Transcript E. coli

ENTEROPATHOGENIC
ESCHERICHIA COLI
II
Hin-chung Wong
Department of Microbiology
Soochow University
SHIGA-LIKE TOXINS
 Enteropathogenic E. coli (EPEC, e.g. O26:H11,
O128:B12, O138:K81) and enterohemorrhagic E.
coli (EHEC, e.g. O157:H7) are not enteroinvasive
and do not produce the classical heat-stable and
heat-labile enterotoxins.
 A cytotoxin known as Shiga-like toxin was
demonstrated in EPEC and EHEC.
 A cytotoxin which could be neutralized by antishiga toxin is known as Shiga-like toxin I (SLT-I),
and the non-neutralizable one is known as shigalike toxin II (SLT-II)
SHIGA-LIKE TOXINS
In Thailand, SLT-producing E. coli was
isolated from 9% of market beef specimens,
from 8 to 28% of fresh beef specimens at
slaughterhouses, and from 11 to 84% of
fecal specimens from cattle
Vero cell cytotoxin E. coli O157:H7 was
isolated from 3.7% of beef, 1.5% of port,
1.5% of poultry, and 2% of lamb samples in
Canada
SHIGA-LIKE TOXINS
 The SLT-I is also known as verotoxin 1 and
the SLT-II is also known as verotoxin 2.
 E. coli producing large among of SLTs are
also named as VTEC (Verotoxin producing
E. coli)
SHIGA-LIKE TOXINS
 Iron is known to depress Shiga toxin production
by Shigella dysenteriae 1, and temperature has
been shown to regulate several genes required
for Shigella invasiveness and also expression of
virulence plasmid in Yersinia.
 Iron also suppressed SLT-I synthesis in E. coli
lysogenized with phage 933J but did not
demonstrably repress toxin synthesis in E. coli
strains carrying the cloned slt-I genes (Table 3).
SHIGA-LIKE TOXINS
SHIGA-LIKE TOXINS
The SLT-I and SLT-II have been purified to
homogeneity.
The bacteria grown in iron-depleted
medium were disrupted by French press
and the toxins purifed by anti-shiga toxin
affinity chromatography or by conventional
biochemical methods.
The SLT-I A subunit has a MW 32,200 and
the SLT-I B subunit has a MW of 7,700
SHIGA-LIKE TOXINS
The SLT-II was purified from E. coli strain
containing the cloned toxin genes on
recombinant plasmid.
Purification was accomplished by a series
of column chromatography techniques
including monoclonal antibody affinity
chromatography (against SLT-II).
The SLT-II consisted of A and B subunits
with apparent molecular weights of 32,000
and 10,200, respectively.
SHIGA-LIKE TOXINS
 By analogy with Shiga toxin, the most likely
A-to-B subunit ratio for SLT-I is 1:5.
The MW of holotoxin is about 70,000
SHIGA-LIKE TOXINS
 The purified SLTs have the same biological
activities as and comparable specific activities to
purified Shiga toxin.
 The molecular basis is probably the catalytic
inactivation of 60S ribosomes in toxin-sensitive
(receptor-expressing) cells (Fig. 14)
 The receptor for SLT-I and SLT-II has been
identified and is the same as for Shiga toxin; it is
a globotriosyl ceramide containing a galactose-à(1->4)-galactose-á-(1->4)-glucose ceramide
SHIGA-LIKE TOXINS
SHIGA-LIKE TOXINS
 Production of SLT is activated in iron-limited media.
 By a gene fusion experiment, the sltA and sltB genes are
regulated by a fur locus.
 A gene fusion between the promoter and proximal portion
of the SLT gene with gene for bacterial alkaline
phosphatase was made.
 Growth in low-iron conditions resulted in a 13- to 16-fold
increase in alkaline phosphatase activity.
 In the presence of a null mutation in the fur locus, however,
alkaline phosphatase activity was constitutively high
regardless of the iron concentration.
 These data indicate negative regulation of the slt operon
by the fur gene product
SHIGA-LIKE TOXINS
SHIGA-LIKE TOXINS
SHIGA-LIKE TOXINS
Some of the strongest circumstantial
evidence comes from epidemiological
studies of E. coli strains isolated from
humans and animals.
Most of the high level cytotoxin producers
were associated with diarrhea, hemorrhagic
colitis, or hemolytic uremic syndrome (HUS)
(Table 4)
SHIGA-LIKE TOXINS
SHIGA-LIKE TOXINS
 It appears that food is the primary source
of infection in man. E. coli O157 has been
isolated from hamburger meat and
unpasteurised milk which was also
associated with hemorrhagic colitis and
HUS. The O157:H7 were isolated from 1.53.7% of samples of beef, pork, poultry and
lamb
HEMOLYSINS
 E. coli produce cell-free and cell-bound
hemolysins, designated as the α- (AH) and βhemolysin, respectively.
 Both the α- and β-hemolysins cause β-hemolysis
(clear zone of lysis) around colonies on blood
agar plates
 An γ-hemolysin was also produced by mutants
resistant to nalidixic acid and this hemolysin does
not hemolyze human or rabbit RBC but does
hemolyze RBC of other species
HEMOLYSINS
Ah is produced by growing hemolytic
isolates in an alkaline meat extract broth,
casein hydrolysate, or a chemical defined
medium at 37C, aerobic, anaerobic
condition and also in CO2.
Aerobic growth enhances AH production.
Both AH and β-hemolysin are produced
during the log phase of growth
HEMOLYSINS
Iron concentration above 100 μM represses
hemolysin production.
It is suggested that a major function of AH
in vivo may be to provide iron for growth
under iron-limiting conditions
 AH has been purified by various
biochemical methods, affinity column with
monoclonal antibody
HEMOLYSINS
 Complexing of AH proteins to LPS could account
for discrepancies between measurements of the
size of active AH (150,000 to 300,000 daltons)
and the 106,000 to 110,000-dal predicted by the
size of its structural gene
 Treatment of AH with DNase, RNase, lecithinase,
or lysozyme has no effect on AH activity,
indicating that nucleotides, lecithin, or
peptidoglycan do not comprise the active site
 However, enzymatic treatment with lipases
destroys hemolytic activity, suggesting that a lipid
component may be necessary for AH activity
HEMOLYSINS
 Divalent cation calcium, strontium, or
barium was required to demonstrate
hemolytic activity in cultures of E. coli.
Calcium is required for binding of AH to
erythrocyte membranes
Calcium autoradiography of the
recombinant hemolysins separated by
SDS-PAGE and transferred to nitrocellulose
showed that full-length, active hemolysin
bound calcium
HEMOLYSINS
HEMOLYSINS
AH is not a heat-stable protein, and it is
inactivated by heating at 56C for as little as
10 min.
However, some species of AH are relatively
more stable to heat.
Stability to heat is depending on the
medium of treatment.
Also, AH is inactivated by formalin and urea
HEMOLYSINS
The AH gene locates on various
incompatible plasmids.
It was shown that Insertion element (IS)
occur in these plasmids that is the possible
explanation for the finding of hemolysin
determinants on various types of plasmids
HEMOLYSINS
At least three cistrons, designated as hlyA,
hlyB, and hlyC, clustered in the AH
determinant were found to be involved in
synthesis and secretion of AH
 hlyA is responsible for synthesis of
precursor, hlyC is responsible for the
processing, and hlyB is responsible for
export of AH
HEMOLYSINS
HEMOLYSINS
 The gene product of hlyA was found to be a
106,000- to 107,000-dal nonsecreted cytoplasmic
protein which is probably the inactive hemolysin
precursor.
 hlyC codes for a 18,000-dal protein that appears
to be involved in the conversion of the precursor
hemolysin to active hemolysin with a proposed
molecular weight of 58,000.
 The hlyC gene product is believed to have dual
functions of (i) activation and (ii) transport of
hemolysin through the cytoplasmic membrane to
the periplasm
HEMOLYSINS
 hlyB is required for transport of hemolysin from
the periplam to the exterior of the cell.
 The hlyBa cistron codes for a 46,000-dal protein
located in the outer membrane that binds the
hemolysin and transports it through the outer
membrane.
 hlyBb codes for a protein of 62,000-dal, most of
which is found in the outer membrane, and
presumably functions in release of hemolysin
from the outer membrane
HEMOLYSINS
Hemolysin determinant on chromosome
has also been cloned and studied.
As with AH plasmid, at least three cistrons
(A, B, and C) are present on the
chromosomal AH determinant.
Cistron hlyA seems to be most variable,
whereas hlyB and hlyC are highly
conserved.
HEMOLYSINS
The primary structure of E. coli hemolysin
(HlyA) contains a 9-amino-acid sequence
which is tandemly repeated 13 times near
the C terminus and which is essential for
hemolytic activity.
The domain involved in binding calcium
was identified as the tandemly repeated
sequences, since the deletion derivative
missing 11 of the 13 repeats did not bind
calcium
HEMOLYSINS
AH is toxic and lethal when intravenously
injected to animal.
It also shows cytotoxicity and the toxicity
can be neutralized by antiserum treatment.
HEMOLYSINS
 The AH had a rather low activity in membranes formed of
pure lipids, such as phosphatidylcholine or
phosphatidylserine.
 In membranes from asolectin, a crude lipid mixture from
soybean, hemolysin was able to increase the conductance
by many order of magnitude in a steep concentrationdependent fashion.
 The asolectin may contain a receptor needed for
membrane activity of the toxin.
 The results of single-channel records showed that the
membrane activity of hemolysin is due to the formation of
ion-permeable channels with a single-channel
conductance of about 500 pS in 0.15 M KCl
ADHERENCE
In Enterotoxigenic E. coli
In Enteropathogenic E. coli
In Enterohemorrhagic E. coli
ADHERENCE In Enterotoxigenic E. coli
 Adhesion of ETEC to the small intestinal mucosa
is now recognized as an important early event in
colonization and the development of diarrheal
disease
 The colonization factor antigens (CFAs) now
include CFA/I, CFA/II, CFA/III, and CFA/IV
(formerly PCF8775).
 The CFA/I, CFA/III, and PCF0159 are probably
homogenous rodlike fimbrial antigens
 A putative human ETEC colonization factor
(PCF0159:H4) has been described in ETEC
serotype O159:H4.
ADHERENCE In Enterotoxigenic E. coli
ADHERENCE In Enterotoxigenic E. coli
 CFA/II is composed of three surface-associated
antigenic components termed coli surface
antigens (CS), CS1, CS2, and CS3.
 Strains of serotype O6:H16 produce either CS1 or
CS2 in associated with CS3, while CS3 alone is
found in most other CFA/II serotypes.
 CFA/IV also exhibits heterogeneity and currently
consists of three distinct CS antigens, CS4, CS5,
and CS6; CS4 and CS5 are rodlike fimbriae,
where a structure has not been reported for CS6
ADHERENCE In Enterotoxigenic E. coli
The adhesion mediated by the fimbriae is
shown in Fig. 20
ADHERENCE In Enterotoxigenic E. coli
 A new nonfimbrial adhesive factor (antigen 8786),
with mol.Wt. 16,300 Da, was found on the
bacterial surface of enterotoxigenic E. coli
O117:H4
 A plasmid was also demonstrated coding for CS5,
CS6, heat-stable enterotoxin, and colicin in O167
 Infact, the structural genes of colonization factors
are located on high-molecular-weight plasmids,
except CS1 and CS2, which are chromosomal
ADHERENCE In Enterotoxigenic E. coli
 Colonizing factor occurs in ETEC, however,
its role in causing diarrhea remains unclear.
Small bowel colonization by colonizing,
nontoxigenic E. coli impairs water and
electrolyte absorption and sucrase activity
in the absence or recognized enterotoxin,
cytotoxin, invasion, or effacement traits
ADHERENCE In Enterotoxigenic E. coli
Toxin produced by bacteria adherent to
cells are targeted more efficiently and
become relatively inaccessible to
neutralization by toxin inhibitors
(VL645 abd VL647 are isogenic strains
Fim+ and Fim- strains of E. coli, each
harboring LT+ plasmid, H-10407-p is an
enterogenic strain lacking CFA/I but
expressing type 1 fimbriae) (Table 5)
ADHERENCE In Enterotoxigenic E. coli
ADHERENCE In Enteropathogenic E. coli
It has been shown that many EPEC strains
adhere to cells (e.g. HEp-2, HeLa) in
characteristic patterns termed localized
adherence (LA) and diffuse adherence (DA)
ADHERENCE In Enteropathogenic E. coli
 It was demonstrated that hemagglutination
(pattern termed HAIII) factor of EPEC is very
similar to the type 1-fimbriae antigenically.
 Type 1-fimbriae have been shown to mediate
adherence to intestinal mucosa.
 But not all the EPEC strains carry type 1-fimbriae,
so other structures are likely to be involved in the
adhesion process.
 Electron microscopy failed to show fimbriae or
pilus-like structures on the bacteria which
exhibited adherence to HEp-2 and HeLa cells
ADHERENCE In Enteropathogenic E. coli
 This so called localized adherence (LA) is
associated with the presence of a plasmid of 50
to 70 MDa. Such plasmids encode the so-called
EPEC adherence factor (EAF)
 The fragment A from pMAR2 was used as probe
in Southern blot analysis, and the result showed
high degree of sequence conservation among
these plasmids.
 Adherence genes from pMAR2 were cloned as
two distinct plasmid regions which confer the
adherence phenotype only when complementing
each other in trans
ADHERENCE In Enteropathogenic E. coli
 A DNA probe has been constructed from
one of the adherence plasmids (pMAR-2)
and has been used in field trials to detect
EPEC
 By comparing the restriction maps, other
plasmids associated with cell adhesion are
not similar to pMAR-2
ADHERENCE In Enteropathogenic E. coli
Another plasmid, pYR111 from serotype
O111:NM, was also associated with
localized adherence (LA) with HeLa cells.
Curing of this plasmid yielded strains which
lost the ability to exhibited LA, resistance to
the antibiotics, and expression of
lipopolysaccharide (LPS) O-antigenic
polysaccharide
ADHERENCE In Enteropathogenic E. coli
ADHERENCE In Enteropathogenic E. coli
The cellular adherence factors were
associated with cell surface structures of
bacteria that were proteinaceous in nature.
So, cellular adherence properties could be
substantially reduced by pronase treatment
and by heat treatment (100C for 5 min) of
bacteria
ADHERENCE In Enteropathogenic E. coli
 However, adherence factor may also exist
in chromosome.
TnphoA insertion mutants of EPEC with
various adherence and pathogenic activity
were obtained.
ADHERENCE In Enteropathogenic E. coli
 By Southern hybridization of plasmid and total
DNA of each strain was performed to determine
the location of each TnphoA insert, and each
TnphoA insert along with flanking EPEC
sequences was also cloned.
 These studies resulted in the grouping of the
mutants into five main categories:



(A) strains with plasmid and chromosomal insertions
that alter adherence,
(B) chromosomal insertions that alter the ability to
induce actin polymerization,
(C) chromosomal insertions that do not affect
adherence or actin polymerization
ADHERENCE In Enteropathogenic E. coli
ADHERENCE In Enteropathogenic E. coli
Studying the adhesion by using electron
microscope, a two-stage model of EPEC
adhesion is proposed
ADHERENCE In Enteropathogenic E. coli
ADHERENCE In Enteropathogenic E. coli
ADHERENCE In Enteropathogenic E. coli
 Fluorescence Actin Staining (FAS) Method
 When bacteria are attached, microvilli are lost.
The underlying cytoskeleton of the epithelial cell
is disorganized, with a proliferation of filamentous
actin.
 The polymerization of actin at the sites of the
attaching and effacing lesion forms the basis of a
recently described diagnostic test for EPEC.
 Fluorescein isothiocyanate (FITC)-phalloidin, the
fluorescein conjugate of a phallotoxin, binds
specifically to polymerized actin
ADHERENCE In Enterohemorrhagic E. coli
 A plasmid of 60 MDa was also found in EHEC
(e.g. O157:H7)
 But experiment in gnotobiotic piglets showed that
the presence of such plasmid is not essential for
expression of virulence
 Such plasmid appears to modify the eukaryotic
cell adherence of E. coli O157:H7 and to confer
that adherence on E. coli HB101 through surface
structures other than pili.
 By electron microscopy, the wildtype strain and
the plasmid cured strain which showed reduced
adherence had pili
ADHERENCE In Enterohemorrhagic E. coli
INVASIVENESS
 Enteroinvasive E. coli (EIEC) are strains with
pathogenicity close to Shigella.
 Epithelial-cell invasiveness as detected by the
ability of the organism to cause
keratoconjunctivitis in the guinea-pig eye (Sereny
test) is absent in enteropathogenic E. coli.
 Strains of Salmonella and Shigella were
internalized after attachment to animal cells.
 Such process also occurs after the adherence of
the E. coli to HEp-2 cells or Henle 407 cells and
multiplication has been seen to take place.
DETECTION
 Using glucuronidase assay
 It was reported in 1976 that β-glucuronidase is
limited to E. coli, Shigella species, and
Salmonella species in the family
Enterobacteriaceae.
 Then, β-glucuronidase substrates have been
incorporated in diverse media to detect E. coli in
samples from a variety of sources, such as
environmental, food, seawater, and clinical
sources.
 Constitutive enzyme test demonstrate that 87 to
97% of E. coli isolates are positive, and inducible
procedures show 91 to 100% positivity.
DETECTION
 Using glucuronidase assay
 A method designated as Colilert system is described as
follows.
 The sample is plated on MacConkey agar, and the
suspected colonies are picked and resuspended in Colilert
tube (Access Analytical Systems, Branford, Conn., U.S.A.)
(medium containing 4-methylumbelliferyl-β-glucuronide,
MUG, as the fluorogenic indicator) rehydrated with 10 ml
of destilled water.
 Tubes are read for fluorescence after 24, 28, and 120 h of
incubation at 35C.
 The tube becomes yellow if total coliforms were present
and fluorescent (under long UV light source) if E. coli is
present
DETECTION
Animal Tissue Culture
DETECTION
Dense concentrations of microfilaments are
present in the apical cytoplasm beneath
attached bacteria.
Such polymerization of actin can by
detected by Fluorescein-labeled phallotoxin
(FAS).
So FAS can be a simple, highly sensitive
diagnostic test for EPEC and EHEC
DETECTION
 Animal Assays
 Rabbit ligated ileal loop assay (RIL) is usually
employed.
 Test using infant mice is a convenient assay for
STa.


Supernatants of cultures (0.1 ml) could be injected with
a no. 30 hypodermic needle into the milk-filled
stomachs of infant mice (1-4 day old) and fluid
accumulation in the intestine was measured after 4 h
by determining the ration of intestine to whole body
weight.
Usually two drops of a 2% solution of pontamine sky
blue 6BX (DuPont) are added to each 1 ml of inoculum.
DETECTION
 Immunological Methods
A hydrophobic grid membrane filter
(HGMF)-enzyme-labeled antibody method
was developed for the rapid detection of
hemorrhagic O157 in food.
An O-antigen-specific monoclonal antibody
was labeled by horseradish peroxidaseprotein A
DETECTION
 Enzyme-linked Immunosorbent Assay (ELISA)
 A competitive ELISA for STa is commercially
available (COLI ST EIA, Denka Seiken)
 Shiga-like toxin (Stx) can be detected by enzyme
immunoassay (EIA) (ProSpecT STEC; Remel,
Lenexa, KS)
 Antibody array constructed on solid supports
using nitrocellulose membrane and poly-l-lysine
(PLL) glass slide was developed for the detection
of E. coli
DETECTION
Latex Agglutination Test
 A simple latex agglutination test (E. coli
O157 latex test, DR620, Oxoid)
VTEC can be detected by VTEC-Screen
'Seiken'.
DETECTION
E. coli in water samples were assayed by
traditional and immunomagnetic
separation/adenosine triphosphate
(IMS/ATP) methods.
Pearson's correlation analysis showed
strong, significant, linear relations between
IMS/ATP and traditional methods for all
sample treatments; strongest linear
correlations were with the direct analysis (r
= 0.62 for E. coli)
DETECTION
DETECTION
 Enzymatic bio-nanotransduction
 It is based on the production and measurement of biological nanosignals (nucleic acid sequences) in response to the biological
recognition of the targeted organism or toxin
 Specifically, biological recognition molecules (such as antibodies) are
linked to DNA templates that code for a T7 RNA polymerase promoter
and a given nucleotide sequence.
 The specific capture and concentration of a target organism or toxin
that is bound to a recognition molecule is followed by an in vitro
transcription reaction of the bound DNA template.
 Detection of the RNA nano-signals on a detection platform is
correlated with the presence or absence of the target in the original
sample.
 By this approach, it is possible to detect multiple targets and target
types (e.g., DNA, RNA, protein, whole cells) in a single sample by
changing the recognition element
DETECTION
DNA methods
Nucleic acid probes
PCR
Real-time PCR
DETECTION