Virulence gene regulation in pathogenic Escherichia coli

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Virulence gene regulation in pathogenic Escherichia coli

Josée Harel, Christine Martin

To cite this version:

Review

article

Virulence

_gene

_regulation

in

_pathogenic

Escherichia coli

Josée Harel

Christine

Martin

b

’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

*

Correspondence

and

_reprints

Tel.: ₍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

_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

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

During

the course of

infection,

pathogenic

micro-organisms

encounter dif-ferent _types of environments and

_they

have to

_adapt

in order to survive and

_multiply.

Thus,

pathogenic

bacteria need to

synthe-size virulence factors that will allow them to

colonize a hostile environment and to

sur-vive the host immune and non-immune defences.

_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 on

intestinal mucosa but becomes

disavanta-geous when the bacteria is in the blood-stream or when the bacteria needs to escape

the bactericidal

_activity

of serum.

There-fore,

micro-organisms

sense the

environ-ment

and,

in response to the

_signals

that

they

receive,

act

_{accordingly by turning}

off

or on the

_expression

of their virulence _genes

factors,

the

_specific

host

_signals

that the

reg-ulatory

protein

detects are not

_yet

under-stood,

although

the environmental

_signals

that modulate the

_expression

of virulence genes have in many cases been identified

in vitro. Parameters such as

_temperature,

osmolarity, pH,

source of

_nitrogen,

con-centrations of

iron,

sugars and amino acids

are known to affect the

_regulation

of

viru-lence genes in vitro. One

challenge

of the

next few _years will be to find out if these

parameters

play

a role in virulence factor

expression

in vivo.

The

_regulation

of

_{pathogenicity}

is

com-plex.

There are interconnections between

regulatory

systems. A

_single

_regulator

can

control the

_expression

of several _genes

(global

regulator) including

virulence genes and

_housekeeping

_{genes. For}

_example,

the leucine

_responsive

_{protein (Lrp),}

the cAMP

receptor

protein

(CRP),

the histone-like

pro-tein

_(H-NS)

and the

_sigma

factor

_RpoS

are

global regulators

that are _{involved in}

regu-latory

networks that control _{virulence genes,}

genes

encoding

metabolic enzymes and

structural

_{components. Often,}

_global

and

specific

regulators simultaneously

affect virulence _gene

_expression

which is

opti-mally

co-ordinated to

_permit

the survival of

a

_pathogen

in a

_particular

ecological

niche.

Here,

we review the well-described

reg-ulatory

mechanisms used to co-ordinate the

expression

of virulence factors

_by

pathogenic

E. coli with a relative

_emphasis

on E coli

causing

diseases in animals. Some

regulatory

mechanisms that have been

described for other

_pathogenic

bacteria

might

also exist in

_pathogenic

E. coli but

are not evoked here

_owing

to the lack of

experimental

results in E. coli. The

regula-tion of

_expression

of virulence determinants

by

global

regulators, including

housekeep-ing regulons

and virulence

_regulons,

spe-cific

_regulators

and then two

_component

regulatory

systems involved in

virulence,

is

_presented.

_{Specific regulation}

mecha-nisms are illustrated

_using

the

regulation

of

genes

encoding

fimbriae,

curli,

haemolysin

or

_capsules

as

_examples.

2. ENVIRONMENTAL

REGULATION OF VIRULENCE GENE EXPRESSION

Not all virulence factors confer a

selective

advantage

to the microbe at the same

_stage

of infection or at the same anatomical site

within the host.

_{Consequently,}

_the expres-sion of certain virulence factors must be

modulated in response to environmental

sig-nals encountered

_throughout

the infectious

cycle

and

_during

the transition from the external milieu to the host. Such

_regulation

allows for the co-ordinated

_expression

of

proteins required

for survival in different environmental 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 evade the host immune _response.

In

vivo,

bacteria first need to sense that

they

have entered into a host.

_Temperature

is an

appropriate signal

since the _temperature

of mammalian bodies is

_generally

around

37 °C,

higher

than the external environment.

Thus,

several virulence factors such as

adhesins,

haemolysin,

or

_{enteropathogenic}

E. coli

_(EPEC)-,

RDEC-1

_(a

rabbit EPEC

strain)-

and STEC

_(shiga-like

toxin

pro-ducing

E.

_{coli)-secreted proteins}

are

specif-ically

expressed

at the normal host

_body

temperature

[1, 34, 71, 78, 89].

Iron

avail-ability

is also an indicator of the host

envi-ronment since iron is

_sequestered

in the

_body

by specific proteins

such as transferrin or

lactoferrin. 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 of omnivorous mammals are

_subjected

to a

low

_pH

stress in the

stomach,

followed

_by

a

more

hospitable

environment with

_respect

to

pH

in the intestinal lumen

_varying

from

_pH

6.5 to 7.5

_[38].

The

_regulation

_{of gene}

is

_complex

and is associated with

_pH

varia-tions,

ion

_{concentration,}

_{proton-motive}

force and membrane

_potential

_{[26, 100].}

Acid

tol-erance is

_highly

_dependent

on the

_growth

phase.

Maximal acid resistance of the E. coli

strain 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

_high

pH. By

using

promoter

fusions to measure

the response

according

to

_changes

in

_pH,

it

was observed that the

_expression

of fim-brial genes such

_{as fas}

_(987P)

_{and foo}

(F165

1

)

responded

to

_changes

in

_pH

_[36].

High

concentrations of

_monoamines,

most

notably norepinephrine,

may be one of the

signals

of the small intestine environment.

Indeed,

norepinephrine

can increase the

growth

and

_production

of virulence factors of ETEC

_(K99)

and EHEC

_(SLT-1,

SLT-II),

and this effect is non-nutritional in nature

[81].

Osmolarity,

carbon source,

concen-tration of

₀

₂

_,

ammonia,

sodium bicarbonate and amino acids can combine to

_precisely

indicate the location of the bacteria inside an

organ

[35].

As an

_example,

Edwards et al.

[36]

proposed

that carbon and/or

_nitrogen

gradients

in the _gut

_provide

a mechanism

that allows

_preferential

colonization of dif-ferent _segments

_by

various

_{enteropathogens.}

Glucose concentrations are

_higher

in the

proximal

small intestine than in the distal

portion,

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

_limiting

carbon 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 source and is

_{repressed by}

ammonium

[1 10, 35].

Consequently,

987P fimbriated E. coli will

colonize the distal small intestine. If

_Bfp

expression

is activated at an

_early

_stage

of

infection,

Bfp

fimbriated EPEC will colo-nize

_proximal

as well as distant _segments of 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 thus adhesin

_expression

is

_suppressed.

Effects of the environmental factors described above have been studied in vitro.

Very

few studies have been

_reported

con-cerning

the

_regulation

of E. coli virulence factors

_expression

in

_vivo,

i.e. in the ani-mal. The

_question

is: are the

_regulations

depicted

in vitro indeed

_occurring

in the host?

_Experimental

infections followed

_by

the detection of virulence factor

_expression

in the _organs or tissues are _necessary to

answer this concern. Pourbakhsh et al.

_{[ 109]}

inoculated chicken air sacs with

_septicaemic

avian E. coli isolates and showed that a

much

_{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

_systemic

organs,

suggesting

that the bacteria

undergo

a

_phase

variation with _respect to Fl fim-briae in vivo. A similar Fl fimbrial

_phase

variation also occurs in vivo

_during

the

experimental

induction of E. coli

_peritonitis

and lower

_urinary

tract infection in mice

_[4,

62, 97]

and

_meningitis

in rats

[116].

In the

same

_study

Pourbakhsh et al. showed that

P fimbriae are

_expressed

in bacteria _present

in air sacs and

_systemic

_{organs, but} not in

trachea,

suggesting

that P fimbriae also

undergo phase

variation in vivo

[109].

The nature 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 in

vivo,

the

_sensing

molecules

involved,

the mechanism of

_signal

_{transduction,}

and to

determine their activities on virulence factors

in a

given

location within the host.

3. GLOBAL REGULATORS OF GENE EXPRESSION

Global

_regulators

are involved in _the reg-ulation of virulence gene

expression.

In

that modulate the

_synthesis

of virulence

fac-tors. Some of

them,

such as

_CRP,

_recognize

specific

nucleotide _sequences to which

_they

bind. 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 levels

of

_supercoiling

_{vary in response} to

envi-ronmental

_changes;

in

_{particular, growth}

of bacterial cells under anaerobic

conditions,

at

a

_high

_osmolarity,

at different _{temperatures,}

or in a

_{nutrient-poor}

medium

[29].

Topoi-somerases are _enzymes that act _upon DNA to alter the level of

_{supercoiling,}

as well as

catenate and decatenate chromosomes. In the bacterial

cell,

DNA is

_negatively

super-coiled

_[33].

It is

_thought

that

_negative

super-helical tension facilitates the

_melting

of DNA _necessary for

_replication

and

tran-scription

[79].

This _{mechanism may be} used

by

pathogenic

E.coli to

_regulate

_genes

nec-essary for virulence.

E. _{coli possesses low molecular}

_weight

proteins

which contribute to the

_higher

order

organization

of the bacterial nucleoid and

to the

_expression

of the

_genetic

information.

Frequently,

small architectural

_proteins

such

as

_protein

HU

_(a

histone-like

_protein),

the

related

_protein

IHF

_(integration

host

fac-tor),

H-NS and FIS

_(factor

for inversion

stimulation)

contribute to the control of

tran-scription

of genes whose

products play

a role in environmental

_adaptation

and thus

in the

_expression

of the virulence.

3.1. H-NS

H-NS is one of the two most abundant

histone-like

_proteins

in E. coli

(the

first one

being

HU).

It is a 16-kDa neutral

_protein

present at 20 000

_copies

_{per cell under} a dimeric form

_(for

a

review,

see

_{[ 137]).}

H-NS

can affect the overall structure of DNA

_by

strongly

compacting

DNA,

altering

DNA

superhelicity 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

of the

chromosome,

but also a rather

_dynamic

role in the

_regulation

_{of gene}

_expression

[56, 63, 129].

Many

of the _genes

_regulated

by

H-NS are involved in bacterial

_adaptation

to environmental stress _{in response} to

_signals

such as

_osmolarity,

_temperature

and

oxy-gen

availability

[6].

Transcription

of many

virulence _{genes in E. coli is}

_{repressed by}

H-NS,

and different

_{transcriptional}

regula-tors act on _gene

_{expression by alleviating}

and/or

_{counteracting}

the effect of H-NS. In

some cases, H-NS seems to be

_implicated

in the

_{thermoregulation}

of virulence factor

expression.

The mechanisms

_by

which H-NS affects _gene

_{transcription}

are not

com-pletely

understood. Correlation between

modifications of the overall _DNA

super-coiling

induced

_by

_{H-NS and gene} expres-sion is not clear. In some cases, H-NS can

bind DNA to

_directly

inhibit _gene

tran-scription

[27, 129],

and it has been

_suggested

that H-NS _prevents the RNA

_polymerase

from

_productively

_interacting

with the pro-moter

_[129].

G6ransson et al.

[47]

have

pro-posed

that H-NS could act as ’silencers’ of DNA

_{regions by forming nucleoprotein}

structures similar to

_{transcriptionally}

inactive chromatine. However, these effects are not

indiscriminate as not all

_promoters

are H-NS

_repressible.

H-NS also

_participates

in the control of

_sigma

S

_synthesis

_[10].

Sev-eral

_examples

of such

_regulations

involved in fimbriae and

_{haemolysin synthesis}

as well l as in bacterial invasiveness are

_given

below.

3.1.1.

_Regulatory

role

_{of H-NS}

in

_fimbrial

_gene

_expression

The

_sfa

determinant codes for S fimbrial adhesins of extraintestinal E. coli. SfaA is

the

_major

structural

subunit,

whereas SfaB and SfaC are

_{positive regulators of sfa}

tran-scription.

Most

_.</

_M

_{transcription}

initiates at

the

_slaB

_promoter, the

_!!faA

_promoter

_being

very

poorly

active. Frameshift mutations in

4

aB

or

₄

_f

_h

_C

result in very low levels of

_sfa

transcription. Expression

of S fimbriae from

hns mutants, where

_pA

_{transcription}

is

strongly

activated

[87].

The authors

con-clude that the

_negative

control exerted

_by

H-NS on

_sfa

transcription

is counteracted

by

the

_sfa-specific

activators SfaB and SfaC

(figure

1).

P fimbriae are

_{produced by}

cells

grow-ing

at 37 °C but not at lower _temperatures such as 25 °C. H-NS

_plays

an

_important

role in this

_{thermoregulation}

_{by repressing}

the

transcription

of a

_regulatory

cistron in the

pap gene cluster.

PapB

and

Papl

are

posi-tive

_{regulators homologous}

to SfaB and

SfaC,

respectively.

Papl transcription

is

dere-pressed

in hns mutants at both 37 and 26 °C.

Transcription

of

_{the papBA}

_{operon is also}

derepressed

at both _temperatures

_although

to a lesser extent

_{[47, 136].}

_By

_competitive

gel

retardation _{assays it} was shown that

H-NS

_specifically

binds _{to pap} DNA

contain-ing

pap GATC sites and was able to block

methylation

of these sites in vitro. It was

suggested

that the

_ability

of H-NS to act as a

methylation blocking

factor is

_dependent

upon the formation of a

_{specific complex}

of

H-NS

_{with pap regulatory}

DNA.

Transcrip-tion of _pap is enhanced

_{by binding}

of the cAMP-CRP

_complex

in the

_intergenic

papl-papB

DNA. In hns mutants,

the pap

operon

is

_expressed

in the absence of the

_PapB

and

cAMP-CRP

_{transcriptional}

activators. It has been

_suggested

that the cAMP-CRP

com-plex

would

_play

a role as an

_{anti-repressor}

alleviating

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 that

promote

the inversion of a

_314-bp

DNA

segment

containing

the

_fimA

_promoter.

FimA is the

_major

structural subunit of _type 1 fimbriae. In hns mutants,

transcription

of

fimB and fimE

is increased at 30 °C and to a

lesser extent at 37

°C,

leading

to an

increased inversion rate

_{of the fimA}

pro-moter. Donato et al.

[27]

have shown that

H-NS

_specifically

and

_{co-operatively}

binds to the

_fimB

_promoter

_region

and represses

transcription. They

propose that H-NS

rec-ognizes

a

_specific

DNA feature rather than

a

_specific

consensus _{sequence. This feature}

could be a

_specific

conformation such as an

intrinsic curvature due to the known

_affinity

of H-NS for curved DNA

_[137].

This was

recently

confirmed

_by

_competitive

_gel

retar-dation _{assays where} it was shown that H-NS binds to the

_regions

_{containing fimB}

and

fimE

promoters

and does not affect the

switching frequency

in vitro

_[105].

Curli are thin fimbriae

_expressed

at 26 °C

in medium or low

osmolarity.

Curli expres-sion is

_dependent

on

_RpoS,

the

_sigma

S

fac-tor.

_{Transcription}

of

_{csgA (encoding}

the

major

structural

_subunit)

can be activated

in

_rpoS

mutants

_by

inactivation of

hns,

but

the

_temperature

and

_osmolarity

control is maintained.

_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

or

indirectly by

H-NS can be relieved

_{by RpoS,}

or

_by

a non-identified

regulatory protein

positively

controlled

_{by RpoS.}

3.1.2.

_Regulatory

role

_{of H-NS}

in enteroinvasive E. coli

_(EIEC)

virulence _gene

_expression

The genes

required

for EIEC invasive-ness are carried on a

_large

virulence

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

repressed

at 30 °C and to a lower extent at 37 °C, whereas

_{transcription}

of virF is

poorly

affected.

Thus,

the

_higher

content of

H-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

_{transcription}

via 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 counteracts

the effect of

H-NS,

as

cAMP-CRP,

SfaB

and

SfaC,

or

_RpoS

in the case of the _pap,

.!fa,

and _{csg operons,}

_{respectively.}

3.1.3.

_{Effect of H-NS}

on

_haemolysin

production

Haemolysin synthesis

is

_repressed

at

_high

osmolarities and at low _{temperatures.}

Car-mona et al.

_[20]

have shown that the

muta-tion in the hha gene,

encoding

a

_putative

histone-like

_{protein, derepresses}

_haemolysin

production

when _{cells grow either} at low temperature or in a

_high

_osmolarity

medium. In a hns mutant as _{in the hha mutant,}

_hlv

_’v

transcription

increases about 2-fold

com-pared

to the

_wild-type

strain. However, in a hha-hns double mutant,

haemolysin

expres-sion shows a 10-fold increase.

Thus,

H-NS

participates

in the modulation of

_expression

of the

_hly

_{genes. The}

_regulatory

mechanism

by

which this is achieved is not clear but it

does not seem to involve modification in

the level of DNA

_supercoiling

_[93].

3.1.4.

_{Effect of typA}

on H-NS

_expression

In E. coli

K12,

the inactivation of the

typA

gene

encoding

the

TypA

protein

(for

tyrosine

phosphoprotein)

alters _the

expres-sion and the modification of _several

pro-teins,

such as the

disappearance

of the

uni-versal stress

_protein

_UspA,

carbon starvation

protein CsplS

and the increased

_synthesis

of H-NS

_{!39, 42].}

The

_TypA

_protein

is

phos-phorylated

on

_tyrosine

residues in vivo and

in vitro in the EPEC strain MAR001 but not in K12 E. coli strains. The _{sequence of}

_TypA

from the E. coli K-12 strain differs from that of an EPEC strain

_by

six amino acid residues

and the

_protein

is three amino acids shorter. Freestone et al.

_[42]

concluded that

_TypA

could be a candidate for

_studying

the role

of

_tyrosine

_{phosphorylation}

in the

_global

regulatory

network.

_BipA

of Salmonella

typhimurium

is the

_homologue

of

_TypA

and is

_implicated

in

_{pathogenesis.}

_{Another team,}

Farris et at.

_[39]

_reported

that

_BipA/TypA

of EPEC is a

_{tyrosine-phosphorylated}

GTPase that _presents a new class of

viru-lence

_regulators

as it controls several

in host

_epithelial

_cells,

_{flagella-mediated}

cell

motility

and resistance to the antibacterial

effects of a human host defence

_protein.

3.2. IHF

_(integration

host

_factor)

IHF is an abundant

_{sequence-specific}

DNA

_{binding protein}

that induces a

signif-icant bend in the DNA and is involved in a

wide

_variety

of _{processes in E.} coli

includ-ing regulation

of _gene

_expression.

himA and

himD _{genes encode} the subunits of IHF.

There is an absolute

_requirement

of IHF for

phase

variation of _type 1 fimbriae which uses

_{site-specific}

recombination to switch

phases.

This recombination has an absolute

requirement

for IHF

_[37].

IHF also

_plays

a

role in the

_expression

of

_region

_{I genes} of

the _type II

_capsule

_{K5 operon}

_[119J.

Bind-ing

site consensus _sequences were

identi-fied in the

_vicinity

of the

_{transcription}

start

site of

_region

1 of the

_kps

cluster. Mutations in himA and himD led to a 5-fold reduction

in the

_expression

of the

_capsular

_gene

_kpsE

at 37 °C in

_region

1. This indicates a role of IHF in

_mediating

the

_expression

of

_region

I of the

_capsular

_{gene cluster}

_[119J.

_J.

3.3. CRP

The

_global

_regulator

CRP

_regulates

a

variety

of genes in E. coli in _response to the level of cAMP which is

_{synthesized by}

the

adenylate cyclase

in response to the carbon

source. Catabolite

_repression

is due to the inactivation of

adenylate cyclase

when

glu-cose is

_transported

into the cell. The

pro-tein CRP when

_complexed

with cAMP binds to a

_specific

DNA _sequence named the

_CRP-binding

site. _{This sequence} occurs

at different distances _upstream of the

tran-scriptional

start site in different operons and

leads to the activation of

_{transcription.}

It is known that cAMP-CRP modulates expres-sion of certain E. coli virulence factors such

as enterotoxins STa and STb

_{[3, 17J}

and

fimbriae such as

_Pap,

_F165,

and

_F165

_z

_pili,

colonization factor

_antigen

II,

987P and K99

[22, 36, 46].

The

_expression

of other

fim-briae,

such as K88

_{expressed by}

ETEC or

BFP

_expressed

_by

_EPEC,

is, however,

not

under CRP-cAMP

_regulation.

From that

observation,

a

_working

model for the

tem-poral

and

_{spatial regulation}

of fimbrial

expression

in the small intestine

_by

the

car-bon source was

_{suggested by}

Edwards and

Schifferli

[36],

where the

_regulation

of fim-brial

_expression

_by

the carbon source is a

major

factor in

_determining

the initial site of intestinal

_binding

_(see

section

_2).

3.4.

_RpoS

rpoS

encodes an RNA

_{polymerase sigma}

factor

_(sigma

_S,

_sigma

38 or

_KatF).

It con-trols a

_regulon

of 30 or more _{genes in}

response to starvation and

_during

transition to the

_{stationary phase. During}

transition into the

_{stationary phase,}

the

_expression

of

sigma S-dependent

genes is activated in a

certain

_temporal

order. The

_sigma

S

_regulon

can be divided into subfamilies which

include genes

regulated

by

specific

stresses

and/or additional

_{global regulatory proteins.}

Subsets of

_{sigma-dependent}

_genes include those _{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

the

Hpl

and

_HpII

_{hydroxyperoxidases}

which

confer resistances to

_H

₂

₀

₂

_,

as well as

_CsgA,

the main subunit of surface

_protein

curli (see

section

8.4) [82].

Maximal acid resistance of

E. coli MC4100

_(and

base

resistance)

is

exhibited at the

_{stationary phase}

and

_depends

on

_RpoS

_{although RpoS}

is not an absolute

requirement

under all

_growth

conditions

[ 122].

For acid

resistance,

and in _part base

resistance,

the

_{rpoS requirement}

can, how-ever, be overcome

_by

anaerobic

_growth

in moderate acid.

3.5.

_Lrp

The leucine

_{responsive protein, Lrp,}

affects the

_{transcription}

of a

_large

number of genes,

increasing

the

expression

of some

are turned on or off

_by

_{exogeneous leucine}

[92].

It is

_noteworthy

that

_Lrp

is involved in all

_regulatory

mechanisms of fimbriae

expression

described so far

_(see

section

_8).

The

_{physiological}

role of

_Lrp

is unclear. Calvo and Matthews

_[19]

_suggest that

_Lrp

positively regulates

genes that function

dur-ing

famine and

_{negatively regulates}

those

working during

a feast.

_They

_propose that

pili

may be

expressed

at

_higher

levels

_during

growth

in a nutrient-deficient medium where an abundance

_{of pili}

_may

_help

bacterial

adherence to

_epithelial

cells,

thus

_helping

the maintenance of bacteria in the environ-ment. Under conditions of nutrient excess,

bacteria can _grow

_rapidly

and should not have any

difficulty

in

_maintaining

their

pres-ence.

_Thus,

abundant

_synthesis

of fimbriae

would not be so critical for survival.

3.6. Fur

The

_exceedingly

low

_availability

of iron

in mammalian tissues is an environmental

signal indicating

to the bacteria _entry into

a host. This

_signal

_triggers

the co-ordinate

expression

of bacterial virulence

determi-nants

_through

the Fur

_protein

which

_plays

a

general

role as a sensor of iron

_availability

in the cell. The

_apoprotein

binds

Fe

z+

as a

cofactor,

and the cofactor-bound

_protein

binds to various

sites,

termed iron-boxes.

This

_{complex negatively}

_regulates

_many

genes involved in iron

uptake

as well as

toxin _genes

_hly

and others. The

_synthesis

of

the 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 to

specific

DNA sequences, the Fur

boxes,

located in the operator

regions

of the

iron-regulated

operons

[18, 25].

Fur does not bind to its DNA _target in the absence of iron and genes are thus

_derepressed

in conditions

of low iron

_{availability.}

It is

_interesting

to note that _{slt-l genes} are carried

_by

a

prophage

and are nevertheless controlled

by

a

_{chromosomally}

encoded

_protein

in a

way similar to aerobactin

_(plasmid

_encoded)

and enterobactin

_{(chromosomally}

_encoded).

In contrast to the slt-I

_promoter,

the slt-II

and slt-llv _promoters do not contain Fur boxes and are not controlled

_by

iron

avail-ability

[ 125].

] .

4. FAMILY OF THE ARAC TRANSCRIPTIONAL ACTIVATOR

AraC is the

_{transcriptional}

_regulator

of

the arabinose operon. Proteins in the

family

of AraC activator contain helix-turn-helix

motifs,

which bind

_specific

DNA _sequences upstream of _{genes that} are

_actively

tran-scribed.

The activation of virulence _gene expres-sion in EPEC

_requires

an

_{AraC-homologous}

transcriptional

activator

_protein

called

PerA/BfpT

[45]

which is encoded

_by

a

vir-ulence

_{plasmid, pMAR2.}

The _genes of the

per locus are

_required

for

_{transcriptional}

activation of _genes whose

_products

are

secreted

_by

the

_type

III secretion _system

(eae,

encoding

intimin,

and

_espB)

and

_they

are also involved in activation

_{of plasmidic}

genes such as _{the adhesin gene}

_bfpA

encod-ing

the bundle

_{forming pilus}

_{[70, 110, 128].}

Insertional inactivation

_{of perA}

led to a

reduced

_expression

of Eae which could be

due to

_specific

mutations in

_perA

or to

_polar

effects on _{genes downstream}

_{of pera.}

Puri-fied

_PerA/BfpT

was shown to bind

_directly

to _{DNA sequences upstream} of

_bfpA

and eae

_[128].

Regulatory proteins

of some fimbrial

operons 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 the

CS I fimbrial genes. It was

_recently

shown that Rns is

_capable

of

_{complementing}

a null

mutation in the S.

_{flexneri virf gene}

encod-ing

the

_homologous

_counterpart

of Rns

expression

in ETEC. It was concluded that

there are differences in the mechanisms

by

which these related

_{transcription}

factors

reg-ulate gene

expression.

It is not known

whether the Rns class of

_{regulatory proteins}

binds DNA or whether there are additional factors in the

_regulatory

network

_[78].

5. SENSOR REGULATORY

PROCESSES

Environmental

_signals

control virulence

gene

expression

in bacteria. A

developing

research field is

_{understanding}

the _ways in which bacteria sense these

_signals

and

trans-duce them into the cell to

_regulate

gene

expression. Many signal

response systems in

bacteria

_operate

_{by complex pathways}

involving

two-component

regulatory

sys-tems. These _systems consist of:

₁₎

the sen-sor

protein

which _{spans the}

cytoplasmic

membrane 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.

When

the sensor

_component

receives an external

signal,

it

_{undergoes autophosphorylation}

and transfers the

_phosphate

residue to the

regulator

protein

which in turn activates or

represses gene

transcription.

Several two-component systems

controlling

virulence

gene

expression

have been well character-ized in different bacteria

_[40J.

In E.

coli,

the

expression

of some _group IK

_antigens

as well as colonic acid is

_{regulated by}

the

RcsABC

_(regulator

of

_{capsule synthesis)}

system.

A

_temperature

below 25

°C,

the presence of a

_{high phosphate}

concentration

and osmotic induction increase the

synthe-sis of these

_antigens

_{[121]. The}

_integral

inner

membrane RcsC sensor and the

cytoplas-mic RcsB effector show

_homologies

to the

family

of the

_{two-component}

histidine kinase

_signalling

_systems

[48

The

mecha-nisms

_whereby

_{extracytoplasmic signals}

are

sensed 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 the

phosphate

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 regulator}

and is

_subject

to

_{degradation by}

the Lon

protease [68!.

] .

Effective mechanisms for the induction or

repression

of virulence _gene

_expression

involve the

_sensing

of

_{’signature’}

molecules

produced by

host tissues. In this _{way, the}

E. coli

_regulons

SoxRS and

_OxyR

are

designed

to deal with the

_cytotoxic

prod-ucts

_{produced during}

the

_{oxygen-dependent}

respiratory

burst,

with a

_large

number of

proteins being

induced in response to

hydro-g

en

peroxide

(H

2

0

2

)

’

superoxide

anion

(0

2

)

or nitric oxide

(NO)

produced

by neutrophils

and

_macrophages

_{[82, 99].}

The SoxRS reg-ulon controls the

_expression

of

approxi-mately

ten genes. SoxR is an iron

_sulphur

protein

that senses _exposure to

_superoxide

and nitric

_oxide,

and then activates the

tran-scription

of the _{soxS gene. SoxR and SoxS}

are

DNA-binding proteins,

SoxR

being

a

member of the MerR-like

_family

_(MerR

is

involved in the _response to

Hg

2+

)

and SoxS

being

a member of the

_AraC-family.

Tran-scription

of soxS is initiated in a manner

dependent

on the

_rpoS

_gene in response to

entering stationary phase growth

[98].

The SoxS

_protein

then activates the

transcrip-tion of a

_variety

of _genes

_including

sodA,

encoding

the

_{Mn-dependent superoxide}

dis-mutase,

nfo, encoding

the DNA

_repair

enzyme endonuclease

IV,

and micF, which

post-transcriptionally

decreases the pro-duction of

_OmpF

_[82].

OxyR,

a member of the

_LysR

_family

of

autoregulators,

is also involved in resistance

to

_{oxygen-related compounds.}

The

_OxyR

protein

activates the

_{transcription}

_of

approx-imately

nine genes in E. coli in response to

H

z

0

2

.

These include

katG,

encoding

HP1 I

catalase,

and

_ahpFC,

_encoding

encoding

a

_protein

that

_{non-specifically}

binds to DNA to

_protect

cells from

_H

₂

₀

₂

toxicity. OxyR

functions both as a sensor and a transducer and contains a critical redox-sensitive

_Cys

residue that is oxidized

by hydrogen peroxide

[117, 139].

The level of

_oxyR

mRNA or

_OxyR

protein

does not

change significantly following

exposure of the cells to

_H

₂

₀

_2’

Hence, it was

_suggested

that

_{post-translational}

_regulation

_by

an

_H

₂

₀

₂

-generated signal

activates the

_pre-existing

OxyR protein

[80].

P

_{pili-mediated}

_{attachment may} also be

an

_important

_part of sensor

_regulatory

pro-cesses involved in

uropathogenic

E. coli

during urinary

tract infection.

_Zhang

and

Normark

[138]

have shown that airs

tran-scription

is

_specifically

activated

_by

P

_pili

attachment. The AirS

_protein

is a

sensor-regulator

protein

essential for the

iron-star-vation _response of

_{uropathogenic}

E. coli that 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

of virulence factors of E. coli via P

_pili

attach-ment.

_Hultgren

_[61]

_hypothesized

that the

adhesion of P

_pili

to

_epithelial

cells avoids

polymerization

of the

_pilin

fibre and thus

leads to

_aggregation

of misfolded

_pilus

sub-units in the

_periplasm.

Protein

_aggregation

in

the

_periplasm

is sensed

_by

the

_CpxA-CpxR

system,

which activates htrA

_encoding

_DegP

(a

periplasmic

protease

degrading

misfolded

proteins)

[23, 107],

and could be involved in

controlling expression

of virulence factors such as

_haemolysin

or CNF

(cytonecrotizing

factor). Thus,

the interaction between the

pathogen

and its host _receptors mediated

_by

adhesins _{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,

bacterial

attachment to mucosal surfaces via P

_pili

indicates that bacteria have reached an

eco-logical

niche where

_expression

of virulence

factors

_{(siderophores,}

_toxins)

are _necessary

for survival.

6. tRNA REGULATION OF VIRULENCE GENES

Many

virulence genes are located on

’pathogenicity

islands’

_{(Pais), large}

chro-mosomal

_regions

that are often associated

with

_particular

tRNA _genes

_[52].

It has been shown that the Pais modulate _{virulence gene}

expression

through

the action of these tRNA

[112].

In

_Shigella,

it has also been demon-strated that

_expression

of

_tRNA

_/

_yr

_partially

complemented

the virR mutation. virR is an

analogue

of hns and is

_required

for

co-ordinating

temperature-regulated

virulence

gene

expression

of

Shigella

[57].

The

_{uropathogenic}

E. coli strain 536

(06

:K15

_:H31)

carries two

_{pathogenicity}

islands,

one Pai

_comprising

the gene cluster

of

_haemolysin

and the other Pai

compris-ing

the _gene clusters of

_haemolysin

and

P-related

fimbriae,

both Pais

_being

flanked

_by

tRNA genes, leuX and

_selC,

_{respectively.}

In the

_{uropathogenic}

strain

_{536, spontaneous}

deletions

_resulting

in the truncation of leuX

or the

_specific

deletion of the

_tRNA

₅

_l

_eu

gene resulted in the lack of

_expression

of _type 1

fimbriae and other virulence factors such as

haemolysin,

aerobactin

_production,

serum

resistance

_phenotype

and

_motility,

while

trans-complementation

of tRNA loci leads to

a restoration of these

_properties

_{[91, 127].}

tRNA

5

&dquo;

1

is

_specific

for the minor leucine

codon UUG. It has been shown that

tRNA

5

l

eu

is

_required

for efficient transla-tion of FimB whose _gene contains five TTG codons

_recognized

_by

_tRNA

₅

_’

_cu

that in turn

leads to type 1 fimbrial

_expression

[113J.

].

The

_{selenocysteine-specific}

tRNA

_(selC!

directly

influences the

_ability

of the E. coli

strain 536 to _{grow under} anaerobic condi-tions because

_{selenocysteine}

is part of the

formate

_{dehydrogenase}

_(FDH)

_enzyme which is involved in mixed acid fermenta-tion. The

_ability

to _grow under anaerobic conditions via mixed acid fermentation may be

_important

for the colonization of E. coli

in the

_kidney

because

_{oxygen-limiting}

7. BLACK HOLES AND

VIRULENCE GENE EXPRESSION

Not

_only

deletion of _genes such as leuX can affect the

expression

of

virulence,

but

addition of some _genes can also alter the

virulence of bacteria. It was observed that

Shigella

and enteroinvasive E. coli

_displayed

deletions in the cadA

_region

_present in most E. coli strains

_including

E. coli K12

_[84].

These deletions have been termed black holes and

_they

enhance the

_expression

of virulence _{genes. Indeed the} introduction of

the cadA _gene

_{encoding lysine}

decarboxy-lase attenuates the virulence of

_Shigella

flexneri

and the enterotoxin

_activity

was inhibited

_by

cadaverine,

a

_product

of the

reaction

_{catalysed by lysine decarboxylase.}

8. REGULATION OF FIMBRIAL

EXPRESSION

Synthesis

of fimbriae is under

_complex

regulatory

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 membrane pore and

regulatory proteins.

The

major

fim-brial subunit must be

_expressed

at

_higher

levels _{than other accessory}

_proteins.

Fim-brial determinants are

_organized

in operons

and differential

_expression

of _genes is achieved

_{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

_papB

promoter. Differential

_expression

of these

two _{genes results from} RNAse

_E-dependent

endonucleolytic cleavage

of the mRNA in the intercistronic

_papB-papA

_region.

This

cleavage

is followed

_{by rapid decay}

of the

upstream

papB-encoding region

and

accu-mulation of the stable

_{papA-encoding}

mRNA,

leading

to

_high

level

_expression

of

the

_major

subunit,

PapA,

relative to that of

the

_{regulatory protein PapB}

₁₉₄

Differen-tial mRNA

_stability

and mRNA

_processing

are also involved in the control of the daa

operon

(encoding

for the F1845

fimbriae)

[ 12].

Transcriptional organization

of the daa operon is

quite

different from that of pap

since the

_major

_{fimbrial subunit gene} daaE is located at the 3’ _{end of the operon.} A

_large

transcript

encoding

accessory

proteins

in

addition to the

_major

subunit is submitted

to

_{endoribonucleolytic cleavage}

to gener-ate a stable daaE mRNA. This

_cleavage

requires

neither RNAse III nor RNAse E.

Second,

expression

of fimbriae is controlled

by

environmental

_{signals through}

alterations

in

_transcript

levels. The control of fimbriae

expression

is of crucial

_importance

for the survival of bacteria in vivo

_[35].

It allows

fimbriae to be

_{expressed only}

at the

ade-quate site in the host and to evade the host’s s immune response. All fimbrial _operons examined so far encode

_{specific regulatory}

proteins

that either activate or _repress

tran-scription.

In

addition,

global regulators

such

as

_Lrp,

CRP, H-NS, IHF,

RpoS, play

roles in

controlling

the

_expression

of most fimbrial

operons. Four different

regulatory

mecha-nisms

_controlling

the

_expression

of fimbriae have been described.

_Operons

that share

common

_regulatory

_properties

are

classi-fied in table I.

8.1. The _pap

_{regulatory family}

Regulation

of the pap

expression

is the

most

_extensively

studied.

_Expression

of P

fimbriae is

_subject

to

_phase

variation,

i.e. fimbrial

_expression

switches between ON

(fimbriae-positive

cells)

and OFF

(fimbriae-negative

cells)

states. The switch

_frequency

is controlled

_by

environmental factors such

as _temperature or carbon source. Phase vari-ation of P fimbriae

_depends

on the

mcthy-lation status of two GATC sites located in

the intercistronic

_region

between _the

regu-latory

genes

papl

and

_papB

which are tran-scribed

_{divergently (figure}

_3).

The two GATC

sites,

GATC-I and

_GATC-II,

are

located within the

_Lrp-binding

sites.

Schematically,

when

_Lrp

binds the