INFECTION AND IMMUNITY, Sept. 2007, p. 4199–4210
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 9
Enteropathogenic and Enterohemorrhagic Escherichia coli
Virulence Gene Regulation?
Jay L. Mellies,1* Alex M. S. Barron,2and Anna M. Carmona3
Biology Department, Reed College, 3203 S.E. Woodstock Boulevard, Portland, Oregon 972021; Oregon Health Sciences University,
L215, 3181 S.W. Sam Jackson Park Rd., Portland, Oregon 972392; and School of Public Health, University of
California, Berkeley, 140 Warren Hall #7360, Berkeley, California 94720-73603
Enteropathogenic Escherichia coli (EPEC) and enterohem-
orrhagic E. coli (EHEC) cause significant morbidity and mor-
tality worldwide (18, 60, 63). Though these E. coli pathotypes
are genetically related, many features of their epidemiology,
their pathogenesis, and the niches they occupy within the hu-
man host are unique. EPEC causes profuse watery diarrhea,
primarily in children under the age of 2 years, and mostly
affects individuals residing in developing countries. In contrast,
adults and children infected by EHEC bacteria can suffer from
either bloody or nonbloody diarrhea, and in a small percentage
of cases a life-threatening complication known as hemolytic
uremic syndrome (HUS) occurs. Many patients with HUS ex-
perience long-term renal damage, and they often require dial-
ysis or kidney transplantation. EHEC produces Shiga toxins
(Stx), which can cause damage to renal endothelial cells, re-
sulting in HUS, while EPEC bacteria do not possess stx (72).
EHEC disease appears in primarily industrialized nations yet
causes fewer disease outbreaks in developing countries. This
observation has been anecdotally attributed to immunological
cross-protection from the related EPEC bacteria prevalent in
the less developed regions of the world.
There are two additional important differences that distin-
guish these two E. coli pathotypes. Approximately 108to 1010
EPEC bacteria are necessary to cause infection in adult human
volunteers (6, 27), while the infectious dose for EHEC is far
less, estimated to be less than 100 CFU (49). Intriguingly,
EPEC infects the small intestine; EHEC infects the large
bowel, inflicting bloody diarrhea resulting from damage to the
colon. Variants of the outer membrane protein intimin, ex-
pressed by both pathotypes, have been implicated as contrib-
utors to tissue tropism (103), but whether intimin is the initial
adhesin and, secondly, whether other factors contribute to the
ability of EPEC to recognize the small bowel and of EHEC to
colonize the large bowel are not clearly understood.
AE. EPEC and EHEC share genetic and phenotypic simi-
larities, most notably the locus of enterocyte effacement (LEE)
pathogenicity island (PAI), encoding a type III secretion sys-
tem (TTSS), and the ability to form attaching and effacing
(AE) intestinal lesions, intimate attachment to the host cell,
and formation of “pedestals” cupping individual bacteria (86);
for recent reviews, see references 12, 60, and 95. The LEE PAI
is essential for disease for both EPEC and EHEC bacteria
(27, 31, 105). The EPEC LEE expressed from a multicopy
plasmid transformed into a K-12 laboratory strain of E. coli
was necessary and sufficient to form the AE phenotype on
human epithelial cells in culture (80). In contrast, the EHEC
LEE alone was not sufficient to confer the AE phenotype when
expressed in a laboratory strain of E. coli (33), suggesting that
factors and/or regulatory proteins necessary for this phenotype
exist outside the EHEC LEE. Indeed, TccP (Tir-cytoskeleton-
coupling protein [also called EspFu], a protein with 24%
amino acid identity to EspF) is encoded within the CP-933U
cryptic prophage in EHEC, is translocated through the TTSS,
and is necessary for actin accumulation and thus AE lesion
formation by EHEC on human epithelial cells in culture (10,
40). It is now known that at least 39 proteins are translocated
through the LEE-encoded EPEC and EHEC TTSSs into the
host cell cytosol (39, 133). Many of the non-LEE-encoded
TTSS-dependent effector proteins are found within cryptic
Flagellar motility. The role of flagellar motility in EPEC and
EHEC pathogenesis is not clearly defined, though it has been
observed that the flagellum itself can contribute to EPEC ad-
herence to epithelial cells in culture (43), and flagella contrib-
ute to colonization in a chick model of EHEC infection (69).
Quorum sensing controls flagellum expression (15, 17), and
this observation is discussed below.
LA phenotype. Clinical identification of EPEC strains has
classically included their ability to attach to cultured epithelial
cells in what has been termed localized adherence (LA) (110).
This phenotype requires the type IV, bundle-forming pilus
(BFP) (26, 42, 116, 117, 126) and is indicated by the formation
of microcolonies, in general between 5 and 200 individual
bacteria in three-dimensional clusters on the surface of the
epithelial cells. The genes encoding the BFP are located on the
70- to 90-kb E. coli attachment factor (EAF) virulence plasmid.
EPEC strains deleted for bfpA, the gene encoding the major
subunit of the BFP, are attenuated for virulence in human
volunteers (6). Some studies indicate that BFP is the initial
attachment factor conferring adherence to the human intesti-
nal epithelia and tissue specificity (42), but other studies have
indicated that EPEC can adhere to intestinal epithelial cells
and form AE lesions in the absence of BFP (53).
EHEC plasmid pO157. Though the EHEC pathotype does
not possess the BFP, these strains do contain a virulence plas-
mid, pO157. Plasmid pO157 gene-encoded proteins implicated
in EHEC pathogenesis include HlyA, a hemolysin; EspP, an
autotransported serine protease involved in the cleavage of
human coagulation factor V (7); ToxB, a 362-kDa protein
sharing amino acid sequence similarity with the large Clostrid-
* Corresponding author. Mailing address: Biology Department,
Reed College, 3203 S.E. Woodstock Boulevard, Portland, OR 97202.
Phone: (503) 517-7964. Fax: (503) 777-7773. E-mail: jay.mellies@reed
?Published ahead of print on 18 June 2007.
at Reed College on August 22, 2007
ium toxin family, which is involved in adherence to epithelial
cells in culture (128); a catalase; and StcE, a zinc metallopro-
tease. StcE is secreted by the etp type II secretion system,
cleaves the C1 esterase inhibitor (C1-INH) of the complement
pathway, has mucinase activity, and is thought to be involved in
colonization and tissue damage (50, 70).
Over the past decade we have gained considerable knowl-
edge concerning the regulation of EPEC and EHEC virulence
genes and their associated phenotypes. Because of the essen-
tial link between diarrheal disease and expression of the TTSS,
i.e., assembly of the secretion apparatus and translocation of
effector proteins into the host cell cytoplasm leading to altered
cell signaling events and ultimately diarrhea, much focus has
been devoted to understanding regulation of genes located
within the LEE PAI. Certainly these studies help us to better
our understanding of the microbe-host interaction, how EPEC
and EHEC perceive and respond to host-associated environ-
mental cues, and understand the important similarities and
differences between these two E. coli pathotypes. This review
summarizes the current knowledge of virulence gene regula-
tion of EPEC and EHEC, draws together a global regulatory
network, and finally discusses how our understanding of
virulence gene regulation of these two important pathogens
is being utilized to develop novel therapies against E. coli
Control of AE lesion formation and protein secretion.
Pathogenic bacteria must respond properly to their surround-
ing environment to coordinate virulence gene expression and
to survive within a specific niche. Elucidation of the kinetics of
AE lesion formation demonstrated that this complex pheno-
type is tightly regulated in response to temperature and growth
phase. Activation of EPEC at 37°C in tissue culture medium
enhanced the formation of AE lesions on human epithelial
cells in culture (108). AE lesions do not form if the bacteria are
incubated at 28°C prior to infecting host cells at 37°C, and they
are formed more readily by cultures in the early exponential
phase of growth. Consistent with the temperature control of
AE lesion formation, protein secretion via the EPEC TTSS
occurs maximally at host body temperature in tissue culture
medium such as Dulbecco’s modified Eagle’s medium
(DMEM), at pH 7, and at physiological osmolarity (64, 65).
Secretion of EspA, EspB, EspC, and Tir proteins is also stim-
ulated in the presence of iron and sodium bicarbonate,
whereas it was inhibited by ammonium chloride or by omission
of calcium from the growth medium (57).
Genetic organization of the LEE. Studies to elucidate tran-
scriptional control of the TTSS, and thus the AE phenotype,
were assisted by characterization of the genetic organization of
the EPEC LEE (33, 82, 109). The LEE1, LEE2, and LEE3
operons encode the type III apparatus components that span
the inner and outer membranes, including EscC, the outer
membrane porin, and EscN, the ATPase of the system. The
LEE4 operon encodes the EspA protein, the monomer that
polymerizes to form the filament over the EscF needle struc-
ture necessary for injection of effector molecules into the host
cell cytoplasm (68); EspB and EspD, which form a pore in the
host cell membrane (56); and EspF, which is injected into the
host cell and targeted to the mitochondria, where it plays a role
in the cell death pathway (94). EspF has also been demon-
strated to disrupt transepithelial cell resistance, leading to dis-
ruption of tight junctions (81). The LEE5 operon encodes the
Tir and intimin proteins, which are necessary for intimate
attachment to the host epithelium, and CesT, a chaperone for
Tir (33, 74).
Silencing by H-NS. As with many virulence systems of gram-
negative pathogens, H-NS plays an important role in the si-
lencing of genes of the LEE and is responsive to multiple
FIG. 1. Regulation of the LEE PAI in EPEC (A) and EHEC (B). Thin arrows represent positive regulatory signals, and thin blunt arrows
represent negative signals. Solid arrows indicate expression of regulatory proteins. Horizontally acquired regulatory proteins appear in ovals with
a black background, and regulatory proteins endogenous to E. coli are within ovals with a white background. See Tables 1 and 2 for listings of
4200 MINIREVIEWSINFECT. IMMUN.
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environmental signals and regulatory proteins (Fig. 1A). In
EPEC, the LEE1 operon is repressed by H-NS at 27°C and
activated at 37°C (135). H-NS binds to EPEC LEE1, LEE2,
and LEE3 regulatory DNA and thus directly controls LEE
transcription. Genetic and biochemical analyses have indicated
that H-NS silences by binding over extended regions and is
capable of bridging or looping DNA (21, 22, 28).
The Ler regulatory cascade. The LEE1-located Ler (LEE-
encoded regulator) has emerged as a key regulator of the
EPEC AE phenotype, exerting its effect at the level of tran-
scription (82) (Fig. 1A). Ler exerts tight control over the genes
of the LEE, as a ler mutant of the prototypical EPEC strain
E2348/69 is severely diminished in its ability to form AE le-
sions on epithelial cells in culture (34, 37). The predicted
15.1-kDa Ler protein exhibits amino acid sequence similarity
with the H-NS family of DNA-binding proteins and shows
greater similarity to the C terminus of H-NS, which is pre-
dicted to be a DNA-binding domain (120). Ler is clearly dis-
tinct from H-NS because all other members of this protein
family identified to this date silence transcription, either di-
rectly or indirectly. In a cascade fashion, upon reaching host
body temperature the LEE1-encoded Ler protein increases
transcription of the LEE2, LEE3, LEE4, and LEE5 operons as
well as espG, escD, and map of the LEE (9, 34, 51, 73, 82, 109,
120, 135). Ler also increases expression of espC (34, 73). The
EspC protein is an enterotoxin encoded within a second EPEC
PAI; it causes disruption of the cytoskeleton and is translo-
cated into the host cell cytosol by a TTSS-independent mech-
anism involving pinocytosis (84, 90, 125, 137), and transloca-
tion is enhanced by contact with the host cell (Fig. 1A). Since
espC is not located within the LEE PAI, Ler is considered a
global regulator of EPEC virulence.
As with EPEC, Ler is a key regulator of EHEC virulence
genes (Fig. 1B). Elliott et al. (34) demonstrated that EHEC
strain 86-24 deleted for the gene encoding this protein did not
form AE lesions on cultured human intestinal epithelial cells
by fluorescent actin-staining assay and that EHEC ler can func-
tionally substitute for EPEC ler. Additionally, in a rabbit model
of infection (rabbit diarrheagenic E. coli), deletion of ler at-
tenuated virulence (149). Ler regulates expression of the
EHEC EspA, EspB, EspD, Tir, intimin, and EspG proteins
targeted for secretion, and by lacZ fusions Ler regulates tran-
scriptional activity of the EHEC LEE2, LEE3, and LEE5 oper-
ons (25, 34). The pO157 virulence plasmid-located stcE gene of
EHEC is regulated by Ler (34, 70, 73), and thus, as in EPEC,
this protein is a global regulator of EHEC virulence genes (Fig.
1B and 2B).
During infection of HEp-2 cells in culture, Ler expression is
necessary only during the early stages of the EPEC infection
process (71). By real-time PCR it was demonstrated that tran-
scription of the EPEC LEE3, LEE4, and LEE5 operons in-
creased over 3 h postinfection, while the expression of LEE1,
carrying ler, decreased during the same period (71). The EspA,
EspB, EspD, and Tir proteins necessary for AE lesion forma-
tion were visualized by immunofluorescence microscopy at 5 h
postinfection, indicating that Ler was not necessary for main-
tenance of these proteins once they were expressed.
Under certain conditions, Ler has been shown to repress
transcription of the EPEC LEE1 operon (5). That study may
indicate that the local concentration of Ler is important in
proper regulation of LEE virulence genes. However, Ler au-
toregulation remains controversial. To further this point, a
DNA microarray experiment to elucidate the Ler and H-NS
regulons in EHEC showed that Ler altered the expression of
?1,300 genes, but none of these genes were repressed by Ler
(J. Smart and J. B. Kaper, personal communication).
Mechanism of Ler action. Multiple lines of evidence suggest
that the mechanism of Ler action is to disrupt H-NS-depen-
dent nucleoprotein complexes requiring both upstream and
downstream H-NS-binding regions (9, 51, 109, 120, 135). In
EPEC, Ler binds to the same regions upstream of the LEE2
and LEE5 promoters as does H-NS, and thus both of these
regulatory proteins act directly on the LEE (9, 51, 120). This
observation, combined with the dissociation constants (Kd) of
H-NS and Ler being approximately 1 ?M and 100 nM, respec-
tively (135; A. M. Carmona and J. L. Mellies, unpublished
data), suggests that Ler acts to relieve silencing by disrupting
H-NS binding. Ler is not a general antagonist of H-NS, be-
cause Ler does not increase expression of the H-NS-regulated
proU operon (34). Currently there is no evidence to suggest
that Ler increases LEE transcription by direct interaction with
Regulation through Ler. Several regulatory proteins indi-
rectly influence EPEC and EHEC AE lesion formation via
Ler. Fis and integration host factor (IHF) positively regulate
the EPEC LEE1, Ler-encoding operon (37, 44) (Fig. 1A and
B), and the observation that purified IHF protein did not bind
to LEE2 regulatory DNA (37) supported earlier conclusions
concerning the Ler regulatory cascade whereby Ler increases
transcriptional activities of all major operons of the LEE ex-
cept LEE1. BipA increases LEE transcription, most likely in-
directly through activating expression of Ler (47) (Fig. 1A).
BipA shares amino acid sequence similarity with eukaryotic
ribosome-binding elongation factor G and possesses both
GTPase and ATPase activities (36).
The EAF virulence plasmid-encoded regulators PerABC
FIG. 2. Virulence gene regulation in the EAF plasmid of EPEC
(A) and the EHEC pO157 plasmid (B). Thin arrows represent positive
regulatory signals, and thin blunt arrows represent negative signals.
Solid arrows indicate expression of regulatory proteins. Horizontally
acquired regulatory proteins appear in ovals with a black background.
Regulation of bfp in response to NH4
noted by box. The EtrA and EivF proteins of EHEC are encoded in a
second cryptic TTSS, termed ETT2, of the Sakai 813 strain.
?concentrations in EPEC is
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regulate AE lesion formation through Ler, as well as the LA
phenotype (see below) (9, 82, 100). PerC directly activates
transcription of LEE1 and then via Ler increases transcription
of the LEE5 operon, encoding intimin. Intimin, encoded by the
eae gene, is down-regulated upon contact with host epithelial
cells (67), a process that involves the EAF plasmid and pre-
sumably PerC (Fig. 2A).
Through comprehensive mutagenesis of the LEE PAI of the
mouse pathogen Citrobacter rodentium, the positive regulator
GrlA, controlling expression of EspB and Tir, was identified
(25). This regulatory protein was functionally equivalent in the
three AE pathogens C. rodentium, EHEC, and EPEC (Fig. 1A
and B). GrlA is predicted to act at the level of transcription
upstream of Ler, and a subsequent study with C. rodentium
indicated that Ler acts directly to control GrlA expression, part
of a complex positive regulatory loop whereby optimal Ler
expression depends on GrlA, thus controlling spatiotemporal
transcription of the genes of the LEE (3). GrlA shares 37%
and 23% identity to Sgh protein of Salmonella and CaiF of
Enterobacteriaceae, respectively. Another LEE-encoded regu-
latory protein, GrlR, expressed from the same operon as GrlA,
represses LEE1, LEE2, and LEE5 transcription (25, 74). This
repressor was postulated to act upstream of Ler as well (Fig.
1A and B).
In EHEC, several regulatory systems are known to control
the expression of Ler, including the RcsC-RcsD-RcsB phos-
phorelay system and the EHEC-specific GrvA protein (132)
(Fig. 1B). Nakanishi et al. reported that induction of the strin-
gent response increased transcriptional activity of the Ler-
encoding LEE1 operon, requiring relA and spoT (88). This
global regulatory response to starvation and entry into the
stationary phase of growth (for a review, see reference 78), in
the presence of elevated levels of ppGpp, enhanced expression
and secretion of EspB and Tir and adherence to Caco-2 epi-
thelial cells in culture through increased expression of Ler. The
nucleoid-binding protein IHF positively regulates EHEC LEE
gene expression through Ler (146), whereas Hha, implicated in
the regulation of ?-hemolysin (93), negatively regulates Ler
expression (112) (Fig. 1B). Deletion of the gene hha in EHEC
resulted in increased expression of Ler and adherence to
HEp-2 epithelial cells in culture. The purified 8.5-kDa Hha
protein bound to the LEE1 regulatory DNA, demonstrating
that this regulation is direct (111). Research thus indicates that
regulation of the LEE PAIs of EPEC and EHEC is exceedingly
complex, involving multiple environmental signals and multi-
ple regulatory proteins, whereby Ler serves as the central re-
ceiver of regulatory input, ultimately controlling the TTSS and
AE phenotype (Fig. 1A and B).
Other regulators of the LEE. Tatsuno et al. (129) identified
two novel regulators of EHEC adherence, YhiF and YhiE,
controlling secretion of EspB, EspD, and Tir (Fig. 1B). YhiF
and YhiE share amino acid identity (23%) and are members of
the LuxR family of transcriptional regulators, which include
portions of two-component regulatory systems in Xanthomo-
nas axonopodis pv. citri, Pseudomonas putida, and the gram-
positive Staphylococcus aureus. These proteins exert transcrip-
tional control on the LEE2 and LEE4 operons but not on ler
(Fig. 1B). Insertional inactivation of the yhiE locus caused
increased shedding of the O157:H7 Sakai strain in a mouse
model of infection, implicating this gene in the regulation of
Additional regulators of the LEE of EHEC are found in a
second, cryptic TTSS of the Sakai 813 strain (147). Deletion of
either of the regulatory genes etrA or eivF from this second,
nonfunctional TTSS leads to increased secretion of EspA,
EspB, Tir, and the pO157 plasmid-encoded StcE and EspP
proteins by immunoblot analysis and adherence to Int-407
cultured epithelial cells (Fig. 1B and 2B). Overexpression of
EtrA or EivF repressed secretion in a high-secreting O26:H?
strain, emphasizing the role of these proteins in repression of
LEE gene expression. Reporter gene fusions of the five major
LEE operons suggested that EtrA and EivF exerted their effect
at the level of transcription. This study is of special interest
because it illustrates the concept that the functionality of reg-
ulatory genes may outlast the functionality of a decayed gene
cluster in which they are located and that they may regulate
distally located genes, a phenomenon dubbed the “Cheshire
cat effect” after the disappearing cat in Alice in Wonderland.
This may be a particularly apt metaphor, since it is increasingly
apparent that proper regulation of attachment factors is essen-
tial for colonization of bacterial pathogens, including EHEC,
and one sees the Cheshire cat’s teeth (i.e., the regulators) when
he disappears in this classic children’s tale.
Quorum sensing. Cell-to-cell communication, or quorum-
sensing, regulation of EPEC and EHEC LEE PAIs was first
reported by Sperandio et al. (121) and mostly occurs through
the coordinate regulation of Ler (Fig. 3) (122). Subsequent
intense research, beyond this initial discovery, indicates that
quorum-sensing regulation of the AE phenotype and flagellar
motility is remarkably complex.
Three main quorum-sensing systems have been described
for gram-negative bacteria (61, 144). In the LuxIR system,
initially described for Vibrio, the autoinducer synthase LuxI
produces an acylated homoserine lactone (HSL) molecule that
binds to the LuxR regulator, which then modulates gene ex-
pression. In the second LuxS/AI-2 system, also found in gram-
FIG. 3. Quorum-sensing signaling of flagellar motility and AE lesion formation in EPEC and EHEC. The cryptic phage-encoded TccP protein
is necessary for AE lesion formation in EHEC. See the text for details.
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positive bacteria, the LuxS autoinducer synthase produces a
furanone from a precursor derived from the metabolism of
S-adenosyl methionine. The most intriguing discovery to date
concerning cell-to-cell signaling in EHEC bacteria is a luxS-
independent AI-3-signaling molecule (141), which cross talks
with the mammalian hormone epinephrine and activates LEE
and flagellar genes (118, 123). The AI-2 autoinducer is a
furanosyl borate diester that depends on LuxS for biosynthesis
(13, 127), but it is now known that AI-2 does not activate
expression of EHEC virulence genes (123, 140, 141).
Via the AI-3 signal perceived through the QseEF two-com-
ponent system (see below), the quorum-sensing regulator
QseA, a member of the LysR family of proteins, directly acti-
vates transcription of the LEE1 operon (113) (Fig. 3). Flagellar
motility is regulated by the QseB/QseC two-component quo-
rum-sensing regulatory proteins, a response regulator and sen-
sor kinase, respectively (16, 17, 124). QseBC controls flagellum
production by activating transcription of the flhDC master reg-
ulators and motA, and this regulation involves the class 2
flagellar gene fliA, encoding the sigma factor ?28. QseBC
is subject to autoregulation, and identically with function at
flhDC, QseB activates qseBC transcription by binding to high-
and low-affinity sites upstream of a QseBC-responsive pro-
An additional two-component system controlling the EHEC
AE phenotype has been recently described. The QseEF pro-
teins are a sensor kinase and response regulator, respectively,
and are transcribed from a single operon (102). QseF activates
transcription of the EspFu effector protein secreted into the
host cell by the TTSS, and a qseF deletion mutant fails to form
AE lesions. The QseEF system is activated by epinephrine
through the QseC sensor kinase (Fig. 3). The precise mecha-
nism of the sensor kinase/response regulator control of quo-
rum-sensing signaling in EHEC continues to be under intense
Activation of LEE1 and flhDC transcription can be demon-
strated by providing exogenous, partially purified AI-3 or by
the addition of epinephrine or norepinephrine (15). The bac-
terial AI-3 molecule is ?1 kDa and is methanol soluble, con-
sistent with its being similar to epinephrine, but the exact
identity is yet to be established. Epinephrine is not produced
by HeLa epithelial cells in culture but rather is found in the
fetal bovine serum added to the culture medium. The AI-3-
dependent signaling can be blocked by the addition of ?- or
?-adrenergic antagonists. The QseC sensor kinase responds
directly to the AI-3/epinephrine and norepinephrine signal, as
a qseC mutant is blind to both AI-3 and epinephrine, and QseC
has been shown to bind to norepinephrine in vitro (15, 124).
The AI-3/epinephrine/norepinephrine quorum-sensing regu-
lon is thus an interkingdom communication system controlling
the AE phenotype and flagellar motility. This regulon is
thought to be of particular importance for EHEC, as the site of
infection is the large bowel, which contains dense populations
of commensal flora.
Though no HSL signaling molecules exist in E. coli, the
LuxR homolog SdiA directly represses the EHEC EspD- and
intimin-encoding operons LEE4 and LEE5, respectively (59)
(Fig. 1B). It has been hypothesized that the biological function
of SdiA, which activates transcription of the ftsQAZ operon,
encoding proteins essential for cell division (2, 143), is to de-
tect the presence of other species of bacteria that produce HSL
signaling molecules (145).
SOS control of the EPEC LEE. We have recently demon-
strated that genes of the EPEC LEE are regulated by the SOS
response (83) (Fig. 1A). In K-12-derived strains, transcrip-
tional activity from LEE2-lacZ and LEE3-lacZ fusions in-
creased in the presence of the DNA-damaging agent mitomy-
cin C, and this activity was both RecA dependent and LexA
dependent. In wild-type EPEC, transcriptional activity of the
LEE2 and LEE3 operons was also increased in the presence of
mitomycin C, and protein secretion was reduced in the presence
of the lexA1 allele, encoding an uncleavable LexA protein, par-
ticularly in the absence of the Ler regulator. Intriguingly, we
also observed increased transcription of the non-LEE, phage-
encoded effector nleA in the presence of mytomicin C. LexA
protein, identical in the K-12 MG1655 and EPEC E2348/69
strains, bound specifically to a predicted SOS box located
within the overlapping LEE2 and LEE3 promoters. Thus, the
SOS response, a regulon fundamental to bacterial survival and
evolution, controls expression of genes encoding components
of the TTSS and a cryptic phage-encoded effector of EPEC.
Regulation of the EPEC LA phenotype. Similar to observa-
tions on the regulation of AE lesion formation, the LA phe-
notype is influenced by environmental conditions, carbon
source, and phase of growth. EPEC showed increased adher-
ence to HEp-2 human intestinal epithelial cells in culture in
DMEM as opposed to rich media (42, 101, 136, 138), adhering
as microcolonies effectively in the presence of glucose but not
in the presence of galactose (136). Expression of the BFP
occurs maximally at 37°C during the exponential phase of
growth (101). Transcription of bfpA is subject to ammonium
ion regulation; the cation NH4
gene (8, 9, 79, 101) (Fig. 2A). Though NH4
gut, in higher concentrations in the distal versus the proximal
small intestine, the significance of this finding in terms of
EPEC pathogenesis remains to be determined. BFP expression
is also linked to the Cpx two-component phosphorelay system.
The sensor kinase CpxA and response regulator CpxR respond
to potentially lethal insults to the cell envelope. The BFP is not
assembled unless the Cpx system is activated (92), and this
regulation is most likely posttranscriptional.
The PerA protein, encoded within the perABC operon ad-
jacent to bfp on the EAF virulence plasmid, regulates bfp
transcription (134) (Fig. 2A). Several studies have indicated
that PerA is required for expression of the BFP, and indeed
mutation in perA renders EPEC unable to display the LA
phenotype (9, 79, 134). (PerA has also been called BfpT .)
PerA shows amino acid sequence similarity to the AraC family
of transcriptional regulators, and it is most closely related to
the VirF protein of Shigella flexneri, a primary activator of
virulence genes in this pathogen (30). PerA is also similar to
the Rns transcriptional activator in ETEC (87). Transcrip-
tional regulation of bfp by PerA is direct, as this protein was
shown to bind to DNA regulatory sequences upstream of bfpA
in vitro (100, 134) and cis-acting sequences between positions
?85 and ?46 were required for activation (8). The perA gene
is subject to autoregulation, and binding regions upstream of
the perA and bfp promoters share significant sequence similar-
ity (55, 85).
The EAF plasmid-located perABC locus is itself subject to
?represses transcription of this
?exists within the
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higher-order regulation. GadX, an activator of glutamate de-
carboxylase genes involved in acid tolerance, represses the
expression of perABC (114) (Fig. 2A). Consistent with this
observation, because PerC regulates expression of Ler, a mu-
tation in gadX resulted in increased translocation of Tir into
host epithelial cells. GadX is a member of the XylR/AraC
family of transcriptional regulators, and its expression is in-
creased in DMEM culture medium and under acidic condi-
tions (pH 5.5). GadX was deemed a transcriptional regulator
based on gel shift assays showing that this protein binds di-
rectly to perABC and gadA and gadB regulatory sequences, and
it may function to properly regulate acid tolerance and viru-
lence gene expression in response to environmental cues
within the gastrointestinal tract.
Per-like molecules in EHEC. The question of whether Per-
like molecules exist in EHEC was addressed by two indepen-
dent research groups (58, 99). Iyoda and Watanabe (58)
screened a genomic library of the EHEC O157:H7 Sakai strain
for genes that controlled expression of LEE fusions. They
identified a DNA fragment containing a gene with similarity to
perC of EPEC. Upon closer inspection of the genomic DNA
sequences, they identified five perC-like sequences but no se-
quences with similarity to either perA or perB. Deletions in
either perC1-1 (also termed pchA) or perC1-2 (also termed
pchB), or double deletions in perC1-1 and perC1-2 or in
perC1-1 and perC1-3 (also termed pchC), reduced the expres-
sion of EspA, EspB, and EspD proteins and adherence to
HEp-2 cells in culture (Fig. 1B). This group postulated that
multiple perC loci were necessary for full expression of the
LEE and found that these loci exerted their effect through
transcriptional activation of the LEE1 operon encoding Ler
(Fig. 1A and 2A).
PerC-like molecules exist not only in the E. coli O157:H7
strain Sakai (PerC1-1, PerC1-2, PerC1-3, PerC2, and PerC3),
but also in the O157:H7 strain EDL933 (PerC1, PerC2,
PerC3-1, and PerC3-2), in uropathogenic E. coli (PerC2,
PerC3, and YfdN), in the Salmonella enterica serovar Typhi-
murium ST64B phage (YfdN), in the S. flexneri SfV phage
(YfdN), and in the E. coli laboratory strain MG1655 (YfdN)
(99). Many of the genes encoding the PerC-like molecules are
found with lambdoid phage. Porter et al. (99) found that the
PerC1, but not the PerC2, proteins of EHEC strains could
activate expression of LEE1 from both organisms, though in
vitro assays could not demonstrate binding of purified PerC
protein to LEE1 regulatory fragments. They hypothesized that
PerC molecules may bind in the presence of IHF, which is also
a positive regulator of LEE1, and perhaps other unidentified
proteins to activate transcription. The PerC-responsive pro-
moters of EHEC and EPEC are located similarly in these
bacteria, approximately 170 bp upstream of the start codon of
ler of LEE1.
Posttranscriptional regulation. Control of secretion of
translocator molecules, e.g., EspB, and effectors, e.g., Tir, is
complex, involving posttranscriptional and posttranslational
regulation. Heterogeneous secretion of EspD protein in hu-
man and bovine O157:H7 isolates led investigators to study the
underlying mechanism regulating this observation (107). They
found that strains possessing EspA translocons, visualized by
immunofluorescence, correlated with high levels of EspD se-
cretion and that the observed variations in secretion were not
controlled at the level of transcription, as demonstrated by
reporter gene fusions. By Northern analysis, secretion of trans-
locon proteins was inversely related to espADB mRNA tran-
script levels. Evidence suggested that sepL, espA, espD, and
espB are transcribed as a single polycistronic operon but that
mRNA processing must occur, separating the transcript into
two fragments, an approximately 1.2-kb sepL-containing tran-
script and a 2.8-kb espADB transcript. These investigators
clearly established that posttranscriptional regulation is in-
volved in EspA filament or translocon formation, though the
exact mechanism of control remains to be determined. They
postulate that the inability to detect the espADB transcripts in
the high-secretor strains may be explained by engagement of
the molecule in the type III apparatus prior to secretion, a
coupling of translation and secretion as proposed for flagellum
assembly in S. enterica serovar Typhimurium (62).
One issue that has remained controversial in the study of
TTSSs is whether contact-dependent secretion occurs (20).
Certainly environmental signals can cue secretion; Ebel et al.
(32) established that temperature and medium conditions af-
fect secretion of EHEC polypeptides. Calcium-deficient con-
ditions signal secretion of effector Yops of Yersinia spp., and
secretion of Ipa effector proteins of S. flexneri can be triggered
by the dye Congo red (96), but whether these environmental
conditions and/or chemical cues actually relate to contact-de-
pendent secretion remains to be determined. Some studies
have indicated that a rapid increase in EHEC LEE gene ex-
pression occurs upon contact with host epithelial cells; tran-
scriptional activity from an espADB-lacZ fusion increased upon
contact with HeLa cells (4), and tir-egfp and map-egfp fusion
activity increased upon contact with bovine intestinal epithelial
cells (106). Beltrametti et al. (4) also demonstrated that the
addition of Ca2?or Mn2?, but not Mg2?, increased the ex-
pression of the espADB-lacZ fusion.
An elegant study by Deng et al. (24) revealed that SepD and
SepL, encoded by the LEE2 and LEE4 operons, respectively,
constitute a molecular switch controlling secretion of translo-
cators and effector molecules, and they began to clarify the role
of calcium in these processes. Beginning with C. rodentium, but
expanding their studies to EHEC and EPEC, they found that
low-calcium conditions inhibit the secretion of translocators,
such as EspA, EspD, and EspB, but enhance the secretion of
effectors, such as Tir and NleA. This phenotype was similar to
that observed for sepD and sepL nonpolar deletion mutants.
SepD and SepL proteins interact with each other and are
localized to the inner membrane. These authors proposed a
model whereby a high calcium level, about 2.5 mM in the
mammalian extracellular fluid (19), stimulates the secretion of
translocator proteins necessary for assembly of the translocon,
and then, once connected to the host cell, the low-calcium
environment (100 to 300 nM, as most calcium is sequestered in
the endoplasmic reticulum) triggers secretion of the effector
proteins. Thus, SepD and SepL were termed “gatekeepers,”
controlling the switch releasing effector proteins into the host
cell cytoplasm (24). The exact mechanism of how the calcium
signal is perceived from the host cell cytoplasm and sensed by
the bacterium remains to be determined.
Phage control of virulence in EHEC. Several studies have
demonstrated that production of EHEC Stx can be modulated
by antibiotics (48, 66, 142, 148) and that Stx production is
at Reed College on August 22, 2007
linked to the Stx phage growth cycle (91, 139). The Shiga toxins
exist in two subgroups, Stx1and Stx2, and are encoded within
prophages (97), though to date only the prophage BP-933W
encoding Stx2has been shown to produce infectious phage
particles. Stx1expression is subject to regulation by the iron-
responsive Fur repressor (1). Expression of Stx2is under tran-
scriptional control of the Q-dependent late phage promoter,
which is subject to quorum-sensing signaling (123, 139).
Herold et al. (52) used a DNA microarray approach to
monitor changes in transcriptional activity in the presence of
low levels of the DNA gyrase inhibitor norfloxacin, which in-
duces DNA damage and the SOS response. Phage titer and
Stx2production dramatically increased approximately 2 h after
the addition of the antibiotic. Of the 118 spots showing in-
creased expression in the presence of the antibiotic, 85 were
phage borne; 52 were from the Stx phages BP-933W and CP-
933V. The most strongly induced genes were found within the
BP-933W phage; stxA2was induced 158-fold. Regulation of the
stx2and recA genes, indicating induction of the SOS response,
was confirmed by real-time reverse transcriptase PCR. Other
induced genes included those for putative phage integrases not
found in prophages; some stress-induced proteins, including
the heat shock protein IbpB; and DNA damage-inducible pro-
teins associated with the SOS response, including DinB.
There is an intimate connection between DNA damage and
phage induction, and hence in the case of EHEC the release of
the Shiga toxin, which is important in causing serious disease.
To add insult to injury, Gamage et al. (38) demonstrated that
Stx phage conversion of intestinal commensal E. coli led to
increased production of the Stx2toxin in a mouse model and
that ciprofloxacin, also a DNA gyrase inhibitor and a com-
monly prescribed antibiotic in the United States, led to lysis of
phage-infected host E. coli and release of the toxin. There was
indeed a 40-fold increase in Stx2production when the suscep-
tible commensal E. coli was infected with the 933W phage,
indicating that the Shiga toxin phages regulate amplification of
the toxin upon phage induction, which is known to occur in the
presence of DNA-damaging agents. In addition to simply up-
regulating virulence genes, the error-prone replication machin-
ery activated by the SOS response may provide the genetic
variability necessary for generation and selection of spontane-
ously resistant bacterial strains. It seems likely that the treat-
ment of E. coli infections by antibiotics may have unintended
negative consequences; the prevailing thought that for the
treatment of EHEC infections antibiotics are contraindicated
is certainly consistent with this idea.
Through elucidation of specific signaling events, perception
of environmental cues, and identification of regulatory pro-
teins controlling expression of virulence genes in EPEC and
EHEC bacteria, a picture of the global regulation of virulence
genes in response to the pathogen-host interaction emerges.
H-NS plays an important role in the regulation of horizontally
transferred, virulence factor-encoding DNA, an idea that has
reappeared in the literature (28). Because foreign DNA in
gram-negative bacteria tends to have a low G?C content (i.e.,
a high A?T content) in comparison with the recipient’s ge-
nome and since H-NS preferentially binds to AT-rich se-
quences, many researchers have hypothesized that H-NS may
endogenously silence horizontally transferred DNA elements,
which when expressed inappropriately might be deleterious to
the bacterium. In Salmonella, selective silencing of horizontally
acquired, low-G?C-content DNA by the nucleoid-associated
protein H-NS, enables the bacterium to avoid inappropriate
expression of foreign DNA (76, 89). Specifically, in EPEC
H-NS silences expression of the TTSS, BFP, and PerABC, and
in EHEC it silences expression of StcE, all of which are en-
coded on horizontally transferred genetic elements. Thus,
H-NS plays a critical role in disease-causing E. coli and Sal-
monella spp., as well as in the evolution of molecular patho-
genesis in these organisms.
Subsequently, EPEC and EHEC bacteria must activate ex-
pression of H-NS-silenced virulence genes, such as the LEE.
Regulatory proteins that counteract H-NS silencing of the
LEE may be acquired along with the horizontally transferred
DNA (Table 1) or may exist endogenously in the recipient,
located on either the chromosome or a plasmid (Table 2).
Several mobile DNA elements encode proteins that either
interact with or inactivate the function of H-NS (23, 33, 75).
For example, the protein encoded by gene 5.5 of phage T7
disrupts H-NS function, resulting in expression of both host
and viral genes (23, 33, 75). Consistent with this idea, the
horizontally acquired TTSSs of EPEC and EHEC bacteria are
regulated by the LEE-encoded global regulator Ler, disrupting
the silencing activity of endogenous H-NS (predicted also to
occur in EHEC) in response to host-associated environmental
cues. Virulence genes that are repressed by a non-H-NS pro-
tein(s) and derepressed by Ler may also exist, but a mechanism
describing this type of regulation has not been reported.
TABLE 1. Horizontally acquired regulators of the LEE
9, 45, 55, 82, 100
aETTS is a second cryptic TTSS of the O157:H7 strain Sakai 813.
TABLE 2. Endogenous regulators of the LEE
37, 73, 146
VOL. 75, 2007MINIREVIEWS4205
at Reed College on August 22, 2007
Investigation of virulence gene regulation in EPEC, EHEC,
and other members of the Enterobacteriaceae family has fur-
thered our understanding of the mechanism of H-NS-mediated
gene silencing. It has been known for some time that H-NS can
bind in regions that flank promoters and that H-NS can facil-
itate DNA looping (for recent reviews on H-NS, see references
28, 29, 104, and 131). For example, H-NS binds over extended
positions both upstream and downstream of the proU and bgl
promoters of E. coli K-12 and the virF promoter of enteroin-
vasive E. coli (11, 35, 77). Similarly, H-NS-dependent repress-
ing DNA sequences exist both upstream and downstream of
the Ler-induced EPEC LEE2 and LEE5 promoters, and Ler
binds to the same region upstream of the LEE2 and LEE5
promoters to which H-NS binds (9, 51, 120; K. R. Haack and
J. L. Mellies, unpublished data). DNA looping by H-NS has
been demonstrated for the ure operon of the urinary tract
pathogen Proteus mirabilis, which is positively regulated by
UreR, an AraC-like molecule (98). A unifying mechanism
emerges from these data, suggesting that the unrelated AraC-
like regulators and H-NS-like protein Ler increase transcrip-
tion by disruption of H-NS-dependent nucleoprotein com-
plexes requiring H-NS binding that flanks promoter sequences.
This mechanism is most likely conserved in H-NS control of
virulence gene expression in the Enterobacteriaceae.
In addition to silencing and antisilencing by the global reg-
ulators H-NS and Ler, respectively, three global regulatory
systems, the stringent response (ppGpp), quorum sensing, and
the SOS response, regulate expression of the TTSS. Why are
there so many global inputs, environmental signals, and regu-
latory proteins controlling expression of the TTSSs of these
pathogens? The answer may simply be that multiple regulatory
inputs ensure a robust response to the host environment and
that evolution occurs randomly, adding intricate layers of reg-
ulation, feedback loops, and seemingly contradictory inputs of
regulation of the TTSS to establish and maintain this robust-
A resurgence in studying the role of bacteriophages in the
pathogenesis of EPEC and EHEC bacteria has occurred. In
EHEC, DNA damage causes Stx2-encoding 933W ?-like phage
induction, which is essential for biosynthesis and release of the
Stx2toxin. Indeed, Stx2production is enhanced by the presence
of DNA-damaging antibiotics (130, 148). In EPEC the SOS
response regulates transcription of two operons within the
LEE and a cryptic prophage gene encoding the NleA effector.
Thus, along with controlling the response to DNA damage,
repair of stalled replication forks, and the expression of error-
prone polymerases leading to increased frequency of muta-
tion, the SOS response regulates expression of important
virulence determinants from both of these E. coli patho-
types. The observation that EPEC and EHEC virulence is,
at least in part, regulated by the SOS response indicates that
these pathogens experience stress associated with DNA
damage in the environment of the host. A number of effec-
tor proteins, translocated into host cells by the TTSS of
EPEC and EHEC, are encoded within cryptic prophages
(39, 133), and whether these molecules are under control of
the SOS response, Ler, GrlRA (25), or perhaps other reg-
ulators remains unknown.
The study of virulence gene regulation is greatly assisting
our understanding of EPEC and EHEC pathogenesis. The
shared global regulator Ler receives environmental signals and
regulatory input from multiple proteins acting on the LEE1
operon and then proceeds to increase transcriptional activity of
the genes necessary for forming AE lesions. Thus, Ler lies at
the heart of the global regulatory network controlling the AE
phenotype for both pathotypes. Quorum-sensing signaling con-
tributes to EHEC colonization of the large intestine, perceiv-
ing the high density of bacteria in the human intestinal tract
versus the relatively low bacterial density in the proximal small
intestine, the site of EPEC infection. Studies elucidating the
EHEC quorum-sensing network illustrate important regula-
tory differences between EPEC and EHEC. Concerning type
III secretion, we are beginning to see a distinction in the
expression of the apparatus components and the actual se-
cretion or translocation of effector molecules as separable,
differentially regulated events. However, many outstanding
questions remain. Beyond quorum sensing, do regulatory
phenomena play a further role in the site of EPEC and EHEC
infection, or tissue tropism? Does regulation contribute to
their vastly different infectious doses? How are the cryptic
prophage-encoded effector molecules and other effector pro-
teins encoded outside the LEE PAI regulated in order to
facilitate coordinated delivery into host cells by the TTSS?
In addition to elucidating host-microbe interactions, studies
dissecting regulatory circuitry offer opportunities to devise new
effective therapies and protective measures against EPEC,
EHEC, and related pathogens. For example, Gauthier et al.
(41) identified a class of compounds for potential therapeutic
use that inhibited transcriptional activity of EPEC LEE oper-
ons, which they hypothesized to be occurring through repres-
sion of ler. Hung et al. (54) identified a compound, virstatin,
that inhibited transcription of ToxT, a key regulator of cholera
toxin and the toxin-coregulated pilus in Vibrio cholerae. Ad-
ministration of virstatin prevented intestinal colonization of V.
cholerae in the infant mouse model. Finally, Cirz et al. (14)
proposed targeting of the SOS regulon of E. coli for chemical
therapies to minimize the evolution of antibiotic resistance,
because they found that the development of resistance to the
antibiotics ciprofloxacin and rifampin required a functional
SOS response. With the growing problem of pan-resistant bac-
teria, researchers must devise novel means to thwart infectious
disease, to extend our therapeutic arsenal beyond traditional
antibiotics. The study and targeting of bacterial regulatory
circuitry will play an increasingly important role in this en-
Research in the laboratory of J.L.M. is supported by an NIH AREA
grant (R15 AI047802-02) and by HHMI and Merck/AAAS grants
awarded to the Biology and Chemistry Departments of Reed College.
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uated, immunogenic, and protects rabbits from lethal challenge with the
wild-type virulent strain. Vaccine 24:3845–3855.
Editor: J. B. Kaper
4210 MINIREVIEWSINFECT. IMMUN.
at Reed College on August 22, 2007