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Virulence gene regulation in pathogenic Escherichia coliJosée Harel, Christine Martin

To cite this version:Josée Harel, Christine Martin. Virulence gene regulation in pathogenic Escherichia coli. VeterinaryResearch, BioMed Central, 1999, 30 (2-3), pp.131-155. �hal-00902563�

Review article

Virulence gene regulationin pathogenic Escherichia coli

Josée Harel Christine Martinb

’Groupe de recherche sur les maladies infectieuses du porc (GREMIP), département de pathologieet microbiologie, faculté de médecine vétérinaire, université de Montréal,

3200 Sicotte, Saint-Hyacinthe, Quebec J2S 7C6, Canadab 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 importantfor 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 specificregulators 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 Escherichiacoli 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 globalregulators, including housekeeping regulons and virulence regulons, specific regulators and thensensor 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

* Correspondence and reprintsTel.: (33) 4 73 62 42 47; fax: (33) 4 73 62 45 81; c-iiiail: cmartin(a-)clermont.inra.tr

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 propriétéimportante pour sa survie dans l’hôte. Ainsi, des signaux environnementaux influencent, par diversesvoies, l’expression des gènes de virulence. Cette dernière est coordonnée par des facteurs de régulationgénéraux, lesquels sont souvent en association avec des éléments régulateurs spécifiques. Dans cetterevue, 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 plusieursgènes 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

des régulateurs spécifiques ainsi que par des systèmes régulateurs réagissant à la détection d’unsignal, sera présentée. Les mécanismes spécifiques de régulation seront illustrés en présentant, entreautres, des systèmes de régulation génétique concernant l’expression des fimbriae, de curli, del’hémolysine et des capsules. © Inra/Elsevier, Paris.

Escherichia coli / gène de virulence / régulation

1. INTRODUCTION

During the course of infection,pathogenic micro-organisms encounter dif-ferent types of environments and they haveto adapt in order to survive and multiply.Thus, pathogenic bacteria need to synthe-size virulence factors that will allow them tocolonize a hostile environment and to sur-vive the host immune and non-immunedefences. However, the expression of a vir-ulence factor is not advantageous to the bac-

terial survival during all the infectious pro-cess. For example, an adhesin can be advan-tageous for the adhesion of a bacteria onintestinal mucosa but becomes disavanta-

geous when the bacteria is in the blood-stream or when the bacteria needs to escapethe bactericidal activity of serum. There-fore, micro-organisms sense the environ-ment and, in response to the signals thatthey receive, act accordingly by turning offor on the expression of their virulence genes[40, 51 ]. For the majority of the virulence

factors, the specific host signals that the reg-ulatory protein detects are not yet under-stood, although the environmental signalsthat modulate the expression of virulencegenes have in many cases been identifiedin vitro. Parameters such as temperature,osmolarity, pH, source of nitrogen, con-centrations of iron, sugars and amino acidsare known to affect the regulation of viru-lence genes in vitro. One challenge of thenext few years will be to find out if these

parameters play a role in virulence factorexpression in vivo.

The regulation of pathogenicity is com-plex. There are interconnections betweenregulatory systems. A single regulator cancontrol the expression of several genes(global regulator) including virulence genesand housekeeping genes. For example, theleucine responsive protein (Lrp), the cAMPreceptor protein (CRP), the histone-like pro-tein (H-NS) and the sigma factor RpoS areglobal regulators that are involved in regu-latory networks that control virulence genes,genes encoding metabolic enzymes andstructural components. Often, global andspecific regulators simultaneously affectvirulence gene expression which is opti-mally co-ordinated to permit the survival ofa pathogen in a particular ecological niche.

Here, we review the well-described reg-ulatory mechanisms used to co-ordinate theexpression of virulence factors bypathogenic E. coli with a relative emphasison E coli causing diseases in animals. Someregulatory mechanisms that have beendescribed for other pathogenic bacteriamight also exist in pathogenic E. coli butare not evoked here owing to the lack ofexperimental results in E. coli. The regula-tion of expression of virulence determinantsby global regulators, including housekeep-ing regulons and virulence regulons, spe-cific regulators and then two componentregulatory systems involved in virulence,is presented. Specific regulation mecha-nisms are illustrated using the regulation ofgenes encoding fimbriae, curli, haemolysinor capsules as examples.

2. ENVIRONMENTALREGULATION OF VIRULENCEGENE EXPRESSION

Not all virulence factors confer a selectiveadvantage to the microbe at the same stageof infection or at the same anatomical sitewithin the host. Consequently, the expres-sion of certain virulence factors must bemodulated in response to environmental sig-nals encountered throughout the infectiouscycle and during the transition from theexternal milieu to the host. Such regulationallows for the co-ordinated expression ofproteins required for survival in differentenvironmental niches. Virulence factors,such as adhesins, toxins, siderophores, etc.,must be synthesized only in the appropri-ate location in the host so that bacteria can

multiply, colonize host tissues and evadethe host immune response.

In vivo, bacteria first need to sense that

they have entered into a host. Temperatureis an appropriate signal since the temperatureof mammalian bodies is generally around37 °C, higher than the external environment.Thus, several virulence factors such asadhesins, haemolysin, or enteropathogenicE. coli (EPEC)-, RDEC-1 (a rabbit EPECstrain)- and STEC (shiga-like toxin pro-ducing E. coli)-secreted proteins are specif-ically expressed at the normal host bodytemperature [1, 34, 71, 78, 89]. Iron avail-ability is also an indicator of the host envi-ronment since iron is sequestered in the bodyby specific proteins such as transferrin orlactoferrin. Thus, limiting iron concentra-tions often induce the expression of viru-lence factors, as is the case for siderophores,shiga-like toxins or haemolysin [ 18, 49, 75]. ].

Second, bacteria need to sense which

organ they have reached. Bacteria travel-ling through the gastrointestinal lumen ofomnivorous mammals are subjected to alow pH stress in the stomach, followed by amore hospitable environment with respect topH in the intestinal lumen varying from pH6.5 to 7.5 [38]. The regulation of geneexpression in response to changes in the pH

is complex and is associated with pH varia-tions, ion concentration, proton-motive forceand membrane potential [26, 100]. Acid tol-erance is highly dependent on the growthphase. Maximal acid resistance of the E. colistrain MC4100 is exhibited at the station-

ary growth phase and is dependent on RpoS,although RpoS is not an absolute require-ment under all growth conditions [122].Expression of several adhesins and EPEC-secreted proteins is inhibited at low or highpH. By using promoter fusions to measurethe response according to changes in pH, itwas observed that the expression of fim-brial genes such as fas (987P) and foo(F1651) responded to changes in pH [36].High concentrations of monoamines, mostnotably norepinephrine, may be one of thesignals of the small intestine environment.Indeed, norepinephrine can increase thegrowth and production of virulence factorsof ETEC (K99) and EHEC (SLT-1, SLT-II), and this effect is non-nutritional in nature[81]. Osmolarity, carbon source, concen-tration of 02, ammonia, sodium bicarbonateand amino acids can combine to preciselyindicate the location of the bacteria inside an

organ [35]. As an example, Edwards et al.[36] proposed that carbon and/or nitrogengradients in the gut provide a mechanismthat allows preferential colonization of dif-ferent segments by various enteropathogens.Glucose concentrations are higher in theproximal small intestine than in the distalportion, in contrast to nitrogen concentra-tions which are higher in the distal segment.The porcine 987P ETEC adhesin is maxi-mally expressed in conditions of limitingcarbon and in the presence of ammonium,whereas the expression of the bundle form-ing pili (Bfp) EPEC adhesin is optimal dur-ing growth with glucose as a carbon sourceand is repressed by ammonium [1 10, 35].Consequently, 987P fimbriated E. coli willcolonize the distal small intestine. If Bfpexpression is activated at an early stage ofinfection, Bfp fimbriated EPEC will colo-nize proximal as well as distant segmentsof the small intestine. The high concentration

of ammonium in the colon might, however,inform EPEC that it is not in an environ-ment appropriate for survival, and thusadhesin expression is suppressed.

Effects of the environmental factorsdescribed above have been studied in vitro.

Very few studies have been reported con-cerning the regulation of E. coli virulencefactors expression in vivo, i.e. in the ani-mal. The question is: are the regulationsdepicted in vitro indeed occurring in thehost? Experimental infections followed bythe detection of virulence factor expressionin the organs or tissues are necessary toanswer this concern. Pourbakhsh et al. [ 109]inoculated chicken air sacs with septicaemicavian E. coli isolates and showed that amuch higher proportion of bacteria colo-nizing the trachea were Fl (type I fimbriae)fimbriated as compared to bacteria colo-nizing the lungs, air sacs and systemicorgans, suggesting that the bacteria undergoa phase variation with respect to Fl fim-briae in vivo. A similar Fl fimbrial phasevariation also occurs in vivo during theexperimental induction of E. coli peritonitisand lower urinary tract infection in mice [4,62, 97] and meningitis in rats [116]. In thesame study Pourbakhsh et al. showed thatP fimbriae are expressed in bacteria presentin air sacs and systemic organs, but not intrachea, suggesting that P fimbriae alsoundergo phase variation in vivo [109]. Thenature of the signal controlling the expres-sion of these fimbriae is, however, unknown.The aim of the next few years will be to

identify the nature of the signals that work invivo, the sensing molecules involved, themechanism of signal transduction, and todetermine their activities on virulence factorsin a given location within the host.

3. GLOBAL REGULATORS OFGENE EXPRESSION

Global regulators are involved in the reg-ulation of virulence gene expression. Inpathogenic bacteria, H-NS, CRP, RpoS, Lrp,play a role in sorting out complex signals

that modulate the synthesis of virulence fac-tors. Some of them, such as CRP, recognizespecific nucleotide sequences to which theybind. Their effects are confined to those

genes possessing these specific binding sites.Others, such as H-NS, have a wider influ-ence in controlling transcription and con-tribute to the organization of DNA topol-ogy in the cell. It has been shown that levelsof supercoiling vary in response to envi-ronmental changes; in particular, growth ofbacterial cells under anaerobic conditions, ata high osmolarity, at different temperatures,or in a nutrient-poor medium [29]. Topoi-somerases are enzymes that act upon DNAto alter the level of supercoiling, as well ascatenate and decatenate chromosomes. Inthe bacterial cell, DNA is negatively super-coiled [33]. It is thought that negative super-helical tension facilitates the melting ofDNA necessary for replication and tran-scription [79]. This mechanism may be usedby pathogenic E.coli to regulate genes nec-essary for virulence.

E. coli possesses low molecular weightproteins which contribute to the higher orderorganization of the bacterial nucleoid andto the expression of the genetic information.Frequently, small architectural proteins suchas protein HU (a histone-like protein), therelated protein IHF (integration host fac-tor), H-NS and FIS (factor for inversionstimulation) contribute to the control of tran-scription of genes whose products play arole in environmental adaptation and thusin the expression of the virulence.

3.1. H-NS

H-NS is one of the two most abundanthistone-like proteins in E. coli (the first onebeing HU). It is a 16-kDa neutral proteinpresent at 20 000 copies per cell under adimeric form (for a review, see [ 137]). H-NScan affect the overall structure of DNA bystrongly compacting DNA, altering DNAsuperhelicity by introducing negative super-coils, and by the induction of DNA bend-ing. H-NS appears, however, to play not

only a structural role in the organization ofthe chromosome, but also a rather dynamicrole in the regulation of gene expression[56, 63, 129]. Many of the genes regulatedby H-NS are involved in bacterial adaptationto environmental stress in response to signalssuch as osmolarity, temperature and oxy-gen availability [6]. Transcription of manyvirulence genes in E. coli is repressed byH-NS, and different transcriptional regula-tors act on gene expression by alleviatingand/or counteracting the effect of H-NS. Insome cases, H-NS seems to be implicatedin the thermoregulation of virulence factorexpression. The mechanisms by which H-NS affects gene transcription are not com-pletely understood. Correlation betweenmodifications of the overall DNA super-coiling induced by H-NS and gene expres-sion is not clear. In some cases, H-NS canbind DNA to directly inhibit gene tran-scription [27, 129], and it has been suggestedthat H-NS prevents the RNA polymerasefrom productively interacting with the pro-moter [129]. G6ransson et al. [47] have pro-posed that H-NS could act as ’silencers’ ofDNA regions by forming nucleoproteinstructures similar to transcriptionally inactivechromatine. However, these effects are notindiscriminate as not all promoters are H-NS repressible. H-NS also participates inthe control of sigma S synthesis [10]. Sev-eral examples of such regulations involvedin fimbriae and haemolysin synthesis as well las in bacterial invasiveness are given below.

3.1.1. Regulatory role of H-NSin fimbrial gene expression

The sfa determinant codes for S fimbrialadhesins of extraintestinal E. coli. SfaA isthe major structural subunit, whereas SfaBand SfaC are positive regulators of sfa tran-scription. Most .</M transcription initiates atthe slaB promoter, the !!faA promoter beingvery poorly active. Frameshift mutations in4aB or 4fhC result in very low levels of sfatranscription. Expression of S fimbriae from.lfaB/!laC mutants is, however, restored in

hns mutants, where pA transcription isstrongly activated [87]. The authors con-clude that the negative control exerted byH-NS on sfa transcription is counteractedby the sfa-specific activators SfaB and SfaC(figure 1).

P fimbriae are produced by cells grow-ing at 37 °C but not at lower temperaturessuch as 25 °C. H-NS plays an important rolein this thermoregulation by repressing thetranscription of a regulatory cistron in thepap gene cluster. PapB and Papl are posi-tive regulators homologous to SfaB andSfaC, respectively. Papl transcription is dere-pressed in hns mutants at both 37 and 26 °C.Transcription of the papBA operon is alsoderepressed at both temperatures althoughto a lesser extent [47, 136]. By competitivegel retardation assays it was shown that H-NS specifically binds to pap DNA contain-ing pap GATC sites and was able to blockmethylation of these sites in vitro. It wassuggested that the ability of H-NS to act as amethylation blocking factor is dependentupon the formation of a specific complex ofH-NS with pap regulatory DNA. Transcrip-tion of pap is enhanced by binding of thecAMP-CRP complex in the intergenic papl-papB DNA. In hns mutants, the pap operonis expressed in the absence of the PapB andcAMP-CRP transcriptional activators. It hasbeen suggested that the cAMP-CRP com-plex would play a role as an anti-repressoralleviating the H-NS-mediated silencing [41]. ].

H-NS is also involved in the phase vari-ation control of type 1 fimbriae [27, 30,101 ]. FimB and FimE are recombinases thatpromote the inversion of a 314-bp DNAsegment containing the fimA promoter.FimA is the major structural subunit of type1 fimbriae. In hns mutants, transcription offimB and fimE is increased at 30 °C and to alesser extent at 37 °C, leading to anincreased inversion rate of the fimA pro-moter. Donato et al. [27] have shown thatH-NS specifically and co-operatively bindsto the fimB promoter region and repressestranscription. They propose that H-NS rec-ognizes a specific DNA feature rather than

a specific consensus sequence. This featurecould be a specific conformation such as anintrinsic curvature due to the known affinityof H-NS for curved DNA [137]. This wasrecently confirmed by competitive gel retar-dation assays where it was shown that H-NS binds to the regions containing fimB andfimE promoters and does not affect theswitching frequency in vitro [105].

Curli are thin fimbriae expressed at 26 °Cin medium or low osmolarity. Curli expres-sion is dependent on RpoS, the sigma S fac-tor. Transcription of csgA (encoding themajor structural subunit) can be activatedin rpoS mutants by inactivation of hns, butthe temperature and osmolarity control ismaintained. Thus, neither of these two pro-teins is responsible for this control [104].Olsen et al. [104] concluded that this tran-scriptional silencing mediated directly orindirectly by H-NS can be relieved by RpoS,or by a non-identified regulatory proteinpositively controlled by RpoS.

3.1.2. Regulatory role of H-NS inenteroinvasive E. coli (EIEC)virulence gene expression

The genes required for EIEC invasive-ness are carried on a large virulence plas-mid. The ability of invasiveness is temper-

ature dependent, as it is expressed at 37 °Cand not at 30 °C. Dagberg et al. [21 haveshown that H-NS plays a crucial role in thethermoregulation of virulence-associatedgenes in EIEC. The VirF protein is a positiveregulator of virG and virB transcription.VirB activates transcription of ipaBCD andother genes present elsewhere on the plas-mid. IpaB, IpaC and IpaD are secreted pro-teins essential for invasion. Using phoA as areporter gene, these authors have shown thatdeletion of hns results at 30 and 37 °C in anincrease in virG transcription but has noeffect on ipaBCD transcription in theabsence of the VirF and VirB activators. Byincreasing the level of H-NS protein in thecell by cloning hns on a low copy vector,the expression of ipaBCD, virG and virB isrepressed at 30 °C and to a lower extent at37 °C, whereas transcription of virF ispoorly affected. Thus, the higher content ofH-NS in the cell causes enhanced ther-

moregulation. These results suggest that H-NS acts directly on virG and virB transcrip-tion and indirectly on ipaBCD transcriptionvia VirB (fcgure 2). In the absence of H-NS,virG expression may become VirF inde-pendent. Thus, VirF is a transcriptional reg-ulator which alleviates and/or counteractsthe effect of H-NS, as cAMP-CRP, SfaBand SfaC, or RpoS in the case of the pap,.!fa, and csg operons, respectively.

3.1.3. Effect of H-NS on haemolysinproduction

Haemolysin synthesis is repressed at highosmolarities and at low temperatures. Car-mona et al. [20] have shown that the muta-tion in the hha gene, encoding a putativehistone-like protein, derepresses haemolysinproduction when cells grow either at lowtemperature or in a high osmolarity medium.In a hns mutant as in the hha mutant, hlv ’vtranscription increases about 2-fold com-pared to the wild-type strain. However, in ahha-hns double mutant, haemolysin expres-sion shows a 10-fold increase. Thus, H-NSparticipates in the modulation of expression

of the hly genes. The regulatory mechanismby which this is achieved is not clear but itdoes not seem to involve modification inthe level of DNA supercoiling [93].

3.1.4. Effect of typA on H-NS expression

In E. coli K12, the inactivation of thetypA gene encoding the TypA protein (fortyrosine phosphoprotein) alters the expres-sion and the modification of several pro-teins, such as the disappearance of the uni-versal stress protein UspA, carbon starvationprotein CsplS and the increased synthesisof H-NS !39, 42]. The TypA protein is phos-phorylated on tyrosine residues in vivo andin vitro in the EPEC strain MAR001 but notin K12 E. coli strains. The sequence of TypAfrom the E. coli K-12 strain differs from thatof an EPEC strain by six amino acid residuesand the protein is three amino acids shorter.Freestone et al. [42] concluded that TypAcould be a candidate for studying the roleof tyrosine phosphorylation in the globalregulatory network. BipA of Salmonellatyphimurium is the homologue of TypA andis implicated in pathogenesis. Another team,Farris et at. [39] reported that BipA/TypAof EPEC is a tyrosine-phosphorylatedGTPase that presents a new class of viru-lence regulators as it controls several pro-cesses likely to be important for EPEC infec-tion: the formation of actin-rich pedestals

in host epithelial cells, flagella-mediated cellmotility and resistance to the antibacterialeffects of a human host defence protein.

3.2. IHF (integration host factor)

IHF is an abundant sequence-specificDNA binding protein that induces a signif-icant bend in the DNA and is involved in awide variety of processes in E. coli includ-ing regulation of gene expression. himA andhimD genes encode the subunits of IHF.There is an absolute requirement of IHF forphase variation of type 1 fimbriae whichuses site-specific recombination to switchphases. This recombination has an absoluterequirement for IHF [37]. IHF also plays arole in the expression of region I genes ofthe type II capsule K5 operon [119J. Bind-ing site consensus sequences were identi-fied in the vicinity of the transcription startsite of region 1 of the kps cluster. Mutationsin himA and himD led to a 5-fold reductionin the expression of the capsular gene kpsEat 37 °C in region 1. This indicates a roleof IHF in mediating the expression of regionI of the capsular gene cluster [119J. J.

3.3. CRP

The global regulator CRP regulates avariety of genes in E. coli in response to thelevel of cAMP which is synthesized by theadenylate cyclase in response to the carbonsource. Catabolite repression is due to theinactivation of adenylate cyclase when glu-cose is transported into the cell. The pro-tein CRP when complexed with cAMPbinds to a specific DNA sequence namedthe CRP-binding site. This sequence occursat different distances upstream of the tran-

scriptional start site in different operons andleads to the activation of transcription. It isknown that cAMP-CRP modulates expres-sion of certain E. coli virulence factors suchas enterotoxins STa and STb [3, 17J andfimbriae such as Pap, F165, and F165z pili,colonization factor antigen II, 987P and K99[22, 36, 46]. The expression of other fim-

briae, such as K88 expressed by ETEC orBFP expressed by EPEC, is, however, notunder CRP-cAMP regulation. From thatobservation, a working model for the tem-poral and spatial regulation of fimbrialexpression in the small intestine by the car-bon source was suggested by Edwards andSchifferli [36], where the regulation of fim-brial expression by the carbon source is amajor factor in determining the initial site ofintestinal binding (see section 2).

3.4. RpoS

rpoS encodes an RNA polymerase sigmafactor (sigma S, sigma 38 or KatF). It con-trols a regulon of 30 or more genes inresponse to starvation and during transitionto the stationary phase. During transitioninto the stationary phase, the expression ofsigma S-dependent genes is activated in acertain temporal order. The sigma S reguloncan be divided into subfamilies whichinclude genes regulated by specific stressesand/or additional global regulatory proteins.Subsets of sigma-dependent genes includethose genes that are also inducible by anaer-obiosis, oxidative or osmotic stress. Prod-ucts of several of these genes could be con-sidered as virulence factors, including theHpl and HpII hydroxyperoxidases whichconfer resistances to H202, as well as CsgA,the main subunit of surface protein curli (seesection 8.4) [82]. Maximal acid resistance ofE. coli MC4100 (and base resistance) isexhibited at the stationary phase and dependson RpoS although RpoS is not an absoluterequirement under all growth conditions[ 122]. For acid resistance, and in part baseresistance, the rpoS requirement can, how-ever, be overcome by anaerobic growth inmoderate acid.

3.5. Lrp

The leucine responsive protein, Lrp,affects the transcription of a large number ofgenes, increasing the expression of someand decreasing that of others, some of which

are turned on or off by exogeneous leucine[92]. It is noteworthy that Lrp is involvedin all regulatory mechanisms of fimbriaeexpression described so far (see section 8).The physiological role of Lrp is unclear.Calvo and Matthews [19] suggest that Lrppositively regulates genes that function dur-ing famine and negatively regulates thoseworking during a feast. They propose thatpili may be expressed at higher levels duringgrowth in a nutrient-deficient medium wherean abundance of pili may help bacterialadherence to epithelial cells, thus helpingthe maintenance of bacteria in the environ-ment. Under conditions of nutrient excess,bacteria can grow rapidly and should nothave any difficulty in maintaining their pres-ence. Thus, abundant synthesis of fimbriaewould not be so critical for survival.

3.6. Fur

The exceedingly low availability of ironin mammalian tissues is an environmental

signal indicating to the bacteria entry intoa host. This signal triggers the co-ordinateexpression of bacterial virulence determi-nants through the Fur protein which plays ageneral role as a sensor of iron availabilityin the cell. The apoprotein binds Fez+ as a

cofactor, and the cofactor-bound proteinbinds to various sites, termed iron-boxes.This complex negatively regulates manygenes involved in iron uptake as well astoxin genes hly and others. The synthesis ofthe Slt-I toxin, iron-chelating molecules(enterobactin and aerobactin siderophores)and membrane proteins involved in the bind-ing and uptake of iron-siderophore com-plexes is repressed in low iron conditions[ 18, 64, 133]. The Fur-iron complex binds tospecific DNA sequences, the Fur boxes,located in the operator regions of the iron-regulated operons [18, 25]. Fur does notbind to its DNA target in the absence of ironand genes are thus derepressed in conditionsof low iron availability. It is interesting tonote that slt-l genes are carried by aprophage and are nevertheless controlled

by a chromosomally encoded protein in away similar to aerobactin (plasmid encoded)and enterobactin (chromosomally encoded).In contrast to the slt-I promoter, the slt-IIand slt-llv promoters do not contain Furboxes and are not controlled by iron avail-ability [ 125]. ] .

4. FAMILY OF THE ARACTRANSCRIPTIONALACTIVATOR

AraC is the transcriptional regulator ofthe arabinose operon. Proteins in the familyof AraC activator contain helix-turn-helix

motifs, which bind specific DNA sequencesupstream of genes that are actively tran-scribed.

The activation of virulence gene expres-sion in EPEC requires an AraC-homologoustranscriptional activator protein calledPerA/BfpT [45] which is encoded by a vir-ulence plasmid, pMAR2. The genes of theper locus are required for transcriptionalactivation of genes whose products aresecreted by the type III secretion system(eae, encoding intimin, and espB) and theyare also involved in activation of plasmidicgenes such as the adhesin gene bfpA encod-ing the bundle forming pilus [70, 110, 128].Insertional inactivation of perA led to areduced expression of Eae which could bedue to specific mutations in perA or to polareffects on genes downstream of pera. Puri-fied PerA/BfpT was shown to bind directlyto DNA sequences upstream of bfpA andeae [128].

Regulatory proteins of some fimbrialoperons such as coo (CS1), cfa (CFA/1), fap(987P) and (AAF/1) are designated as Rns-like and share sequence identity with AraC.Rns is required for positive activation of theCS I fimbrial genes. It was recently shownthat Rns is capable of complementing a nullmutation in the S. flexneri virf gene encod-ing the homologous counterpart of Rns[108]. The VirF protein cannot, however,complement Rns as an activator of CS 1 gene

expression in ETEC. It was concluded thatthere are differences in the mechanisms bywhich these related transcription factors reg-ulate gene expression. It is not knownwhether the Rns class of regulatory proteinsbinds DNA or whether there are additionalfactors in the regulatory network [78].

5. SENSOR REGULATORYPROCESSES

Environmental signals control virulencegene expression in bacteria. A developingresearch field is understanding the ways inwhich bacteria sense these signals and trans-duce them into the cell to regulate geneexpression. Many signal response systems inbacteria operate by complex pathwaysinvolving two-component regulatory sys-tems. These systems consist of: 1) the sen-sor protein which spans the cytoplasmicmembrane and monitors some environ-mental parameters; and 2) the regulator pro-tein that mediates an adaptive response, usu-ally by a change in gene expression. Whenthe sensor component receives an external

signal, it undergoes autophosphorylationand transfers the phosphate residue to theregulator protein which in turn activates orrepresses gene transcription. Several two-component systems controlling virulencegene expression have been well character-ized in different bacteria [40J. In E. coli, the

expression of some group IK antigens aswell as colonic acid is regulated by theRcsABC (regulator of capsule synthesis)system. A temperature below 25 °C, the

presence of a high phosphate concentrationand osmotic induction increase the synthe-sis of these antigens [121]. The integral innermembrane RcsC sensor and the cytoplas-mic RcsB effector show homologies to thefamily of the two-component histidinekinase signalling systems [48 The mecha-nisms whereby extracytoplasmic signals aresensed and transduced by the RcsC mem-brane sensor are not unknown. Presumably,after receipt of a stimulatory signal, RcsC

first autophosphorylates at a reactive histi-dine residue, then subsequently transfers thephosphate to a conserved aspartate on RcsB.This form of RcsB may form a more stable

complex with RcsA and promotes cps tran-scription perhaps aided by RcsF [48, 50,69]. RcsA is an additional positive regulatorand is subject to degradation by the Lonprotease [68!. ] .

Effective mechanisms for the induction or

repression of virulence gene expressioninvolve the sensing of ’signature’ moleculesproduced by host tissues. In this way, theE. coli regulons SoxRS and OxyR aredesigned to deal with the cytotoxic prod-ucts produced during the oxygen-dependentrespiratory burst, with a large number ofproteins being induced in response to hydro-gen peroxide (H202)’ superoxide anion (02)or nitric oxide (NO) produced by neutrophilsand macrophages [82, 99]. The SoxRS reg-ulon controls the expression of approxi-mately ten genes. SoxR is an iron sulphurprotein that senses exposure to superoxideand nitric oxide, and then activates the tran-scription of the soxS gene. SoxR and SoxSare DNA-binding proteins, SoxR being amember of the MerR-like family (MerR isinvolved in the response to Hg2+) and SoxSbeing a member of the AraC-family. Tran-scription of soxS is initiated in a mannerdependent on the rpoS gene in response toentering stationary phase growth [98]. TheSoxS protein then activates the transcrip-tion of a variety of genes including sodA,encoding the Mn-dependent superoxide dis-mutase, nfo, encoding the DNA repairenzyme endonuclease IV, and micF, which

post-transcriptionally decreases the pro-duction of OmpF [82].

OxyR, a member of the LysR family ofautoregulators, is also involved in resistanceto oxygen-related compounds. The OxyRprotein activates the transcription of approx-imately nine genes in E. coli in response to

Hz02. These include katG, encoding HP1 Icatalase, and ahpFC, encoding NADPH-dependent alkyl hydroxyperoxidase, gorA,encoding a glutathione reductase and dps

encoding a protein that non-specificallybinds to DNA to protect cells from H202toxicity. OxyR functions both as a sensorand a transducer and contains a criticalredox-sensitive Cys residue that is oxidizedby hydrogen peroxide [117, 139]. The levelof oxyR mRNA or OxyR protein does notchange significantly following exposure ofthe cells to H202’ Hence, it was suggestedthat post-translational regulation by an H202-generated signal activates the pre-existingOxyR protein [80].

P pili-mediated attachment may also bean important part of sensor regulatory pro-cesses involved in uropathogenic E. coliduring urinary tract infection. Zhang andNormark [138] have shown that airs tran-scription is specifically activated by P piliattachment. The AirS protein is a sensor-regulator protein essential for the iron-star-vation response of uropathogenic E. colithat activates the synthesis of the

siderophore iron acquisition system. Fur-thermore, the two-component signal trans-duction system, CpxA-CpxR, has been sug-gested to play a role in the expression ofvirulence factors of E. coli via P pili attach-ment. Hultgren [61] hypothesized that theadhesion of P pili to epithelial cells avoidspolymerization of the pilin fibre and thusleads to aggregation of misfolded pilus sub-units in the periplasm. Protein aggregation inthe periplasm is sensed by the CpxA-CpxRsystem, which activates htrA encoding DegP(a periplasmic protease degrading misfoldedproteins) [23, 107], and could be involved incontrolling expression of virulence factorssuch as haemolysin or CNF (cytonecrotizingfactor). Thus, the interaction between thepathogen and its host receptors mediated byadhesins may be a means for bacteria to

sample and consequently elicit the appro-priate response upon their arrival at a poten-tial colonization site. Thereby, bacterialattachment to mucosal surfaces via P piliindicates that bacteria have reached an eco-

logical niche where expression of virulencefactors (siderophores, toxins) are necessaryfor survival.

6. tRNA REGULATION OFVIRULENCE GENES

Many virulence genes are located on’pathogenicity islands’ (Pais), large chro-mosomal regions that are often associatedwith particular tRNA genes [52]. It has beenshown that the Pais modulate virulence geneexpression through the action of these tRNA[112]. In Shigella, it has also been demon-strated that expression of tRNA/yr partiallycomplemented the virR mutation. virR is ananalogue of hns and is required for co-ordinating temperature-regulated virulencegene expression of Shigella [57].

The uropathogenic E. coli strain 536(06 :K15 :H31) carries two pathogenicityislands, one Pai comprising the gene clusterof haemolysin and the other Pai compris-ing the gene clusters of haemolysin and P-related fimbriae, both Pais being flanked bytRNA genes, leuX and selC, respectively.In the uropathogenic strain 536, spontaneousdeletions resulting in the truncation of leuXor the specific deletion of the tRNA5 leu generesulted in the lack of expression of type 1fimbriae and other virulence factors such as

haemolysin, aerobactin production, serumresistance phenotype and motility, whiletrans-complementation of tRNA loci leads toa restoration of these properties [91, 127].tRNA5&dquo;1 is specific for the minor leucinecodon UUG. It has been shown that

tRNA5 leu is required for efficient transla-tion of FimB whose gene contains five TTGcodons recognized by tRNA5’cu that in turnleads to type 1 fimbrial expression [113J. ].

The selenocysteine-specific tRNA (selC!directly influences the ability of the E. colistrain 536 to grow under anaerobic condi-tions because selenocysteine is part of theformate dehydrogenase (FDH) enzymewhich is involved in mixed acid fermenta-tion. The ability to grow under anaerobicconditions via mixed acid fermentation maybe important for the colonization of E. coliin the kidney because oxygen-limiting con-ditions are found in deeper regions of thekidney [112]. ].

7. BLACK HOLES ANDVIRULENCE GENE EXPRESSION

Not only deletion of genes such as leuXcan affect the expression of virulence, butaddition of some genes can also alter thevirulence of bacteria. It was observed that

Shigella and enteroinvasive E. coli displayeddeletions in the cadA region present in mostE. coli strains including E. coli K12 [84].These deletions have been termed blackholes and they enhance the expression ofvirulence genes. Indeed the introduction ofthe cadA gene encoding lysine decarboxy-lase attenuates the virulence of Shigellaflexneri and the enterotoxin activity wasinhibited by cadaverine, a product of thereaction catalysed by lysine decarboxylase.

8. REGULATION OF FIMBRIALEXPRESSION

Synthesis of fimbriae is under complexregulatory controls. First, assembly of fim-briae depends on the appropriate levels rel-ative to one another of major and minor fim-brial subunits, the chaperon, outer membranepore and regulatory proteins. The major fim-brial subunit must be expressed at higherlevels than other accessory proteins. Fim-brial determinants are organized in operonsand differential expression of genes isachieved by post-transcriptional mecha-nisms. In the pap operon, encoding P fim-briae, the two first cistrons, papA and papB,are co-transcribed to mRNA from the papBpromoter. Differential expression of thesetwo genes results from RNAse E-dependentendonucleolytic cleavage of the mRNA inthe intercistronic papB-papA region. Thiscleavage is followed by rapid decay of theupstream papB-encoding region and accu-mulation of the stable papA-encodingmRNA, leading to high level expression ofthe major subunit, PapA, relative to that ofthe regulatory protein PapB 194 Differen-tial mRNA stability and mRNA processingare also involved in the control of the daa

operon (encoding for the F1845 fimbriae)[ 12]. Transcriptional organization of the daaoperon is quite different from that of papsince the major fimbrial subunit gene daaEis located at the 3’ end of the operon. A largetranscript encoding accessory proteins inaddition to the major subunit is submittedto endoribonucleolytic cleavage to gener-ate a stable daaE mRNA. This cleavagerequires neither RNAse III nor RNAse E.Second, expression of fimbriae is controlledby environmental signals through alterationsin transcript levels. The control of fimbriaeexpression is of crucial importance for thesurvival of bacteria in vivo [35]. It allowsfimbriae to be expressed only at the ade-quate site in the host and to evade the host’s simmune response. All fimbrial operonsexamined so far encode specific regulatoryproteins that either activate or repress tran-scription. In addition, global regulators suchas Lrp, CRP, H-NS, IHF, RpoS, play roles incontrolling the expression of most fimbrialoperons. Four different regulatory mecha-nisms controlling the expression of fimbriaehave been described. Operons that sharecommon regulatory properties are classi-fied in table I.

8.1. The pap regulatory family

Regulation of the pap expression is themost extensively studied. Expression of Pfimbriae is subject to phase variation, i.e.fimbrial expression switches between ON(fimbriae-positive cells) and OFF (fimbriae-negative cells) states. The switch frequencyis controlled by environmental factors suchas temperature or carbon source. Phase vari-ation of P fimbriae depends on the mcthy-lation status of two GATC sites located inthe intercistronic region between the regu-latory genes papl and papB which are tran-scribed divergently (figure 3). The twoGATC sites, GATC-I and GATC-II, arelocated within the Lrp-binding sites.

Schematically, when Lrp binds the non-methylated GATC-II site, transcription from

the pB promoter is locked and cells are in theOFF state. Under conditions of high cAMPlevels, Papl is expressed and binds to Lrp,resulting in a shift in Lrp binding from theGATC-11 site to the GATC-1 site. Binding ofLrp-PapI to GATC-I activates transcriptionfrom the pB promoter and switches the cellsto the ON state. The switch between the

OFF and ON states requires DNA replica-tion to alter the methylation status of GATC-I, because binding of Lrp-Papl to this siteis inhibited by full methylation. PapB acti-vates papf transcription and represses papBAtranscription (figure 3) [15, 66, 67, 95, 96,130, 132].

Several other fimbrial operons are con-trolled by analogous mechanisms with slightvariations. sfa (S fimbriae), daa (F1845),clp (31A), fae (K88),,foo (F]651) operonsencode for proteins with significant homolo-gies to Papl and PapB [22, 60, 83, 87]. Theirexpression is controlled by Lrp and Dammethylation. daa, vfa, fbo and clp operonsare under phase variation control but fae isnot. Lrp is required for the OFF to ONswitch of the phase variation-controlledoperons. clp and foo are the only operonsdescribed so far that belong to this regulatoryfamily in which expression is modulated bythe level of leucine and alanine in the growthmedium. It has been shown that these aminoacids totally inhibit the OFF to ON switch ofclp but the precise molecular mechanism isnot 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 transcriptionin phase ON cells or in cells not subjected tophase variation. Martin [83] has shown thatLrp and CIpB repress clp transcription inphase ON cells and that this effect requiresDam methylation. Lrp also acts as a negativeregulator of the fae operon which is notunder 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.Regulatory p!f7 and prfB genes of the pefoperon encoding for P-related fimbriae areable to complement mutations in sfac andsfaB regulatory genes in a uropathogenic E.coli isolate. Prfl and PrfB share 87 and 76 %

homology with SfaC and SfaB, respectively.Moreover, mutations in pr!Z and prfB lead tosignificant reductions in sfa transcription [88].

8.2. Regulation of type I fimbriaeexpression

Phase variation controls the expressionof type I fimbriae (fin2 operon) by site-spe-cific recombination. Recombination leadsto the inversion of a 314-bp element.According to the orientation of the promoter,fimbriae are either produced or not pro-duced. The inversion process is carried out

by two integrase-like proteins, FimB andFimE. FimB is involved in ON to OFF andOFF to ON inversion, FimE only in ON toOFF inversion [2, 73 In addition, fourglobal regulators modulate the fim switch:Lrp, IHF, H-NS and RpoS (figure 4).

Lrp stimulates the,/im inversion in aDam-independent manner. Lrp slightlyincreases the transcription of.fimB, decreasesfimE transcription by 2-fold 113], and bindswith high affinity to the switch element invitro [43!. Leucine potentiates the effect of

Lrp on the switch. The model of Roesch andBlomfield [1 14] assumes that leucine pro-motes the selective dissociation of Lrp fromone DNA binding site within a multimericnucleoprotein complex, resulting in a struc-ture that is more favourable for recombina-tion than that produced by the Lrp bound toall binding sites.

The integration host factor (IHF) is nec-essary for the fim inversion. IHF could playa direct role in site-specific recombination orit could modulate transcription of,fitiibandlor,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 bothfimB and,fiiiie is enhanced [ 101 J. Donatoet al. [27 ! have shown that H-NS interactsdirectly with the fimB promoter sequences.This results in a promoter-specific repres-sion.

The alternative sigma factor RpoS exertsa negative control on,firnA (encoding themajor fimbrial subunit) and firrrB promot-ers by an unknown mechanism and influ-ences DNA inversion negatively in brothcultures [32]. ].

8.3. Regulation of K99 expression

The regulation of K99 expression is lesswell understood. The K99 determinantencodes eight genes organized in three tran-scriptional units. Region I encodes f(inA-D,region 2.fanE-F, region 3,fanG-H [65!. Thejhiac gene product is the major fimbrial sub-unit, whereas products of/o/;/t and /!/tB arerelated to each other and to PapB. None ofthe ’/Ú/1 genes is related to pcrpl [115]. K99expression is not controlled by phase varia-tion but is repressed by leucine and alanine.The global regulators CRP and Lrp are bothrequired for K99 exprcssion [ 15, 65 Lo-Tscng ct al. 177 j demonstrated that Lrpaffects only the transcription of region Igenes, whereas Inoue et al. have shown thatCRP is required for expression of region I

and 2 genes [15, 65]. Differential methyla-tion due to Lrp is, however, not involved inthe regulation of K99 expression, as it is forthe fimbriae of the P regulatory family. Themechanism by which leucine and alanineaffect K99 synthesis remains unknown. Therole of FanA and FanB remains obscure.Frameshift mutations in FanA or FanBreduced the level of K99 production 8- and16-fold, respectively, but did not have mucheffect on transcription from the fanA pro-moter.

8.4. Regulation of curli expression

A new type of fimbriae has beendescribed, whose expression is maximal inconditions which are characteristic of anextraintestinal environment, i.e. low tem-perature (26 °C) and low osmolarity. Thesefimbriae, referred to as curli [102], are syn-thesized by pathogenic and non-pathogenicE. coli as well as by other Gram-negativebacteria [28]. They bind fibronectin, plas-minogen, different sera and tissue proteinsand the dye Congo red. Curli formationinvolves a novel and a so far unique assem-bly mechanism not yet completely under-stood.

Two divergently transcribed operons arenecessary for curli formation, c.sgBA andcsgDEFG [53]. CsgA, the fibre subunit, isfirst secreted in the extracellular milieu as asoluble protein and then polymerized at thecell surface in a CsgB-dependent manner[54]. CsgG is an outer membrane-locatedlipoprotein required for stable maintenanceof CsgA and CsgB [76]. CsgD is a tran-scriptional activator of the csgBA promoterbelonging to the LuxR family [53]. Regu-lation of curli expression is complex andinvolves at least two global regulators, H-NSand RpoS. So far, no report presents evi-dence for a role of Lrp in this regulation.Thus, Lrp seems to be involved in regulatingexpression of fimbriae synthesized in con-ditions that mimic the in vivo environment.Curli are produced in the stationary phase.

Transcription of csgBA and csgDEFG isdependent upon RpoS in wild-type cells,but not in a hns background [53, 104]. Therequirement of RpoS for transcription ofcsgBA may be indirect via CsgD with CsgDbeing necessary for csgBA transcription.Temperature and low osmolarity control offibronectin binding as well as growth phasecontrol of csgBA transcription are main-tained in a rpoSIhns double mutant, indi-cating that neither H-NS nor RpoS is respon-sible for these regulatory responses [5, 53].Considering that CsgD is required for csgBAtranscription and is produced in a hnslrpoSdouble mutant, Hammar et al. [531 hypoth-esized that CsgD may have the capabilityto respond to starvation signals and/or highcell density by activating csgBA transcrip-tion. This is a seductive hypothesis sinceCsgD belongs to the LuxR family of tran-scriptional activators involved in quorumsensing regulation (see in section 11). ).

9. REGULATION OF HAEMOLYSINEXPRESSION

E. coli haemolysin (Hly) is a well-char-acterized member of the RTX family ofcytotoxins associated with urinary tractinfections and other extra-intestinal E. coliinfections [134, 135]. Synthesis of Hly isdirected from an operon consisting of fourcontiguous genes, hIyCABD. The hlyA geneencodes the component of haemolysin,which undergoes HlyC-dependent acyla-tion. Sec-independent haemolysin secretionrequires HlyB and HlyD. Two transcriptsinitiating at the same promoter locatedupstream of hlyC are synthesized: a majorhlyCA mRNA and a minor hIyCABDmRNA. The higher stability of the majortranscript accounts in part for the differentialexpression of HlyC and HIyA relative toHlyB and HlyD. Hly expression is regulatedby several mechanisms involved in mRNAstability, DNA supercoiling, transcriptionelongation 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 represshaemolysin expression. The Hha protein isrepresentative of a new class of modulatorsof gene expression in enterobacteria [24].Carmona et al. have shown that Hha influ-ences DNA topology and suggest that it is ahistone-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. TheHha protein participates in the temperature-and osmolarity-dependent regulation of Hlyexpression in a manner that strikingly resem-bles what has been shown for H-NS withother virulence determinants. The hha muta-tion significantly increases expression ofhLy genes in high osmolarity media and atlow temperatures [90]. The Hha protein ishighly similar to the YmoA protein fromYersdnia enterolitica. The hha gene can com-

plement the yrreoA mutation and vice versa[ 9, 85 The complementation appears to bedependent on gene dosage 19]. ].

9.2. Transcription elongation

hIyCABD transcription elongation ismodulated by the RfaH protein which con-trols a regulon governing the synthesis,export and assembly of cell surface andextracellular components that influenceDNA transfer and virulence: RfaH is a pos-itive regulator of rfa (encoding the LPScore), tra (encoding the F pili) and kps(encoding the production of type 2 capsule)gene expression [8]. RfaH abolishes tran-scriptional polarity within a transcript,increasing transcription of distal gencs inoperons. The non-encoding regions of allRfaH-affected operons contain all or a por-tion of a conserved 39-bp sequence namedJUMP-Start. The ops element is the secondhalf of a direct repeat in the JUMP-Start

sequence. Bailey et al. [8] propose that theops element recruits the RfaH protein to thetranscription complex. Then the RfaH-influ-enced elongating RNA polymerase com-plex resists transcription termination sig-nals downstream of the ops element (figure5). This model is supported by the signifi-cant amino acid similarities between RfaHand NusG, an essential transcription elon-

gation factor required for effective anti-ter-mination by the N protein of bacteriophagelambda. In the hly operon, transcriptionalpolarity is due to a Rho-independent termi-nator between hlyA and hlyB genes. Co-operation between RfaH and the cis actingops element enables the RNA polymerasecomplex to proceed through the hlyA-hlyBintergenic terminator [7].

9.3. Stabilization of the activeconformation of the toxin

LPS may play a role in protecting thesecreted HlyA protein from degradation.Removing the core sugars from LPS affectsthe kinetics and stability of secretedhaemolytic activity. The model proposedby Bauer et al. [11] suggests that Hly existsas a complex including LPS. Thesemolecules combine to form an active toxinwhose stability is aided by the LPS innercore. When the inner core is incomplete,LPS and Hly form large, inactive aggre-gates, and render Hly more susceptible todecay.

10. REGULATION OF CAPSULESYNTHESIS

Group I K antigens are characterized bya high molecular mass (over 100 kDa) anda low charge density. The coexpression ofthe group IK antigens and LPS is restrictedto a few LPS serotypes (primarily 08, 09and 020) [111 Expression of some group 1K antigens as well as colonic acid is regu-lated by the RcsABC (regulator of capsulesynthesis) system (see section 5). A mem-brane-anchored DjIA protein, a member ofthe DnaJ ’J-domain’ family, acts in concertwith RcsB/C two-component system toincrease induction of the cps (capsularpolysaccharide) operon [69]. Moreover, thecps operon activation by DjIA is dependentupon DnaK (Hsp70) and GrpE, which areparts of a chaperon machine.

Group II K antigens have a lower molec-ular mass and a higher charge density thangroup I K antigens, are coexpressed with avariety of LPS and expressed at 37 °C (atthe physiological temperature). K 1 and K5kps clusters have been determined. The syn-thetic, regulatory and export componentsfor capsule expression are encoded in threefunctionally distinct gene blocks [1111. Thekps cluster of K1 is functionally divided intothree regions. These regions are organized astwo convergently transcribed operonsinserted into a monocistronic tRNA genepheV. The central region, region 2, containsa biosynthetic cassette that is flanked oneither side by regions I and 3 whose genesfunction in more general aspects of capsulebiosynthesis. Mutations in either region 1or region 3 tend to cause accumulation ofintracellular polysaccharides within the cell.This suggests that these regions are impor-tant for the transport of polysaccharides tothe cell surface. The six genes of the regionI operon are transcribed in the same direc-tion as pheV.

Region I of the K5 capsular gene clusterin which kpsFEDUCS genes were identi-fied is transcribed as a single transcriptionalunit that is processed to yield a smaller tran-script specific for the kpsS gene which is atthe 3’ end of the transcript 1119 Processingof mRNA appears to be implicated in a dif-ferential expression of kp.r genes. Mutationsin the himA and himD genes which encodethe subunits of IHF led to a reduction in the

expression of KpsE at 37 °C [119]. RegionI is submitted to a thermoregulation withno transcription at 18 °C. Expression of theregion 1 operon is thermoregulated by tran-scriptional control of its first gene, kp.sF[ 119J. It appears that regulation of region 1

expression in response to temperature ismediated neither by the has, rimJ nor hhagene products, all of which have been impli-cated in temperature-dependent regulationgene expression in other systems [ 119].A mutation in the rfaH gene abolishes

the K5 capsule expression at 37 °C [123].Expression of region I is not mediated by

RfaH, which is required for expression ofregion 3 at 37 °C and the expression of rfagenes [1 19, 123]. Stevens et al. [124], pro-posed a model where RfaH regulates expres-sion of the E. coli group II capsule geneclusters by allowing readthrough transcrip-tion to proceed from region 3 into region 2.The non-encoding region upstream of theclusters involved in the production of vari-ous polysaccharide antigens contains a 39-bp sequence (a sequence upstream of kspMof region 3 of the kps cluster, outer core rfa,0-antigens rfb of LPS). This was referredto as the JUMP-Start sequence and it hasbeen postulated to be involved in the tran-scriptional regulation of bacterial gene clus-ters encoding surface proteins. The JUMP-Start sequence could cause RfaH-dependentantitermination at other Rho-dependent ter-minators suggesting that the JUMP-Startsequence may function, in a manner anal-ogous to a lambda nut site, in the regulationof bacterial gene clusters encoding surfacepolysaccharides (see section 9.2) (figure 5).

11. PERSPECTIVES

The regulation of the expression of viru-lence genes has mainly been studied in vitro.Nowadays, with the advent of new geneticmethods, the identification of the regulationmechanisms that are induced in vivo as wellas the determination of their activities onvirulence factors in a given location withinthe host are now ongoing. New methodolo-gies for the study of in vivo gene expres-sion allow the isolation of bacterial genesexpressed during infection [55, 106]. Usinggenetic reporter fusions, genetic expressionat the cellular level within infected animalscould be examined [40]. Through the useof reporter molecules such as the green flu-orescent protein and others under develop-ment, it will be possible to exploremicrobe-host interaction in real time in the

living host [40].Effective mechanisms for the induction of

virulence genes could involve the sensing

of ’signature’ molecules produced by hosttissues [82]. Probing the host environmentwith tools that define the genes expressedby bacteria during infection will provide abetter definition of the microbial-host inter-action at both ends of the spectrum. At a

higher level, one needs to understand howsignals are detected and interpreted to int7u-ence gene expression and what are the effec-tor signals produced by the host tissues.More specifically, since several type IIIsecretion systems of attaching and effacingE. coli are activated by the contact of thebacteria with the surface of eukaryotic cells,the questions raised are: what is the nature ofeukaryotic cell surface signals that activatethe type III secretion systems, and what arethe regulatory proteins involved in the reg-ulation of these secretion systems [58]?

Another aspect of E. coli virulence factor

expression that is under scrutiny is the roleof the population density detection in E. cnlivirulence. In pathogenic bacteria, such asf.sfM!/M!na.s, quorum sensing plays adefined role in virulence. Quorum sensing isa phenomenon by which bacteria sense andrespond to their own population density byreleasing and sensing pheromones. In Gram-negative bacteria, quorum sensing is oftenperformed by the LuxR family of tran-scriptional regulators, which affect pheno-types as diverse as conjugation, biolumi-nescence and expression of virulence genes.In Gram-negative bacteria the most com-mon form of quorum sensing is mediatedby the production and subsequent percep-tion of autoinducers, the acylated homoser-ine lactones (acyl HSLs). By sensing thedensity of a secreted autoinducer, bacteriacan sense if there is a quorum of their pop-ulation sufficiently present to initiate theappropriate biochemical reaction. Homo-logues of LuxR, such as SdiA (suppressor ofcell division inhibition) and CsgD have beenidentified in E. coli [53, 120[. SdiA-medi-ated autoinduction with RpoS regulatesf’tsQA whose products are involved in celldivision [ 120]. Recently, by using the Vib-rio hcirveyi mutant that responds exclusively

to an uncharacterized signal molecule AI-2, Surette and Bassler [126] demonstratedthat cell-free culture fluids of E. coli and S.

typhimurium can contain high levels of AI-2-like factors. The role of this putativeautoinducer and SdiA in quorum sensingfor pathogenic E coli and the regulation ofthe expression of virulence factors remainsto be determined.

Given that most virulence genes appear tobe regulated, regulatory systems are obvioustargets for the development of new thera-peutic drugs. The identification of molecu-lar targets including regulators associatedwith adaptation/survival and pathogenesisoffers the prospect of expanding the hori-zons of anti-infective therapy beyond theconfines of agents that are merely bacteri-cidal or bacteriostatic [74]. It could increasethe level of therapy beyond that currentlypossible with antibiotics alone. Thus, a bet-ter understanding of the regulation mecha-nisms that govern the expression of viru-lence factors not only in vitro but alsotemporally and spatially within the host will l lallow the development of new therapeuticapproaches.

ACKNOWLEDGEMENTS

We express our gratitude to F. Daigle,G. Szatmari and M.C. Tessier for criticallyreviewing the manuscript, and to E. Roussetand S. Dutilloy for graphical assistance. Thiswork was in part supported by grants fromthe Natural Sciences and EngineeringResearch Council of Canada (OGP0025120)and Fonds pour la Formation de Chercheurset 1’Aide a la Recherche (93-ER-0214) (toJ.H.) and OTAN (to C. M.).

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