic E. coli respond to environmental conditions. The involvement of global regulators, including housekeeping regulons and virulence regulons, specific regulators and then sensor regulatory systems involved in virulence, is described. Specific regulation mechanisms are illus- trated using the regulation of genes encoding for fimbriae, curli, haemolysin and capsules as exam- ples. © lnra/Elsevier, Paris.

Escherichia coli / virulence gene / regulation

Résumé - Régulation de l’expression des gènes de virulence chez les Escherichia coli pathogènes. Le contrôle de l’expression génétique d’un pathogène au cours d’une infection s’avère une propriété importante pour sa survie dans l’hôte. Ainsi, des signaux environnementaux influencent, par diverses voies, l’expression des gènes de virulence. Cette dernière est coordonnée par des facteurs de régulation généraux, lesquels sont souvent en association avec des éléments régulateurs spécifiques. Dans cette revue, des mécanismes de régulation bien étudiés impliqués dans l’expression des facteurs de viru- lence chez les Escherichia coli pathogènes sont présentés, une certaine emphase étant mise sur les E. cnli responsables des maladies animales. Chez les E. cnli pathogènes, l’expression de plusieurs gènes codant pour des déterminants de virulence est soumise aux conditions du milieu environnant. La régulation de l’expression des facteurs de virulence des E. coli pathogènes par des régulateurs glo- baux, lesquels incluent les régulons de gènes de ménage de même que des régulons de virulence, par

* Correspondence and reprints Tel.: (33) 4 73 62 42 47; fax: (33) 4 73 62 45 81; c-iiiail: cmartin(a-)clermont.inra.tr des régulateurs spécifiques ainsi que par des systèmes régulateurs réagissant à la détection d’un signal, sera présentée. Les mécanismes spécifiques de régulation seront illustrés en présentant, entre autres, des systèmes de régulation génétique concernant l’expression des fimbriae, de curli, de l’hémolysine et des capsules. © Inra/Elsevier, Paris. Escherichia coli / gène de virulence / régulation

1. INTRODUCTION terial survival during all the infectious pro- cess. For example, an adhesin can be advan- During the course of infection, tageous for the adhesion of a on pathogenic micro-organisms encounter dif- intestinal mucosa but becomes disavanta- ferent types of environments and they have geous when the bacteria is in the blood- to adapt in order to survive and multiply. stream or when the bacteria needs to escape Thus, pathogenic bacteria need to synthe- the bactericidal activity of serum. There- size virulence factors that will allow them to fore, micro-organisms sense the environ- colonize a hostile environment and to sur- ment and, in response to the signals that vive the host immune and non-immune they receive, act accordingly by turning off defences. However, the expression of a vir- or on the expression of their virulence genes ulence factor is not advantageous to the bac- [40, 51 ]. For the majority of the virulence factors, the specific host signals that the reg- 2. ENVIRONMENTAL ulatory protein detects are not yet under- REGULATION OF VIRULENCE stood, although the environmental signals GENE EXPRESSION that modulate the expression of virulence genes have in many cases been identified Not all virulence factors confer a selective in vitro. Parameters such as temperature, advantage to the microbe at the same stage osmolarity, pH, source of nitrogen, con- of infection or at the same anatomical site centrations of iron, sugars and amino acids within the host. Consequently, the expres- are known to affect the regulation of viru- sion of certain virulence factors must be lence genes in vitro. One challenge of the modulated in response to environmental sig- next few years will be to find out if these nals encountered throughout the infectious parameters play a role in virulence factor cycle and during the transition from the expression in vivo. external milieu to the host. Such regulation allows for the co-ordinated of The regulation of pathogenicity is com- expression for survival in different plex. There are interconnections between proteins required environmental niches. Virulence factors, regulatory systems. A single regulator can such as adhesins, toxins, etc., control the expression of several genes siderophores, must be in the (global regulator) including virulence genes synthesized only appropri- ate location in the host so that bacteria can and housekeeping genes. For example, the leucine responsive protein (Lrp), the cAMP multiply, colonize host tissues and evade the host immune receptor protein (CRP), the histone-like pro- response. tein (H-NS) and the sigma factor RpoS are In vivo, bacteria first need to sense that global regulators that are involved in regu- they have entered into a host. Temperature latory networks that control virulence genes, is an appropriate signal since the temperature genes encoding metabolic enzymes and of mammalian bodies is generally around structural components. Often, global and 37 °C, higher than the external environment. specific regulators simultaneously affect Thus, several virulence factors such as virulence gene expression which is opti- adhesins, haemolysin, or enteropathogenic mally co-ordinated to permit the survival of E. coli (EPEC)-, RDEC-1 (a rabbit EPEC a pathogen in a particular ecological niche. strain)- and STEC (shiga-like toxin pro- E. are Here, we review the well-described reg- ducing coli)-secreted proteins specif- at the normal host ulatory mechanisms used to co-ordinate the ically expressed body expression of virulence factors by temperature [1, 34, 71, 78, 89]. Iron avail- is also an indicator of the host envi- pathogenic E. coli with a relative emphasis ability ronment since iron is in the body on E coli causing diseases in animals. Some sequestered regulatory mechanisms that have been by specific proteins such as transferrin or described for other pathogenic bacteria lactoferrin. Thus, limiting iron concentra- the of viru- might also exist in pathogenic E. coli but tions often induce expression lence factors, as is the case for are not evoked here owing to the lack of siderophores, experimental results in E. coli. The regula- shiga-like toxins or haemolysin [ 18, 49, 75].]. tion of expression of virulence determinants Second, bacteria need to sense which by global regulators, including housekeep- organ they have reached. Bacteria travel- ing regulons and virulence regulons, spe- ling through the gastrointestinal lumen of cific regulators and then two component omnivorous mammals are subjected to a regulatory systems involved in virulence, low pH stress in the stomach, followed by a is presented. Specific regulation mecha- more hospitable environment with respect to nisms are illustrated using the regulation of pH in the intestinal lumen varying from pH genes encoding fimbriae, curli, haemolysin 6.5 to 7.5 [38]. The regulation of gene or capsules as examples. expression in response to changes in the pH is complex and is associated with pH varia- of ammonium in the colon might, however, tions, ion concentration, proton-motive force inform EPEC that it is not in an environ- and membrane potential [26, 100]. Acid tol- ment appropriate for survival, and thus erance is highly dependent on the growth adhesin expression is suppressed. Maximal acid resistance of the E. coli phase. Effects of the environmental factors strain MC4100 is exhibited at the station- described above have been studied in vitro. ary and is on growth phase dependent RpoS, Very few studies have been reported con- although RpoS is not an absolute require- cerning the regulation of E. coli virulence ment under all growth conditions [122]. factors expression in vivo, i.e. in the ani- of several adhesins and EPEC- Expression mal. The question is: are the regulations secreted proteins is inhibited at low or high depicted in vitro indeed occurring in the pH. By using promoter fusions to measure host? Experimental infections followed by the response according to changes in pH, it the detection of virulence factor expression was observed that the of fim- expression in the organs or tissues are necessary to brial such genes as fas (987P) and foo answer this concern. Pourbakhsh et al. [ 109] to in (F1651) responded changes pH [36]. inoculated chicken air sacs with septicaemic High concentrations of monoamines, most avian E. coli isolates and showed that a notably norepinephrine, may be one of the much higher proportion of bacteria colo- of the small intestine environment. signals nizing the trachea were Fl (type I fimbriae) can increase the Indeed, norepinephrine fimbriated as compared to bacteria colo- and of virulence factors growth production nizing the lungs, air sacs and systemic of ETEC (K99) and EHEC (SLT-1, SLT- organs, suggesting that the bacteria undergo and this in II), effect is non-nutritional nature a phase variation with respect to Fl fim- [81]. Osmolarity, carbon source, concen- briae in vivo. A similar Fl fimbrial phase tration of 02, ammonia, sodium bicarbonate variation also occurs in vivo during the and amino acids can combine to precisely experimental induction of E. coli peritonitis indicate the location of the bacteria inside an and lower urinary tract infection in mice [4, organ [35]. As an example, Edwards et al. 62, 97] and meningitis in rats [116]. In the [36] proposed that carbon and/or nitrogen same study Pourbakhsh et al. showed that gradients in the gut provide a mechanism P fimbriae are expressed in bacteria present that allows preferential colonization of dif- in air sacs and systemic organs, but not in ferent segments by various enteropathogens. trachea, suggesting that P fimbriae also Glucose concentrations are higher in the undergo phase variation in vivo [109]. The proximal small intestine than in the distal nature of the signal controlling the expres- portion, in contrast to nitrogen concentra- sion of these fimbriae is, however, unknown. tions which are higher in the distal segment. The aim of the next few years will be to The porcine 987P ETEC adhesin is maxi- identify the nature of the signals that work in mally expressed in conditions of limiting vivo, the sensing molecules involved, the carbon and in the presence of ammonium, mechanism of signal transduction, and to whereas the expression of the bundle form- determine their activities on virulence factors ing pili (Bfp) EPEC adhesin is optimal dur- in a given location within the host. ing growth with glucose as a carbon source and is repressed by ammonium [1 10, 35]. 3. GLOBAL REGULATORS OF Consequently, 987P fimbriated E. coli will GENE EXPRESSION colonize the distal small intestine. If Bfp expression is activated at an early stage of Global regulators are involved in the reg- infection, Bfp fimbriated EPEC will colo- ulation of virulence gene expression. In nize proximal as well as distant segments pathogenic bacteria, H-NS, CRP, RpoS, Lrp, of the small intestine. The high concentration play a role in sorting out complex signals that modulate the synthesis of virulence fac- only a structural role in the organization of tors. Some of them, such as CRP, recognize the chromosome, but also a rather dynamic specific nucleotide sequences to which they role in the regulation of gene expression bind. Their effects are confined to those [56, 63, 129]. Many of the genes regulated genes possessing these specific binding sites. by H-NS are involved in bacterial adaptation Others, such as H-NS, have a wider influ- to environmental stress in response to signals ence in controlling and con- such as osmolarity, temperature and oxy- tribute to the organization of DNA topol- gen availability [6]. Transcription of many ogy in the cell. It has been shown that levels virulence genes in E. coli is repressed by of supercoiling vary in response to envi- H-NS, and different transcriptional regula- ronmental changes; in particular, growth of tors act on gene expression by alleviating bacterial cells under anaerobic conditions, at and/or counteracting the effect of H-NS. In a high osmolarity, at different temperatures, some cases, H-NS seems to be implicated or in a nutrient-poor medium [29]. Topoi- in the thermoregulation of virulence factor somerases are enzymes that act upon DNA expression. The mechanisms by which H- to alter the level of supercoiling, as well as NS affects gene transcription are not com- catenate and decatenate chromosomes. In pletely understood. Correlation between the bacterial cell, DNA is negatively super- modifications of the overall DNA super- coiled [33]. It is thought that negative super- coiling induced by H-NS and gene expres- helical tension facilitates the melting of sion is not clear. In some cases, H-NS can DNA necessary for replication and tran- bind DNA to directly inhibit gene tran- scription [79]. This mechanism may be used scription [27, 129], and it has been suggested by pathogenic E.coli to regulate genes nec- that H-NS prevents the RNA polymerase essary for virulence. from productively interacting with the pro- moter [129]. G6ransson et al. [47] have E. coli low molecular pro- possesses weight that H-NS could act as ’silencers’ of which contribute to the order posed proteins higher DNA of the bacterial nucleoid and regions by forming nucleoprotein organization structures similar to inactive to the of the information. transcriptionally expression genetic chromatine. However, these effects are not Frequently, small architectural proteins such indiscriminate as not all promoters are H- as HU histone-like the protein (a protein), NS H-NS also in related IHF host fac- repressible. participates protein (integration the control of S Sev- H-NS and for inversion sigma synthesis [10]. tor), (factor eral examples of such regulations involved stimulation) contribute to the control of tran- in fimbriae and haemolysin synthesis as welll scription of genes whose products play a as in bacterial invasiveness are given below. role in environmental adaptation and thus in the expression of the virulence. 3.1.1. Regulatory role of H-NS 3.1. H-NS in fimbrial gene expression

H-NS is one of the two most abundant The sfa determinant codes for S fimbrial histone-like proteins in E. coli (the first one adhesins of extraintestinal E. coli. SfaA is being HU). It is a 16-kDa neutral protein the major structural subunit, whereas SfaB present at 20 000 copies per cell under a and SfaC are positive regulators of sfa tran- dimeric form (for a review, see [ 137]). H-NS scription. Most .operon is also derepressed at both temperatures although to a lesser extent [47, 136]. By competitive a consensus This feature gel retardation assays it was shown that H- specific sequence. NS specifically binds to pap DNA contain- could be a specific conformation such as an intrinsic curvature due to the known ing pap GATC sites and was able to block affinity of H-NS for curved DNA This was methylation of these sites in vitro. It was [137]. confirmed retar- suggested that the ability of H-NS to act as a recently by competitive gel methylation blocking factor is dependent dation assays where it was shown that H- NS binds to the and upon the formation of a specific complex of regions containing fimB H-NS with pap regulatory DNA. Transcrip- fimE promoters and does not affect the in vitro tion of pap is enhanced by binding of the switching frequency [105]. cAMP-CRP complex in the intergenic papl- Curli are thin fimbriae expressed at 26 °C papB DNA. In hns mutants, the pap operon in medium or low osmolarity. Curli expres- is expressed in the absence of the PapB and sion is dependent on RpoS, the sigma S fac- cAMP-CRP transcriptional activators. It has tor. Transcription of csgA (encoding the been suggested that the cAMP-CRP com- major structural subunit) can be activated plex would play a role as an anti-repressor in rpoS mutants by inactivation of hns, but alleviating the H-NS-mediated silencing [41].]. the temperature and osmolarity control is H-NS is also involved in the phase vari- maintained. Thus, neither of these two pro- ation control of type 1 fimbriae [27, 30, teins is responsible for this control [104]. Olsen et al. concluded that this tran- 101 ]. FimB and FimE are recombinases that [104] mediated or promote the inversion of a 314-bp DNA scriptional silencing directly segment containing the fimA promoter. indirectly by H-NS can be relieved by RpoS, FimA is the major structural subunit of type or by a non-identified regulatory protein 1 fimbriae. In hns mutants, transcription of positively controlled by RpoS. fimB and fimE is increased at 30 °C and to a lesser extent at 37 °C, leading to an 3.1.2. Regulatory role of H-NS in increased inversion rate of the fimA pro- enteroinvasive E. coli (EIEC) moter. Donato et al. [27] have shown that virulence gene expression H-NS specifically and co-operatively binds to the fimB promoter region and represses The genes required for EIEC invasive- transcription. They propose that H-NS rec- ness are carried on a large virulence plas- ognizes a specific DNA feature rather than mid. The ability of invasiveness is temper- ature dependent, as it is expressed at 37 °C and not at 30 °C. Dagberg et al. [21 have shown that H-NS plays a crucial role in the thermoregulation of virulence-associated genes in EIEC. The VirF protein is a positive regulator of virG and virB transcription. VirB activates transcription of ipaBCD and other genes present elsewhere on the plas- mid. IpaB, IpaC and IpaD are secreted pro- teins essential for invasion. Using phoA as a reporter gene, these authors have shown that deletion of hns results at 30 and 37 °C in an increase in virG transcription but has no effect on ipaBCD transcription in the absence of the VirF and VirB activators. By increasing the level of H-NS protein in the cell by cloning hns on a low copy vector, the expression of ipaBCD, virG and virB is of the hly genes. The regulatory mechanism which this is achieved is not clear but it repressed at 30 °C and to a lower extent at by 37 °C, whereas transcription of virF is does not seem to involve modification in the level of DNA poorly affected. Thus, the higher content of supercoiling [93]. H-NS in the cell causes enhanced ther- 3.1.4. on H-NS moregulation. These results suggest that H- Effect of typA expression NS acts directly on virG and virB transcrip- In E. coli K12, the inactivation of the tion and indirectly on ipaBCD transcription via VirB (fcgure 2). In the absence of H-NS, typA gene encoding the TypA protein (for alters the virG expression may become VirF inde- tyrosine phosphoprotein) expres- sion and the modification of several pendent. Thus, VirF is a transcriptional reg- pro- such as the ulator which alleviates and/or counteracts teins, disappearance of the uni- versal stress carbon starvation the effect of H-NS, as cAMP-CRP, SfaB protein UspA, and the increased and SfaC, or RpoS in the case of the pap, protein CsplS synthesis of H-NS The is .!fa, and csg , respectively. !39, 42]. TypA protein phos- phorylated on tyrosine residues in vivo and in vitro in the EPEC strain MAR001 but not 3.1.3. on Effect of H-NS haemolysin in K12 E. coli strains. The sequence of TypA production from the E. coli K-12 strain differs from that of an EPEC strain by six amino acid residues Haemolysin synthesis is repressed at high and the protein is three amino acids shorter. osmolarities and at low temperatures. Car- Freestone et al. [42] concluded that TypA mona et al. [20] have shown that the muta- could be a candidate for studying the role tion in the hha gene, encoding a putative of tyrosine phosphorylation in the global histone-like protein, derepresses haemolysin regulatory network. BipA of Salmonella production when cells grow either at low typhimurium is the homologue of TypA and temperature or in a high osmolarity medium. is implicated in pathogenesis. Another team, In a hns mutant as in the hha mutant, hlv’v Farris et at. [39] reported that BipA/TypA transcription increases about 2-fold com- of EPEC is a tyrosine-phosphorylated pared to the wild-type strain. However, in a GTPase that presents a new class of viru- hha-hns double mutant, haemolysin expres- lence regulators as it controls several pro- sion shows a 10-fold increase. Thus, H-NS cesses likely to be important for EPEC infec- participates in the modulation of expression tion: the formation of actin-rich pedestals in host epithelial cells, flagella-mediated cell briae, such as K88 expressed by ETEC or motility and resistance to the antibacterial BFP expressed by EPEC, is, however, not effects of a human host defence protein. under CRP-cAMP regulation. From that observation, a working model for the tem- 3.2. IHF (integration host factor) poral and spatial regulation of fimbrial expression in the small intestine by the car- bon source was Edwards and IHF is an abundant sequence-specific suggested by Schifferli where the of fim- DNA binding protein that induces a signif- [36], regulation brial the carbon source is a icant bend in the DNA and is involved in a expression by factor in the initial site of wide variety of processes in E. coli includ- major determining intestinal (see section ing regulation of gene expression. himA and binding 2). himD genes encode the subunits of IHF. There is an absolute requirement of IHF for 3.4. RpoS phase variation of type 1 fimbriae which uses site-specific recombination to switch rpoS encodes an RNA polymerase sigma phases. This recombination has an absolute factor (sigma S, sigma 38 or KatF). It con- requirement for IHF [37]. IHF also plays a trols a regulon of 30 or more genes in role in the expression of region I genes of response to starvation and during transition the type II capsule K5 operon [119J. Bind- to the stationary phase. During transition ing site consensus sequences were identi- into the stationary phase, the expression of fied in the vicinity of the transcription start sigma S-dependent genes is activated in a site of region 1 of the kps cluster. Mutations certain temporal order. The sigma S regulon in himA and himD led to a 5-fold reduction can be divided into subfamilies which in the expression of the capsular gene kpsE include genes regulated by specific stresses at 37 °C in region 1. This indicates a role and/or additional global regulatory proteins. of IHF in mediating the expression of region Subsets of sigma-dependent genes include I of the capsular gene cluster [119J.J. those genes that are also inducible by anaer- obiosis, oxidative or osmotic stress. Prod- 3.3. CRP ucts of several of these genes could be con- sidered as virulence factors, including the and which The global regulator CRP regulates a Hpl HpII hydroxyperoxidases confer resistances to as well as variety of genes in E. coli in response to the H20, CsgA, level of cAMP which is synthesized by the the main subunit of surface protein curli (see section Maximal acid resistance of adenylate cyclase in response to the carbon 8.4) [82]. E. coli MC4100 (and base resistance) is source. Catabolite repression is due to the inactivation of adenylate cyclase when glu- exhibited at the stationary phase and depends on is not an absolute cose is transported into the cell. The pro- RpoS although RpoS tein CRP when complexed with cAMP requirement under all growth conditions [ For acid resistance, and in base binds to a specific DNA sequence named 122]. part resistance, the can, how- the CRP-binding site. This sequence occurs rpoS requirement be overcome anaerobic in at different distances upstream of the tran- ever, by growth moderate acid. scriptional start site in different operons and leads to the activation of transcription. It is known that cAMP-CRP modulates expres- 3.5. Lrp sion of certain E. coli virulence factors such as enterotoxins STa and STb [3, 17J and The leucine responsive protein, Lrp, fimbriae such as Pap, F165, and F165z pili, affects the transcription of a large number of colonization factor antigen II, 987P and K99 genes, increasing the expression of some [22, 36, 46]. The expression of other fim- and decreasing that of others, some of which are turned on or off by exogeneous leucine by a chromosomally encoded protein in a [92]. It is noteworthy that Lrp is involved way similar to (plasmid encoded) in all regulatory mechanisms of fimbriae and enterobactin (chromosomally encoded). expression described so far (see section 8). In contrast to the slt-I promoter, the slt-II The physiological role of Lrp is unclear. and slt-llv promoters do not contain Fur Calvo and Matthews [19] suggest that Lrp boxes and are not controlled by iron avail- positively regulates genes that function dur- ability [ 125].] . ing famine and negatively regulates those working during a feast. They propose that pili may be expressed at higher levels during 4. FAMILY OF THE ARAC growth in a nutrient-deficient medium where TRANSCRIPTIONAL an abundance of pili may help bacterial ACTIVATOR adherence to epithelial cells, thus helping the maintenance of bacteria in the environ- AraC is the transcriptional regulator of ment. Under conditions of nutrient excess, the arabinose operon. Proteins in the family bacteria can grow rapidly and should not of AraC activator contain helix-turn-helix have any difficulty in maintaining their pres- motifs, which bind specific DNA sequences ence. Thus, abundant synthesis of fimbriae upstream of genes that are actively tran- would not be so critical for survival. scribed. The activation of virulence gene expres- 3.6. Fur sion in EPEC requires an AraC-homologous transcriptional activator protein called The exceedingly low availability of iron PerA/BfpT [45] which is encoded by a vir- in mammalian tissues is an environmental ulence plasmid, pMAR2. The genes of the locus are for signal indicating to the bacteria entry into per required transcriptional activation of whose are a host. This signal triggers the co-ordinate genes products expression of bacterial virulence determi- secreted by the type III secretion system and and nants through the Fur protein which plays a (eae, encoding intimin, espB) they are also involved in activation general role as a sensor of iron availability of plasmidic such as the adhesin encod- in the cell. The apoprotein binds Fez+ as a genes gene bfpA cofactor, and the cofactor-bound protein ing the bundle forming pilus [70, 110, 128]. binds to various sites, termed iron-boxes. Insertional inactivation of perA led to a reduced of Eae which could be This complex negatively regulates many expression due to mutations in or to genes involved in iron uptake as well as specific perA polar effects on downstream Puri- toxin genes hly and others. The synthesis of genes of pera. the Slt-I toxin, iron-chelating molecules fied PerA/BfpT was shown to bind directly (enterobactin and aerobactin siderophores) to DNA sequences upstream of bfpA and and membrane proteins involved in the bind- eae [128]. ing and uptake of iron-siderophore com- Regulatory proteins of some fimbrial plexes is repressed in low iron conditions operons such as coo (CS1), cfa (CFA/1), fap [ 18, 64, 133]. The Fur-iron complex binds to (987P) and (AAF/1) are designated as Rns- specific DNA sequences, the Fur boxes, like and share sequence identity with AraC. located in the operator regions of the iron- Rns is required for positive activation of the regulated operons [18, 25]. Fur does not CS I fimbrial genes. It was recently shown bind to its DNA target in the absence of iron that Rns is capable of complementing a null and genes are thus derepressed in conditions mutation in the S. flexneri virf gene encod- of low iron availability. It is interesting to ing the homologous counterpart of Rns note that slt-l genes are carried by a [108]. The VirF protein cannot, however, prophage and are nevertheless controlled complement Rns as an activator of CS 1 gene expression in ETEC. It was concluded that first autophosphorylates at a reactive histi- there are differences in the mechanisms by dine residue, then subsequently transfers the which these related transcription factors reg- phosphate to a conserved aspartate on RcsB. ulate gene expression. It is not known This form of RcsB may form a more stable whether the Rns class of regulatory proteins complex with RcsA and promotes cps tran- binds DNA or whether there are additional scription perhaps aided by RcsF [48, 50, factors in the regulatory network [78]. 69]. RcsA is an additional positive regulator and is subject to degradation by the Lon protease [68!.] . 5. SENSOR REGULATORY Effective mechanisms for the induction or PROCESSES repression of virulence gene expression involve the sensing of ’signature’ molecules In the Environmental signals control virulence produced by host tissues. this way, E. coli SoxRS and OxyR are gene expression in bacteria. A developing regulons to deal with the research field is understanding the ways in designed cytotoxic prod- ucts the which bacteria sense these signals and trans- produced during oxygen-dependent burst, with a number of duce them into the cell to regulate gene respiratory large induced in to response systems in proteins being response hydro- expression. Many signal anion bacteria operate by complex pathways gen peroxide (H20)’ superoxide (02) or nitric oxide (NO) neutrophils involving two-component regulatory sys- produced by and [82, 99]. The SoxRS reg- tems. These systems consist of: 1) the sen- macrophages ulon controls the of approxi- sor protein which spans the cytoplasmic expression ten SoxR is an iron membrane and monitors some environ- mately genes. sulphur that senses to mental parameters; and 2) the regulator pro- protein exposure superoxide and nitric oxide, and then activates the tran- tein that mediates an adaptive response, usu- of the soxS gene. SoxR and SoxS ally by a change in gene expression. When scription are SoxR a the sensor receives an external DNA-binding proteins, being component MerR-like is signal, it undergoes autophosphorylation member of the family (MerR involved in the to and SoxS and transfers the phosphate residue to the response Hg2+) a member of the Tran- regulator protein which in turn activates or being AraC-family. of soxS is initiated in a manner represses gene transcription. Several two- scription on the in to component systems controlling virulence dependent rpoS gene response [98]. The gene have been well character- entering stationary phase growth expression then activates the ized in different bacteria [40J. In E. coli, the SoxS protein transcrip- tion of a of genes sodA, of some group IK antigens as variety including expression the dis- well as colonic acid is regulated by the encoding Mn-dependent superoxide the DNA RcsABC (regulator of capsule synthesis) mutase, nfo, encoding repair endonuclease IV, and micF, which system. A temperature below 25 °C, the enzyme decreases the pro- presence of a high phosphate concentration post-transcriptionally and osmotic induction increase the synthe- duction of OmpF [82]. sis of these antigens [121]. The integral inner OxyR, a member of the LysR family of membrane RcsC sensor and the cytoplas- autoregulators, is also involved in resistance mic RcsB effector show homologies to the to oxygen-related compounds. The OxyR family of the two-component histidine protein activates the transcription of approx- kinase signalling systems [48 The mecha- imately nine genes in E. coli in response to nisms whereby extracytoplasmic signals are Hz02. These include katG, encoding HP1I sensed and transduced by the RcsC mem- catalase, and ahpFC, encoding NADPH- brane sensor are not unknown. Presumably, dependent alkyl hydroxyperoxidase, gorA, after receipt of a stimulatory signal, RcsC encoding a glutathione reductase and dps encoding a protein that non-specifically 6. tRNA REGULATION OF binds to DNA to protect cells from H20 VIRULENCE GENES toxicity. OxyR functions both as a sensor and a transducer and contains a critical Many virulence genes are located on redox-sensitive Cys residue that is oxidized ’pathogenicity islands’ (Pais), large chro- by hydrogen peroxide [117, 139]. The level mosomal regions that are often associated of oxyR mRNA or OxyR protein does not with particular tRNA genes [52]. It has been change significantly following exposure of shown that the Pais modulate virulence gene the cells to H202’ Hence, it was suggested expression through the action of these tRNA that post-translational regulation by an H20- [112]. In Shigella, it has also been demon- generated signal activates the pre-existing strated that expression of tRNA/yr partially OxyR protein [80]. complemented the virR mutation. virR is an analogue of hns and is required for co- P pili-mediated attachment may also be ordinating temperature-regulated virulence an of sensor pro- important part regulatory gene expression of Shigella [57]. cesses involved in uropathogenic E. coli The E. coli strain 536 during urinary tract infection. Zhang and uropathogenic :K15 carries two Normark [138] have shown that airs tran- (06 :H31) pathogenicity scription is specifically activated by P pili islands, one Pai comprising the gene cluster of and the other Pai attachment. The AirS protein is a sensor- haemolysin compris- regulator protein essential for the iron-star- ing the gene clusters of haemolysin and P- related fimbriae, both Pais flanked vation response of uropathogenic E. coli being by that activates the synthesis of the tRNA genes, leuX and selC, respectively. In the strain siderophore iron acquisition system. Fur- uropathogenic 536, spontaneous deletions in the truncation of leuX thermore, the two-component signal trans- resulting or the deletion of the duction system, CpxA-CpxR, has been sug- specific tRNA5 eul gene resulted in the lack of of 1 gested to play a role in the expression of expression type virulence factors of E. coli via P pili attach- fimbriae and other virulence factors such as aerobactin serum ment. Hultgren [61] hypothesized that the haemolysin, production, resistance and while adhesion of P pili to epithelial cells avoids phenotype motility, polymerization of the pilin fibre and thus trans-complementation of tRNA loci leads to a restoration of these 127]. leads to aggregation of misfolded pilus sub- properties [91, units in the periplasm. Protein aggregation in tRNA5&dquo;1 is specific for the minor leucine the periplasm is sensed by the CpxA-CpxR codon UUG. It has been shown that is for efficient transla- system, which activates htrA encoding DegP tRNA5 eul required tion of FimB whose contains five TTG (a periplasmic protease degrading misfolded gene proteins) [23, 107], and could be involved in codons recognized by tRNA5’cu that in turn controlling expression of virulence factors leads to type 1 fimbrial expression [113J.]. such as haemolysin or CNF (cytonecrotizing The selenocysteine-specific tRNA (selC! factor). Thus, the interaction between the directly influences the ability of the E. coli pathogen and its host receptors mediated by strain 536 to grow under anaerobic condi- adhesins may be a means for bacteria to tions because selenocysteine is part of the sample and consequently elicit the appro- formate dehydrogenase (FDH) enzyme priate response upon their arrival at a poten- which is involved in mixed acid fermenta- tial colonization site. Thereby, bacterial tion. The ability to grow under anaerobic attachment to mucosal surfaces via P pili conditions via mixed acid fermentation may indicates that bacteria have reached an eco- be important for the colonization of E. coli logical niche where expression of virulence in the kidney because oxygen-limiting con- factors (siderophores, toxins) are necessary ditions are found in deeper regions of the for survival. kidney [112].]. 7. BLACK HOLES AND operon (encoding for the F1845 fimbriae) VIRULENCE GENE EXPRESSION [ 12]. Transcriptional organization of the daa operon is quite different from that of pap since the fimbrial subunit daaE Not only deletion of genes such as leuX major gene is located at the 3’ end of the A can affect the expression of virulence, but operon. large in addition of some genes can also alter the transcript encoding accessory proteins virulence of bacteria. It was observed that addition to the major subunit is submitted Shigella and enteroinvasive E. coli displayed to endoribonucleolytic cleavage to gener- deletions in the cadA region present in most ate a stable daaE mRNA. This cleavage E. coli strains including E. coli K12 [84]. requires neither RNAse III nor RNAse E. These deletions have been termed black Second, expression of fimbriae is controlled holes and they enhance the expression of by environmental signals through alterations in of virulence genes. Indeed the introduction of transcript levels. The control fimbriae is of the cadA gene encoding lysine decarboxy- expression crucial importance for the lase attenuates the virulence of Shigella survival of bacteria in vivo [35]. It allows fimbriae to be at the ade- flexneri and the enterotoxin activity was expressed only site in the host and to evade the host’ss inhibited by cadaverine, a product of the quate reaction catalysed by lysine decarboxylase. immune response. All fimbrial operons examined so far encode specific regulatory proteins that either activate or repress tran- In such 8. REGULATION OF FIMBRIAL scription. addition, global regulators as roles in EXPRESSION Lrp, CRP, H-NS, IHF, RpoS, play controlling the expression of most fimbrial operons. Four different regulatory mecha- Synthesis of fimbriae is under complex nisms controlling the expression of fimbriae regulatory controls. First, assembly of fim- have been described. Operons that share briae on the levels rel- depends appropriate common regulatory properties are classi- ative to one another of major and minor fim- fied in table I. brial subunits, the chaperon, outer membrane pore and regulatory proteins. The major fim- brial subunit must be expressed at higher 8.1. The levels than other accessory proteins. Fim- pap regulatory family brial determinants are organized in operons and differential expression of genes is Regulation of the pap expression is the achieved by post-transcriptional mecha- most extensively studied. Expression of P nisms. In the pap operon, encoding P fim- fimbriae is subject to phase variation, i.e. briae, the two first cistrons, papA and papB, fimbrial expression switches between ON are co-transcribed to mRNA from the papB (fimbriae-positive cells) and OFF (fimbriae- promoter. Differential expression of these negative cells) states. The switch frequency two genes results from RNAse E-dependent is controlled by environmental factors such endonucleolytic cleavage of the mRNA in as temperature or carbon source. Phase vari- the intercistronic papB-papA region. This ation of P fimbriae depends on the mcthy- cleavage is followed by rapid decay of the lation status of two GATC sites located in upstream papB-encoding region and accu- the intercistronic region between the regu- mulation of the stable papA-encoding latory genes papl and papB which are tran- mRNA, leading to high level expression of scribed divergently (figure 3). The two the major subunit, PapA, relative to that of GATC sites, GATC-I and GATC-II, are the regulatory protein PapB 194 Differen- located within the Lrp-binding sites. tial mRNA stability and mRNA processing Schematically, when Lrp binds the non- are also involved in the control of the daa methylated GATC-II site, transcription from OFF and ON states requires DNA replica- tion to alter the methylation status of GATC- I, because binding of Lrp-Papl to this site is inhibited by full methylation. PapB acti- vates papf transcription and represses papBA transcription (figure 3) [15, 66, 67, 95, 96, 130, 132]. Several other fimbrial operons are con- trolled by analogous mechanisms with slight variations. sfa (S fimbriae), daa (F1845), clp (31A), fae (K88),,foo (F]651) operons encode for proteins with significant homolo- gies to Papl and PapB [22, 60, 83, 87]. Their expression is controlled by Lrp and Dam methylation. daa, vfa, fbo and clp operons are under phase variation control but fae is not. Lrp is required for the OFF to ON switch of the phase variation-controlled operons. clp and foo are the only operons described so far that belong to this regulatory family in which expression is modulated by the level of leucine and alanine in the growth medium. It has been shown that these amino acids totally inhibit the OFF to ON switch of clp but the precise molecular mechanism is not yet known [83]. Lrp and the specific reg- ulators are not only involved in phase vari- ation control but also in the regulatory mech- anisms controlling the level of transcription in phase ON cells or in cells not subjected to phase variation. Martin [83] has shown that Lrp and CIpB repress clp transcription in phase ON cells and that this effect requires Dam methylation. Lrp also acts as a negative regulator of the fae operon which is not under phase variation control [60]. Several E. coli strains possess determi- nants for the synthesis of two or more fim- briae. Cross regulation of two unlinked fim- brial operons belonging to the same regulatory family has been demonstrated. the pB promoter is locked and cells are in the Regulatory p!f7 and prfB genes of the pef OFF state. Under conditions of high cAMP operon encoding for P-related fimbriae are levels, Papl is expressed and binds to Lrp, able to complement mutations in sfac and resulting in a shift in Lrp binding from the sfaB regulatory genes in a uropathogenic E. GATC-11 site to the GATC-1 site. Binding of coli isolate. Prfl and PrfB share 87 and 76 % Lrp-PapI to GATC-I activates transcription homology with SfaC and SfaB, respectively. from the pB promoter and switches the cells Moreover, mutations in pr!Z and prfB lead to to the ON state. The switch between the significant reductions in sfa transcription [88].

Lrp on the switch. The model of Roesch and Blomfield [1 14] assumes that leucine pro- motes the selective dissociation of Lrp from one DNA binding site within a multimeric nucleoprotein complex, resulting in a struc- ture that is more favourable for recombina- tion than that produced by the Lrp bound to all binding sites. The integration host factor (IHF) is nec- essary for the fim inversion. IHF could play a direct role in site-specific recombination or it could modulate transcription of,fitiib andlor,lii7iE [31, 37].] . The histone-like protein H-NS has a neg- ative influence on the rate of recombina- tion. In a hns mutant, transcription of both fimB and,fiiiie is enhanced [ 101 J. Donato et al. [27 ! have shown that H-NS interacts directly with the fimB promoter sequences. This results in a promoter-specific repres- sion. The alternative sigma factor RpoS exerts a negative control on,firnA (encoding the major fimbrial subunit) and firrrB promot- ers by an unknown mechanism and influ- ences DNA inversion in broth 8.2. of I fimbriae negatively Regulation type cultures [32].]. expression

Phase variation controls the expression 8.3. Regulation of K99 expression of type I fimbriae (fin2 operon) by site-spe- cific recombination. Recombination leads The of K99 is less to the inversion of a element. regulation expression 314-bp well understood. The K99 determinant According to the orientation of the promoter, encodes eight genes organized in three tran- fimbriae are either produced or not pro- scriptional units. Region I encodes f(inA-D, duced. The inversion process is carried out two FimB and region 2.fanE-F, region 3,fanG-H [65!. The by integrase-like proteins, is the fimbrial sub- FimE. FimB is involved in ON to OFF and jhiac gene product major unit, whereas are OFF to ON inversion, FimE in ON to products of/o/;t and /!/tB only related to each other and to None of OFF inversion [2, 73 In addition, four PapB. is related to K99 modulate switch: theÚ/1 ’/ genes pcrpl [115]. global regulators the fim is not controlled varia- Lrp, IHF, H-NS and RpoS (figure 4). expression by phase tion but is repressed by leucine and alanine. Lrp stimulates the,/im inversion in a The global regulators CRP and Lrp are both Dam-independent manner. Lrp slightly required for K99 exprcssion [ 15, 65 Lo- increases the transcription of.fimB, decreases Tscng ct al. 177 j demonstrated that Lrp fimE transcription by 2-fold 113], and binds affects only the transcription of regionI with high affinity to the switch element in genes, whereas Inoue et al. have shown that vitro [43!. Leucine potentiates the effect of CRP is required for expression of regionI and 2 genes [15, 65]. Differential methyla- Transcription of csgBA and csgDEFG is tion due to Lrp is, however, not involved in dependent upon RpoS in wild-type cells, the regulation of K99 expression, as it is for but not in a hns background [53, 104]. The the fimbriae of the P regulatory family. The requirement of RpoS for transcription of mechanism by which leucine and alanine csgBA may be indirect via CsgD with CsgD affect K99 synthesis remains unknown. The being necessary for csgBA transcription. role of FanA and FanB remains obscure. Temperature and low osmolarity control of Frameshift mutations in FanA or FanB fibronectin binding as well as growth phase reduced the level of K99 production 8- and control of csgBA transcription are main- 16-fold, respectively, but did not have much tained in a rpoSIhns double mutant, indi- effect on transcription from the fanA pro- cating that neither H-NS nor RpoS is respon- moter. sible for these regulatory responses [5, 53]. Considering that CsgD is required for csgBA transcription and is produced in a hnslrpoS 8.4. Regulation of curli expression double mutant, Hammar et al. [531 hypoth- esized that CsgD may have the capability to to starvation and/or A new type of fimbriae has been respond signals high described, whose expression is maximal in cell density by activating csgBA transcrip- conditions which are characteristic of an tion. This is a seductive hypothesis since extraintestinal environment, i.e. low tem- CsgD belongs to the LuxR family of tran- perature (26 °C) and low osmolarity. These scriptional activators involved in quorum in section fimbriae, referred to as curli [102], are syn- sensing regulation (see 11).). thesized by pathogenic and non-pathogenic E. coli as well as by other Gram-negative bacteria [28]. They bind fibronectin, plas- 9. REGULATION OF HAEMOLYSIN different sera and tissue minogen, proteins EXPRESSION and the dye Congo red. Curli formation involves a novel and a so far unique assem- mechanism not under- bly yet completely E. coli is a well-char- stood. haemolysin (Hly) acterized member of the RTX family of Two divergently transcribed operons are cytotoxins associated with urinary tract necessary for curli formation, c.sgBA and infections and other extra-intestinal E. coli csgDEFG [53]. CsgA, the fibre subunit, is infections [134, 135]. Synthesis of Hly is first secreted in the extracellular milieu as a directed from an operon consisting of four soluble protein and then polymerized at the contiguous genes, hIyCABD. The hlyA gene cell surface in a CsgB-dependent manner encodes the component of haemolysin, [54]. CsgG is an outer membrane-located which undergoes HlyC-dependent acyla- lipoprotein required for stable maintenance tion. Sec-independent haemolysin secretion of CsgA and CsgB [76]. CsgD is a tran- requires HlyB and HlyD. Two transcripts scriptional activator of the csgBA promoter initiating at the same promoter located belonging to the LuxR family [53]. Regu- upstream of hlyC are synthesized: a major lation of curli expression is complex and hlyCA mRNA and a minor hIyCABD involves at least two global regulators, H-NS mRNA. The higher stability of the major and RpoS. So far, no report presents evi- transcript accounts in part for the differential dence for a role of Lrp in this regulation. expression of HlyC and HIyA relative to Thus, Lrp seems to be involved in regulating HlyB and HlyD. Hly expression is regulated expression of fimbriae synthesized in con- by several mechanisms involved in mRNA ditions that mimic the in vivo environment. stability, DNA supercoiling, transcription Curli are produced in the stationary phase. elongation and activity of the product. 9.1. DNA supercoiling

Hly expression is modulated by envi- ronmental factors such as osmolarity, tem- perature and anaerobiosis [89]. Low tem- perature and high osmolarity repress haemolysin expression. The Hha protein is representative of a new class of modulators of gene expression in enterobacteria [24]. Carmona et al. have shown that Hha influ- ences DNA topology and suggest that it is a histone-like protein [20]. They have estab- lished a relationship between the hha muta- tion and an increase in haemolysin synthe- sis through changes in DNA topology. The Hha protein participates in the temperature- and osmolarity-dependent regulation of Hly expression in a manner that strikingly resem- bles what has been shown for H-NS with other virulence determinants. The hha muta- tion significantly increases expression of hLy genes in high osmolarity media and at low temperatures [90]. The Hha protein is highly similar to the YmoA protein from Yersdnia enterolitica. The hha gene can com- plement the yrreoA mutation and vice versa [ 9, 85 The complementation appears to be dependent on gene dosage 19].].

9.2. Transcription elongation

hIyCABD transcription elongation is modulated by the RfaH protein which con- trols a regulon governing the synthesis, export and assembly of cell surface and extracellular components that influence DNA transfer and virulence: RfaH is a pos- itive regulator of rfa (encoding the LPS core), tra (encoding the F pili) and kps (encoding the production of type 2 capsule) sequence. Bailey et al. [8] propose that the recruits the RfaH to the gene expression [8]. RfaH abolishes tran- ops element protein scriptional polarity within a transcript, transcription complex. Then the RfaH-influ- increasing transcription of distal gencs in enced elongating RNA polymerase com- operons. The non-encoding regions of all plex resists transcription termination sig- RfaH-affected operons contain all or a por- nals downstream of the ops element (figure tion of a conserved 39-bp sequence named 5). This model is supported by the signifi- JUMP-Start. The ops element is the second cant amino acid similarities between RfaH half of a direct repeat in the JUMP-Start and NusG, an essential transcription elon- gation factor required for effective anti-ter- Group II K antigens have a lower molec- mination by the N protein of bacteriophage ular mass and a higher charge density than lambda. In the hly operon, transcriptional group I K antigens, are coexpressed with a polarity is due to a Rho-independent termi- variety of LPS and expressed at 37 °C (at nator between hlyA and hlyB genes. Co- the physiological temperature). K 1 and K5 operation between RfaH and the cis acting kps clusters have been determined. The syn- ops element enables the RNA polymerase thetic, regulatory and export components complex to proceed through the hlyA-hlyB for capsule expression are encoded in three intergenic terminator [7]. functionally distinct gene blocks [1111. The kps cluster of K1 is functionally divided into three regions. These regions are organized as 9.3. Stabilization of the active two convergently transcribed operons conformation of the toxin inserted into a monocistronic tRNA gene pheV. The central region, region 2, contains a cassette that is flanked on LPS may play a role in protecting the biosynthetic secreted HlyA protein from degradation. either side by regions I and 3 whose genes function in more of Removing the core sugars from LPS affects general aspects capsule the kinetics and stability of secreted biosynthesis. Mutations in either region 1 haemolytic activity. The model proposed or region 3 tend to cause accumulation of intracellular within the cell. by Bauer et al. [11] suggests that Hly exists polysaccharides This that these are as a complex including LPS. These suggests regions impor- tant for the of to molecules combine to form an active toxin transport polysaccharides whose stability is aided by the LPS inner the cell surface. The six genes of the region I are transcribed in the same direc- core. When the inner core is incomplete, operon tion LPS and Hly form large, inactive aggre- as pheV. gates, and render Hly more susceptible to Region I of the K5 capsular gene cluster decay. in which kpsFEDUCS genes were identi- fied is transcribed as a single transcriptional unit that is processed to yield a smaller tran- 10. REGULATION OF CAPSULE script specific for the kpsS gene which is at SYNTHESIS the 3’ end of the transcript 1119 Processing of mRNA appears to be implicated in a dif- ferential of Mutations Group I K antigens are characterized by expression kp.r genes. in the himA and himD which encode a high molecular mass (over 100 kDa) and genes the subunits of IHF led to a reduction in the a low charge density. The coexpression of of at 37 °C the group IK antigens and LPS is restricted expression KpsE [119]. Region I is submitted to a with to a few LPS serotypes (primarily 08, 09 thermoregulation no at 18 °C. of the and 020) [111 Expression of some group 1 transcription Expression is tran- K antigens as well as colonic acid is regu- region 1 operon thermoregulated by lated by the RcsABC (regulator of capsule scriptional control of its first gene, kp.sF It that of 1 synthesis) system (see section 5). A mem- [ 119J. appears regulation region in to is brane-anchored DjIA protein, a member of expression response temperature mediated neither the rimJ nor hha the DnaJ ’J-domain’ family, acts in concert by has, all of which have been with RcsB/C two-component system to gene products, impli- cated in increase induction of the cps (capsular temperature-dependent regulation in other polysaccharide) operon [69]. Moreover, the gene expression systems [ 119]. cps operon activation by DjIA is dependent A mutation in the rfaH gene abolishes upon DnaK (Hsp70) and GrpE, which are the K5 capsule expression at 37 °C [123]. parts of a chaperon machine. Expression of region I is not mediated by RfaH, which is required for expression of of ’signature’ molecules produced by host region 3 at 37 °C and the expression of rfa tissues [82]. Probing the host environment genes [1 19, 123]. Stevens et al. [124], pro- with tools that define the genes expressed posed a model where RfaH regulates expres- by bacteria during infection will provide a sion of the E. coli group II capsule gene better definition of the microbial-host inter- clusters by allowing readthrough transcrip- action at both ends of the spectrum. At a tion to proceed from region 3 into region 2. higher level, one needs to understand how The non-encoding region upstream of the signals are detected and interpreted to int7u- clusters involved in the production of vari- ence gene expression and what are the effec- ous polysaccharide antigens contains a 39- tor signals produced by the host tissues. bp sequence (a sequence upstream of kspM More specifically, since several type III of region 3 of the kps cluster, outer core rfa, secretion systems of attaching and effacing 0-antigens rfb of LPS). This was referred E. coli are activated by the contact of the to as the JUMP-Start sequence and it has bacteria with the surface of eukaryotic cells, been postulated to be involved in the tran- the questions raised are: what is the nature of scriptional regulation of bacterial gene clus- eukaryotic cell surface signals that activate ters encoding surface proteins. The JUMP- the type III secretion systems, and what are Start sequence could cause RfaH-dependent the regulatory proteins involved in the reg- antitermination at other Rho-dependent ter- ulation of these secretion systems [58]? minators suggesting that the JUMP-Start Another of E. coli virulence factor sequence may function, in a manner anal- aspect that is under is the role ogous to a lambda nut site, in the regulation expression scrutiny of the detection in E. cnli of bacterial gene clusters encoding surface population density virulence. In such as polysaccharides (see section 9.2) (figure 5). pathogenic bacteria, f.sfM!/M!na.s, quorum sensing plays a defined role in virulence. Quorum sensing is a which bacteria sense and 11. PERSPECTIVES phenomenon by respond to their own population density by releasing and sensing pheromones. In Gram- The of the of viru- regulation expression bacteria, quorum is often lence has been studied in vitro. negative sensing genes mainly performed by the LuxR family of tran- with the advent of new Nowadays, genetic scriptional regulators, which affect pheno- methods, the identification of the regulation types as diverse as conjugation, biolumi- mechanisms that are induced in vivo as well nescence and expression of virulence genes. as the determination of their activities on In Gram-negative bacteria the most com- virulence factors in a location within given mon form of quorum sensing is mediated the host are now New methodolo- ongoing. by the and subsequent percep- for the of in vivo production gies study gene expres- tion of autoinducers, the acylated homoser- sion allow the isolation of bacterial genes ine lactones (acyl HSLs). By sensing the expressed during infection [55, 106]. Using density of a secreted autoinducer, bacteria reporter fusions, genetic genetic expression can sense if there is a quorum of their pop- at the cellular level within infected animals ulation sufficiently present to initiate the could be examined the use [40]. Through appropriate biochemical reaction. Homo- of reporter molecules such as the green flu- logues of LuxR, such as SdiA (suppressor of orescent protein and others under develop- cell division inhibition) and CsgD have been it will be to ment, possible explore identified in E. coli [53, 120[. SdiA-medi- microbe-host in time in the interaction real ated autoinduction with RpoS regulates host [40]. living f’tsQA whose products are involved in cell Effective mechanisms for the induction of division [ 120]. Recently, by using the Vib- virulence genes could involve the sensing rio hcirveyi mutant that responds exclusively to an uncharacterized signal molecule AI- controls phase variation of type I fimbriae of 2, Surette and Bassler demonstrated E.scherichia coli, Proc. Natl. Acad. Sci. USA [126] 82 (1985) 5724-5727. that cell-free culture fluids of E. coli and S. 131[ Alderette J.F., Robertson D.C., Repression of typhimurium can contain high levels of AI- heat-stable enterotoxin synthesis in enterotox- 2-like factors. The role of this putative igenic Escherichin coli, Infect. Immun. 177 ( 1977) 629-633. autoinducer and SdiA in quorum sensing for E coli and the of [4] Alkan M.L., Wong L., Silverblatt F.J., Change pathogenic regulation in degree of type I piliation of Escherichia coli the expression of virulence factors remains during experimental peritonitis in the mouse, to be determined. Infect. Immun. 54 (1986) 549-554. [5] Arnqvist A., Olsen A., Normark S., S- Given that most Sigma virulence genes appear to dependent growth-phase induction of the csgBA be regulated, regulatory systems are obvious promoter in Escherichia coli can be achieved in vivo 70 in the targets for the development of new thera- by sigma absence of the nucleoid-associated protein H-NS, Mol. Micro- peutic drugs. The identification of molecu- biol. 13 (1994) 1021-1032. lar associated targets including regulators [6! Atlung T., Ingmer H., H-NS: a modulator of with adaptation/survival and pathogenesis environmentally regulated gene expression, offers the prospect of expanding the hori- Mol. Microbiol. 24 (1997) 7-17. M.J., Koronakis zons of anti-infective therapy beyond the [71 Bailey Hughes C., V., Increased distal gene transcription by the elon- confines of that are bacteri- agents merely gation factor RfaH, a specialized homologue cidal or bacteriostatic [74]. It could increase of NusG, Mol. Microhiol. 22 (1996) 729-737. the level of therapy beyond that currently [8J Bailey M.J., Hughes C., Koronakis V., RfaH and the element, of a novel possible with antibiotics alone. Thus, a bet- op.s components system controlling bacterial transcription elon- ter understanding of the regulation mecha- gation, Mol. Microbiol. 26 (1997) 845-851.1. nisms that govern the expression of viru- [9] Balsalobre C., Juarez A., Madrid C., Mourino lence factors not only in vitro but also M., Prenafeta A., Munoa F., Coiriplementation temporally and spatially within the host willl l of the hhn mutation in Escherichia coli by the ymoA gene from Yersinin fn