ENTEROPATHOGENIC
ESCHERICHIA COLI
I
Hin-chung Wong
Department of Microbiology
Soochow University
Content
 INTRODUCTION
 HEAT-LABILE ENTEROTOXINS



General Characteristics
Gene and Regulation
Mode of Action
 HEAT-STABLE ENTEROTOXINS


General Characteristics
Mode of Action and Regulation
 ENTEROTOXIN PLASMIDS






SHIGA-LIKE TOXINS
Purification and Structure
Mode of Action
Production and Regulation
Genetics
Role in Disease
 HEMOLYSINS
 Production and Purification



Characteristics
Genetics
Role in Virulence and Mode of Action
Content
 ADHERENCE



In Enterotoxigenic E. coli
In Enteropathogenic E. coli
In Enterohemorrhagic E. coli
 INVASIVENESS
 DETECTION







Using glucuronidase assay
Animal Tissue Culture
Animal Assays
Immunological Methods
Enzymatic bio-nanotransduction
Nucleic Acid Probes
Using polymerase chain reaction
INTRODUCTION
E. coli is usually considered to be an
opportunistic pathogen which constitutes a
large portion of the normal intestinal flora of
humans
This organism can, however, contaminate,
colonize, and subsequently cause infection
of extraintestinal sites and is a major cause
of septicemia, peritonitis, abscesses,
meningitis, and urinary tract infections in
humans
INTRODUCTION
 E. coli was first incriminated as an
enteropathogen in 1945, responsible for an
outbreak of infantile diarrhea
 Enteropathogenic E. coli (EEC) has been
associated with diarrhea in developing countries
and localities having poor sanitation
 In the developed countries, EEC has been
historically associated primarily with infantile
diarrhea, but it was later recognized that adults
also may suffer from the illness
 E. coli are also enteropathogenic in animals
INTRODUCTION
E. coli O157:H7 causes severe illneses
(hemorrhagic) and it does possess
distinguishing characteristics, e.g. does not
ferment sorbitol within 24 h, does not
possess α-glucuronidase activity, and does
not grow well at all at 44-45.5C
INTRODUCTION
 There are several subgroups of EEC




(A) enterotoxigenic (ETEC)
(B) enteroinvasive (EIEC)
(C) hemorrhagic (EHEC)
(D) enteropathogenic (EPEC) strains
 Some authors classify them into six
different pathotypes: ETEC, EIEC, EPEC,
enteroaggregative E. coli, diffusely
adherent E. coli, and Shiga toxin-producing
E. coli (STEC)
INTRODUCTION
 In 1995, the enteropathogenic E. coli (EPEC)
pathotype is divided into two groups, typical
EPEC (tEPEC) and atypical EPEC (aEPEC)
 The property that distinguishes these two groups
is the presence of the EPEC adherence factor
plasmid (pEAF), which is only found in tEPEC
 aEPEC strains are emerging enteropathogens
that have been detected worldwide
 The large variety of serotypes and genetic
virulence properties of aEPEC strains from
nonclassical EPEC serogroups makes it difficult
to determine which strains are truly pathogenic
INTRODUCTION
 Humans are thought to be the principal if not the only
reservoir of toxigenic and invasive strains of E. coli,
contaminating foods via contact with food or via contact of
processing equipment with water contaminated by human
feces
 In contrast, animals are reservoirs of the hemorrhagic
strain (O157:H7); hence, foods of animal origin may
become contaminated via slaughter procedures or postprocessing recontamination
 However, when E. coli is isolated from foods, pathogenic
serotypes are usually absent or represent a very low
percentage of the total isolates
HEAT-LABILE ENTEROTOXINS
 Probably the most common type of EEC strains is
the enterotoxigenic type. Heat-labile (LT) and
heat-stable (ST) enterotoxins are produced.
 Two partially cross-reacting antigenic variants of
plasmid-coded LT, designated LTh and LTp, have
been described in E. coli
 LTh is associated with E. coli isolates from
humans, and LTp is associated with E. coli
isolates from pigs
 The LT family from restricted geographical region
exhibited a segregated pattern of dissemination
that was revealed by a restriction enzyme site
polymorphism
HEAT-LABILE ENTEROTOXINS
Another heat-labile enterotoxin was
discovered in extracts of E. coli SA53, a
strain isolated from water buffalo
It activated adenylate cyclase.
Hyperimmune antisera prepared against
LTh and LTp or CT do not neutralize the
crude LT-like toxin in Y1 adrenal cell
assays
HEAT-LABILE ENTEROTOXINS
Subcloning and minicell experiments
indicated that the toxin is composed of two
polypeptide subunits that are encoded by
two genes
The two toxin subunits exhibited mobilities
on PAGE gels that are similar to those of
CT and LT
HEAT-LABILE ENTEROTOXINS
HEAT-LABILE ENTEROTOXINS
The LT gene was cloned into E. coli and
two proteins of molecular weights 11,500 (B
subunit) and 25,500 (A subunits) were
produced
The LT A subunit structureal gene (eltA)
was sequenced and the amino acid
sequence deduced
HEAT-LABILE ENTEROTOXINS
It is proposed by Pickett et al. that the LTp
and LTh (antigenic variants of LT will both
be included in serogroup I and should be
designated LTp-I and LTh-I and the LT-like
toxin will be the prototype for serogroup II
enterotoxins and should be renamed LT-II.
Two distinct members of the LT-II family,
LT-IIa and LT-IIb, are now known, and both
have A and B subunits which are similar in
size to those of CT and LT-I
HEAT-LABILE ENTEROTOXINS
 It starts with methionine, ends with leucine, and comprises
254 amino acids
 The computed molecular weight of LT A is 29,673.
 The A subunit genes of CT and LT (LT-I) are 78.6%
homologous, and the B subunit genes are 78%
homologous
 The NH2-terminal regions exhibit the highest degree of
homology (91%) as compared with CT subunit A, while
the COOH-terminal region, containing the sole cystine
residue in each toxin is less conserved (52%)
 Alignment of homologous residues in the COOH-terminal
regions of LT A and CT A indicates that a likely site for
proteolytic cleavage of LT A is after Arg residue 188
HEAT-LABILE ENTEROTOXINS
 The gene of LT-IIa was studied. It is organized in
a transcriptional unit similar to those of CT and
LT-I
 The A subunit gene of LT-IIa was found to be
57% homologous with the A subunit gene of LTh-I
and 55% homologous with the A gene of CT
 Most of the homology derived from the region of
the A gene which encodes the A1 fragment
 The B gene of LT-IIa was not homologous with
the B gene of LTh-I or CT
HEAT-LABILE ENTEROTOXINS
 The LT-IIb gene was also cloned and analysed.
 The A genes of LT-IIa and LT-IIb exhibited 71% DNA
sequence homology with each other and 55 to 57%
homology with the A genes of CT and LT-I.
 The B subunits of LT-IIa and LT-IIb differ from the LT-I in
their carbohydrate-binding specificities.
 The B genes of LT-IIa and LT-IIb were 66% homologous,
but neither had significant homology with the B genes of
CT and LT-Is.
 The A subunits of the heat-labile enterotoxins also have
limited homology with other ADP-ribosylating toxins,
including pertussis toxin, diphtheria toxin, and
Pseudomonas aeruginosa exotoxin A
ADP-ribosylation is the addition of one or more
ADP-ribose moieties to a protein
HEAT-LABILE ENTEROTOXINS
 The carboxy-terminal domain of EtxB (encodes B
subunit) mediates A subunit-B subunit interaction
 The gene encoding the B subunit of LT was
mutated at its 3' end by targeted addition of
random nucleotide sequences
 The functional and structural properties of the
gene products were analysed, that these mutants
were defective in their ability to associate stably
with A subunits and form holotoxin
HEAT-LABILE ENTEROTOXINS
Mode of Action
LT from E. coli is a protein of approximately
86,000 daltons that consists of one A
polypeptide and five B polypeptides held
together by noncovalent bonds
LT is closely related to cholera enterotoxin
(CT) in structure, antigenicity, and mode of
action. Both LT and CT bind to ganglioside
GM1 receptors on eukaryotic target cells
via their B subunits
Ganglioside
http://en.wikipedia.org/wiki/Ganglioside
HEAT-LABILE ENTEROTOXINS
 The A subunit of LT like CT undergoes a
proteolytic cleavage that produces two fragments
designated A1 and A2
 The A1 fragment catalyzes the NAD-linked ADP
ribosylation of a regulatory subunit of adenylate
cyclase in the plasma membrane of eucaryotic
target cells, resulting in stimulation of adenylate
cyclase activity
 The activation of adenylate cyclase in mucosal
cells in the small intestine causes secretion of
fluid and electrolytes into the lumen and produces
watery diarrhea.
HEAT-LABILE ENTEROTOXINS
 The LT-II, similar to CT and LT-I, increases
cAMP by activating adenylate cyclase
through the GTP-dependent ADPribosylation of specific membrane.
HEAT-LABILE ENTEROTOXINS
Fibroblasts incubated with LT-II had an
increased cAMP content (Fig. 2)
HEAT-LABILE ENTEROTOXINS
as well as a fourfold elevation of membrane
adenylate cyclase activity (Fig. 3).
HEAT-LABILE ENTEROTOXINS
 The B subunit of LTh (Human) is also hemagglutinating
 Very strong hemagglutination of both neuraminidase- and
pronase-treated human erythrocytes was induced by the
B subunit of LTh.
 Different blood groups reacted differently to such
enhancement
 Combining site of the B subunit may gain access to the
receptor exposed on erythrocytes more easily by enzyme
treatment.
 Neuraminidase and pronase are suppose to convert major
gangliosides to GM1 and/or expose masked receptors for
the B subunit. Both CT and LT strongly react with
ganglioside GM1.
HEAT-LABILE ENTEROTOXINS
 Separation of ETEC bacteria from target intestinal
epithelial monolayers by semipermeable filters
prevented activation of adenylate cyclase
suggesting that pathogen-host cell contact is
required for efficient toxin delivery
 Likewise, a non-motile strain bearing a mutation
in the flagellar fliD gene was deficient in delivery
of LT relative to the ETEC prototype
 Although LT secretion via the type II secretion
system (T2SS) was responsive to a variety of
environmental factors, neither toxin release nor
delivery depended on transcriptional activation of
genes encoding LT or the T2SS
HEAT-LABILE ENTEROTOXINS
Enterotoxigenic E. coli LT-induced diarrhea
is the leading cause of infant death in
developing countries.
Ginger significantly blocked the binding of
LT to cell-surface receptor G M1 (Fig. 4),
resulting in the inhibition of fluid
accumulation in the closed ileal loops of
mice (Fig. 5).
HEAT-LABILE ENTEROTOXINS
HEAT-LABILE ENTEROTOXINS
HEAT-LABILE ENTEROTOXINS
 Biological-activity-guided searching for active
components showed that zingerone
(vanillylacetone) was the likely active constituent
responsible for the antidiarrheal efficacy of
ginger.
 Further analysis of chemically synthesized
zingerone derivatives revealed that compound
31 (2-[(4-methoxybenzyl)oxy] benzoic acid)
significantly suppressed LT-induced diarrhea in
mice via an excellent surface complementarity
with the B subunits of LT(Fig. 6)
HEAT-STABLE ENTEROTOXINS
 The heat-stable enterotoxins are low-molecularweight, heat-stable, nonantigenic proteins which
do not cause intestinal secretion by activation of
adenylate cyclase
 At least two types have been described, one with
biological activity in suckling mice and piglets
(STa, or named as STh, or ST-I) due to
stimulation of particulate intestinal guanylate
cyclase and a second which induces secretion by
an unknown mechanism only in piglets (STb, or
known as STp, or ST-II).
HEAT-STABLE ENTEROTOXINS
 Generally STs are known to be small peptide toxins
consisting of 18 (STb) or 19 (STa) amino acids.
 The different STa (M.W. about 2,000) from different
animal origins are remarkably homogeneous.
 The amino acid composition of STa of porcine, bovine,
and human origins were identical and consisted of 10 of
the 18 amino acids usually present in proteins.
 Six of the 18 amino acids were half-cystines which appear
to be present as three disulfide bonds in the native form of
the toxin. These disulfide bonds are important for toxic
activity.
HEAT-STABLE ENTEROTOXINS
STb is a heat-stable enterotoxin which does
not cause intestinal fluid secretion in the
suckling mouse as STa does, but does
cause intestinal fluid secretion in pig
intestinal loop assays.
It is insoluble in methanol, while STa is
methanol-soluble
HEAT-STABLE ENTEROTOXINS
 Since STs are small peptides and are
nonantigenic, fusion proteins (e.g. STa with
outer membrane protein C, etc.) (Saarilahti
et al., 1989) or synthetic ST peptide
conjugated with ovalbumin could be use as
the immunoprophylactic agents against
diarrhea caused by STs.
HEAT-STABLE ENTEROTOXINS
 In a study, these two toxins were examined in terms of
importance for piglets >1 week old with the construction of
isogenic single- and double-deletion mutants and
inoculation of 9-day-old F4ac receptor-positive gnotobiotic
piglets.
 Based on the postinoculation percent weight change per h
and serum bicarbonate concentrations, the virulence of
the STb- mutant (Delta estB) did not significantly differ
from that of the parent.
 However, deletion of the LT genes (Delta eltAB) in the
STb(-) mutant resulted in a complete abrogation of weight
loss, dehydration, and metabolic acidosis in inoculated
pigs, and LT complementation restored the virulence of
this strain.
HEAT-STABLE ENTEROTOXINS
 The cysteine residues were substituted in vivo by
oligonucleotide-directed site-specific
mutangenesis to dissociate each disulfide bond
and examined the biological activitites of the
resulting mutants (Fig.7, 8).
 All three disulfide bonds formed at fixed positions
are required for full expression of the biological
activity of STb.
 It has some fexibilities in its conformation to exert
toxic activity and that the role of each disulfide
bond in exerting toxic activity is not quite the
same
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
 The STs share biologically active sequences
which reside in the C-terminal 13 amino acid
residues. Substitution of the asparagines residue
at position 11 of STb by other amino acids
resulted in significant decrease in enterotoxic
activities, although the conformation was not
changed (Okamoto et al., 1988).
 The amino acid sequences and disulfide bonds of
the heat-stable enterotoxins of E. coli, Yersinia
enterocolitica, and Vibrio cholerae non-O1 are
shown in Fig. 9
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
Analogs of ST were made, including the
native 18-amino-acid ST, the 14-amino-acid
carboxy terminus of this native peptide with
a proline at position 12, and the 14-aminoacid carboxy terminus of in which the
proline at position 12 was substituted with
glycine (Table 1).
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
Each analog bound to the receptor in a
dose-dependent fashion, native ST with the
highest adherence (Fig. 10).
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
Similarly, these peptides maximally
activated particulate guanylate cyclase and
stimulate intestinal secretion in suckling
mice, and native ST with the highest
potency (Fig. 11, Table 2).
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
HEAT-STABLE ENTEROTOXINS
 It demonstrats that the four amino-terminal
residues contribute significantly to the
potency of these peptides.
In addition, the turn imposed by the proline
residue at position 12 is not absolutely
required for receptor occupancy or
activation of the biochemical cascade that
results in intestinal secretion.
However, it significantly increases the
potency of the toxin
HEAT-STABLE ENTEROTOXINS
 The STb structural gene (estA) was cloned
into high-expression vector pKC30
downstream from the strong PL promoter
and the expression was studied
10-20-fold increase in mRNA was produced
by the recombinant strain.
HEAT-STABLE ENTEROTOXINS
 Both STs are synthesized as precursor
proteins and are then converted to the
active forms with intramolecular disulfide
bonds after being released into the
periplasm.
The active STs are finally translocated
across the outer membrane through a
tunnel made by TolC.
HEAT-STABLE ENTEROTOXINS
 Several transporters in the inner membrane and their
periplasmic accessory proteins are known to combine with
TolC and form a tripartite transport system.
 Pulse-chase experiments using E. coli BL21(DE3)
mutants in which various transporter genes (acrAB, acrEF,
emrAB, emrKY, mdtEF, macAB, and yojHI) had been
knocked out and analyzed the secretion of STs in those
strains.
 The results revealed that the extracellular secretion of STII
was largely decreased in the macAB mutant and the toxin
molecules were accumulated in the periplasm, although
the secretion of STI was not affected in any mutant
HEAT-STABLE ENTEROTOXINS
 The periplasmic stagnation of STII in the
macAB mutant was restored by the
introduction of pACYC184, containing the
macAB gene, into the cell (Fig. 12).
These results indicate that MacAB, an ATPbinding cassette transporter of MacB and
its accessory protein, MacA, participates in
the translocation of STII from the periplasm
to the exterior.
HEAT-STABLE ENTEROTOXINS
ENTEROTOXIN PLASMIDS
 Enterotoxin plasmids from classical strains
(frequently associated with diarrhea, e.g. O6, O25,
O27, O128, and O159) did not transfer by
conjugation from clinical isolates, whereas those
from rare strains (rarely associated with diarrhea,
e.g. O7, O17, O80, O98, O139, and O153)
transferred almost always from the clinical
isolates by conjugation.
 Analyses of enterotoxin plasmids by restriction
endonucleases and hybridization with the
enterotoxin probes revealed that the strains with
the same O serotype and toxigenicity carry
closely related enterotoxin plasmids