Binding of intimin with Tir on the bacterial surface is prerequisite for the barrier disruption induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) infects intestinal epithelial cells and perturbs the intestinal barrier that limits the paracellular movement of molecules. The disruption of the barrier is mediated by the effectors translocated into the host cells through the bacterial type III secretion system (TTSS). A previous report has described the importance of a bacterial outer membrane protein, intimin, in EPEC-mediated disruption of the barrier, and proposed that intimin, in concert with a host intimin receptor, controls the activity of the translocated barrier-disrupting effectors [P. Dean, B. Kenny, Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein, Mol. Microbiol. 54 (2004) 665-675]. In this study, we found that the importance of intimin is in its ability to bind a bacterial intimin receptor, Tir. Additionally, the impaired ability of an intimin-negative mutant was not restored by co-infection with intimin-expressing TTSS mutants. Collectively, the results in this study favor an alternative scenario explaining the importance of intimin, that the binding of intimin with Tir on the bacterial surface triggers or promotes the translocation of factors required for the efficient disruption of the barrier. Thus, the interaction of intimin with Tir may serve as a molecular switch that controls the delivery of virulence factors into the host cells.     

A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.     

Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN

Type III secretion is a transport mechanism by which bacteria secrete proteins across their cell envelope. This protein export pathway is used by two different bacterial nanomachines: the flagellum and the injectisome. An indispensable component of these secretion systems is an ATPase similar to the F₁-ATPase β subunit. Here we characterize EscN, an enteropathogenic Escherichia coli type III ATPase. A recombinant version of EscN, which was fully functional in complementation tests, was purified to homogeneity. Our results demonstrate that EscN is a Mg²⁺-dependent ATPase (k_cat 0.35 s⁻¹). We also define optimal conditions for the hydrolysis reaction. EscN displays protein concentration-dependent activity, suggesting that the specific activity changes with the oligomeric state of the protein. The presence of active oligomers was revealed by size exclusion chromatography and native gel electrophoresis.     

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escherichia coli from calves

AIMS: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. METHODS AND RESULTS: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx(1) exhibited stx(1c) in 13 (31.70%) isolates, while that of stx(2) revealed stx(2c) in eight (24.24%) and stx(2d) in two (6.06%) isolates. Subtyping of eae gene showed the presence of eae-beta, eae-eta and eae-zeta in two, three and four isolates respectively. None of the E. coli isolates possessed stx(2e), stx(2f), eae-alpha, eae-delta, eae-epsilon and eae-xi. All EPEC isolates were atypical. CONCLUSIONS: stx(1), stx(1c), stx(2), stx(2c), stx(2d), eae-beta, eae-eta and eae-zeta subtypes are prevalent in STEC and EPEC isolates in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first subtype analysis of stx(2) and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans.     

Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. It produces a characteristic intestinal histopathological lesion on enterocytes known as ‘attaching and effacing’ (A/E), and these two steps are mediated by a type-III secretory system. In the present study, we evaluated the effect on the initial host cell attachment step produced by bovine lactoferrin (bLF) and three synthetic peptides: lactoferricin (LFcin), lactoferrampin (LFampin) and LFchimera. A special focus was given to the hemolytic activity and EPEC-induced actin polymerization in HEp-2 cells, as well as to the espA gene expression, which produces the protein responsible for primary contact with the host cells. Results show that EPEC attachment to HEp-2 cells was significantly suppressed by bLF and LFchimera at 125 and 40 μM, respectively. EPEC-mediated actin polymerization was blocked by bLF and LFchimera at 88 and 99%, respectively. LFchimera inhibited the attachment and A/E lesion caused by EPEC in a dose-dependent manner. In the presence of 125 μM bLF, the expression level of the espA gene was decreased by 50% compared to the untreated control. LFchimera at concentrations of 20 μM and 40 μM diminished the level of espA gene expression 100 and 1000 fold, respectively (P < 0.001). Although bLF, LFchimera, LFcin, and LFampin all significantly blocked the hemolysis produced by EPEC (P < 0.001), the two former compounds produced this effect at lower concentrations. These two compounds, bLF and LFchimera, were able to inhibit the first steps of the mechanism of the damage used by EPEC. This data suggests that LFchimera could provide protection against enteropathogens that share this mechanism.     

LamB-mediated adherence of enteropathogenic Escherichia coli to HEp-2 cells

Aims:  To establish the role of maltoporin (LamB) in adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells in vitro.
Methods and Results:  Three strains, wild type (WT) EPEC, a maltoporin (LamB) mutant ΔlamB , and DH5α were used to study adherence to cultured HEp-2 cells. Mutant ΔlamB was found to be deficient in adherence compared to WT EPEC. Adherence of ΔlamB was restored to wild type levels when complemented with the cloned lamB gene. The non–adherent strain DH5α also adhered to HEp-2 cells when it harboured the cloned lamB gene. The LamB protein was isolated from WT EPEC by electroelution and antibodies were raised in rabbits. The specificity of the antibodies was analysed by Western blotting. Anti-LamB antiserum reduced adherence of WT EPEC to HEp-2 cells. The LamB protein was coated on latex beads and the beads adhered to HEp-2 cells. Anti-LamB antiserum prevented bead adherence to HEp-2 cells. Multiple sequence alignment showed that the L9 loop of EPEC LamB had four amino acids different from the L9 loop of LamB from several other related pathogens.
Conclusions:  LamB serves as an alternative or additional adherence factor for EPEC.
Significance and Impact of the Study:  Adherence is an important component of the pathogenesis of noninvasive pathogens like EPEC. A putative adhesin such as LamB, which has already been found to be co-expressed with virulence factor EspB may be a potential vaccine candidate for control of EPEC and related pathogens.     

An overview of atypical enteropathogenic Escherichia coli

The enteropathogenic Escherichia coli (EPEC) pathotype is currently 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, which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. Herein, we review the serotypes, virulence properties, genetic relationships, epidemiology, reservoir and diagnosis of aEPEC, including those strains not belonging to the classical EPEC serogroups (nonclassical EPEC serogroups). 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.     

Investigation of diarrhoeic faecal samples for enterotoxigenic, Shiga toxin-producing and typical or atypical enteropathogenic Escherichia coli in Kashmir, India

Three hundred and twenty-six Escherichia coli isolates recovered from 326 human faecal specimens from sporadic cases of diarrhoea in Kashmir valley, India, were investigated for the presence of stx(1), stx(2), eaeA, hlyA and lt virulence genes. None of the samples was positive for stx genes or Shiga toxins by PCR or enzyme-linked immunosorbent assay. Twenty-three E. coli isolates showed the presence of the eaeA gene, whereas three isolates harboured the lt gene. Enteropathogenic E. coli (EPEC) belonged to 10 different serogroups. Out of 23 EPEC isolates, the majority (78.26%) were atypical while five (21.73%) were typical. Only one of the typical EPEC harboured the EAF plasmid. Subtyping of the eaeA gene showed the presence of eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta in one, two, four and two isolates, respectively. None of the E. coli isolates possessed eaeA-delta, eaeA-epsilon and eaeA-zeta. This study further upholds the opinion that Shiga toxin-producing E. coli do not pose a major threat to human health in India and eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta could be common EPEC subtypes prevalent in humans with diarrhoea in India. The present study appears to be the first report of subtype analysis of the eaeA gene from India and also records the isolation of EPEC with the eaeA-xi gene from humans.     

Fimbrial adhesins produced by atypical enteropathogenic Escherichia coli strains

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA (86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains, respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.     

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10^?8 mol l^?1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.     

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

Type III secretion (T3S), a protein export pathway common to Gram‐negative pathogens, comprises a trans‐envelope syringe, the injectisome, with a cytoplasm‐facing translocase channel. Exported substrates are chaperone‐delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first “translocators”, then “effectors”. We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane‐associated pseudo‐effector SepL and its chaperone SepD. This renders SepL a high‐affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD‐coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.     

Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India

Aims: To study the prevalence and characterize atypical enteropathogenic Escherichia coli (EPEC) and Shiga toxin producing E. coli (STEC) in avian species in India. Methods and Results: Two hundred and twelve faecal samples collected from 62 chickens, 50 ducks and 100 pigeons were investigated for the presence of stx₁, stx₂, eae and ehxA virulence genes by multiplex PCR. In all, 42 E. coli isolates (25 chicken, 2 duck and 15 pigeon) possessed at least one virulence gene. Out of these, nine (4·24%) isolates were STEC and 33 (15·56%) were EPEC. All isolates from duck and chicken were EPEC while among 15 pigeon isolates nine (60%) were STEC and six (40%) were EPEC. Among the STEC isolates four each carried stx₁ or stx₂ and one possessed both stx₁ and stx₂. Subtype analysis of stx revealed the presence of stx_2f in four STEC isolates. None of the STEC isolates carried stx_1c, stx_2c, stx_2d or stx_2e. Isolates carrying stx_2f demonstrated vero cell toxicity. One each belonged to serogroup O17 and O78, while one was rough and the other untypeable. All EPEC isolates were atypical as they lacked bfpA. This appears to be the first report of detection of stx_2f from India. Conclusions: The study established the presence of stx₁ and stx_2f containing E. coli in pigeons and atypical EPEC in poultry in India. Pigeons might serve as vectors for transmission of STEC to environment and humans. Significance and Impact of the Study: Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons and humans.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Aims: To develop a duplex real‐time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2‐encoding genes which are pivotal to EHEC‐mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non‐E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H^?, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H^?, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real‐time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real‐time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real‐time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.     

Structure of the cyclomodulin Cif from pathogenic Escherichia coli

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.     

Characterization of the α-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding α-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire α- hly CABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited α- hly determinants. The α- hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli α-hly plasmid pHly152, in particular, the structural α- hly CABD genes (99.2% identity) and the regulatory hly R regions (98.8% identity). pEO5 and α-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural α- hly CABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS 2 element upstream of the hly C gene in pHly152. The presence of transposon-like structures at both ends of the α- hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.     

A self-transmissible plasmid of an enteropathogenic Escherichia coli strain codes for mannose-resistant haemagglutinin and for antibiotic resistance

Abstract Four enteropathogenic Escherichia coli strains were studied with respect to their antibiotic resistance characters, plasmid patterns, toxin production and haemagglutination properties. Two of these strains showed multiple antibiotic resistance characters, although all possessed several plasmids of varying sizes. One of the strains DD-41 showed the presence of a non-fimbrial cell-associated mannose-resistant haemagglutinin (MRHA) which was encoded by a 70 MDa plasmid. Conjugation experiments demonstrated that this MRHA-containing plasmid also coded for ampicillin and tetracycline resistance factors and was self-transmissible.     

Cytolethal distending toxin (CLDT) production by enteropathogenic Eschrichia coli (EPEC)

Culture supernates of 16 strains of EPEC belonging to 6 different serogroups, when assayed on Chinese Hamster Ovary (CHO) cells up to 96-120 h, induced distinct morphological changes. The supernate activities were heat-labile, unrelated to heat-labile enterotoxin (LT), verotoxin (VT), or hemolysin, did not show necrosis in rabbit skin and caused no fluid secretion in the rabbit ileal loop assay (RILA). Simultaneous production of CLDT and heat-stable enterotoxin (ST) were detected in four EPEC strains.     

Comparison of Tir from enterohemorrahgic and enteropathogenic Escherichia coli strains: two homologues with distinct intracellular properties

Summary Tir of enteropathogenic Escherichia coli (EPEC) or enterohemorrahgic E. coil (EHEC) is translocated by a type III secretion system to the host cell membranes where it serves as a receptor for the binding of a second bacterial membrane protein. In response to the binding, EPEC Tir is phosphorylated at Tyr₄₇₄, and this phosphorylation is necessary for the signaling of pedestal formation. Tir of EHEC has no equivalent phosphorylation site but it is similarly needed for cytoskeleton rearrangement. How these two Tir molecules achieve their function by apparently different mechanisms is not completely clear. To examine their intrinsic differences, the two Tirs were expressed in HeLa cells and compared. Actin in complexes could be pelleted down from the lysate of cells expressing EHEC Tir but not EPEC Tir. By immunostaining, neither Tir molecule was found in phosphorylated state. In the cytoplasm, EHEC Tir was frequently found in fibrous structures whereas EPEC Tir was observed completely in a diffusive form. The determinant critical for the EHEC Tir fibrous formation was mapped to the C-terminal region of the molecule that deviates from the EPEC counterpart. This region may play a role in taking an alternative route different from Tyr₄₇₄ phosphorylation to transduce signals.