enteropathogenic escherichia coli ii

ENTEROPATHOGENICENTEROPATHOGENIC ESCHERICHIA COLIESCHERICHIA COLI

II II

Hin-chung Wong Department of Microbiology Soochow

University

SHIGA-LIKE TOXINS SHIGA-LIKE TOXINS Enteropathogenic E. coli (EPEC, e.g. O26:H11, O

128: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 anti-shiga toxin is known as Shiga-like toxin I (SLT-I), and the non-neutralizable one is known as shiga-like toxin II (SLT-II)

SHIGA-LIKE TOXINS 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


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 SHIGA-LIKE TOXINS Iron is known to depress Shiga toxin production b

y 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).


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 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.


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 SHIGA-LIKE TOXINS The purified SLTs have the same biological activit

ies 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 r

egulated 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


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 TOXINSSHIGA-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 1.5-3.7% of samples of beef, pork, poultry and lamb

HEMOLYSINS HEMOLYSINS E. coli produce cell-free and cell-bound hemolysin

s, designated as the α- (AH) and β-hemolysin, 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 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 dur

ing the log phase of growth


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 HEMOLYSINS Complexing of AH proteins to LPS could account f

or 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 HEMOLYSINS Divalent cation calcium, strontium, or bariu

m 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


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


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


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

HEMOLYSINSHEMOLYSINS

HEMOLYSINS HEMOLYSINS The gene product of hlyA was found to be a 106,0

00- 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

HEMOLYSINSHEMOLYSINS hlyB is required for transport of hemolysin from th

e periplam to the exterior of the cell. The hlyBa cistron codes for a 46,000-dal protein l

ocated 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


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.

HEMOLYSINSHEMOLYSINSThe 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


AH is toxic and lethal when intravenously injected to animal.

It also shows cytotoxicity and the toxicity can be neutralized by antiserum treatment.

HEMOLYSINSHEMOLYSINS 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 concentration-dependent 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 ADHERENCE

In Enterotoxigenic E. coli In Enteropathogenic E. coli In Enterohemorrhagic E. coli

ADHERENCE In Enterotoxigenic ADHERENCE In Enterotoxigenic E. coliE. 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.


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


The adhesion mediated by the fimbriae is shown in Fig. 20


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


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


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 Enteropathogenic ADHERENCE In Enteropathogenic E. coliE. 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)


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


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


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


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


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


However, adherence factor may also exist in chromosome.

TnphoA insertion mutants of EPEC with various adherence and pathogenic activity were obtained.


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


Studying the adhesion by using electron microscope, a two-stage model of EPEC adhesion is proposed


Fluorescence Actin Staining (FAS) Method When bacteria are attached, microvilli are lost. Th

e 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 ADHERENCE In Enterohemorrhagic E. coliEnterohemorrhagic 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 ADHERENCE In Enterohemorrhagic E. coliEnterohemorrhagic E. coli

INVASIVENESS 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 DETECTION Using glucuronidase assay It was reported in 1976 that β-glucuronidase is lim

ited 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 DETECTION Using glucuronidase assay A method designated as Colilert system is described as fol

lows. The sample is plated on MacConkey agar, and the suspec

ted 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 DETECTION

Animal Tissue Culture

DETECTION 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 DETECTION

Animal Assays Rabbit ligated ileal loop assay (RIL) is usually em

ployed. Test using infant mice is a convenient assay for S

Ta. 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 DETECTION

Immunological Methods A hydrophobic grid membrane filter (HGM

F)-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 peroxidase-protein A

DETECTION DETECTION

Enzyme-linked Immunosorbent Assay (ELISA) A competitive ELISA for STa is commercially avai

lable (COLI ST EIA, Denka Seiken) Shiga-like toxin (Stx) can be detected by enzyme i

mmunoassay (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 DETECTION

Latex Agglutination Test A simple latex agglutination test (E. coli O1

57 latex test, DR620, Oxoid) VTEC can be detected by VTEC-Screen 'S

eiken'.

DETECTION 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

DETECTION DETECTION Enzymatic bio-nanotransduction It is based on the production and measurement of biological nano-sig

nals (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 DETECTION

DNA methodsNucleic acid probesPCRReal-time PCR

DETECTION DETECTION

enteropathogenic escherichia coli ii

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