Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.     

Modulation of tight junction barrier function by outer membrane proteins of enteropathogenic Escherichia coli: role of F-actin and junctional adhesion molecule-1

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea. In this work we investigated the effect of outer membrane proteins (OMP) of EPEC on barrier integrity and the role of actin, junctional adhesion molecule (JAM) and signaling pathways contributing to these changes. Barrier function was assessed by transepithelial electrical resistance (TER). OMP of wild type EPEC, eaeA and maltoporin mutants decreased TER levels of Caco-2 cells. The OMP of espB mutant was deficient in decreasing TER of Caco-2 cells. The proteinase K-digested wild type OMP and EAF mutant OMP did not cause any change in barrier function. Our previous studies have demonstrated that EPEC OMP induced changes in cadherin junctions of Caco-2 cells. Immunofluorescence revealed disruption in actin cytoskeleton by EPEC OMP. However, no change in expression of junctional adhesion molecule-1 was observed. NF-kappaB inhibitor slightly blocked the decrease in TER and protected against actin disruption while ERK1/2 inhibitor had no effect in blocking these changes. In conclusion, our data suggest that the OMP of EPEC alter intestinal barrier function by disrupting actin cytoskeleton and signaling pathways like NF-kappaB may have a role in regulating barrier changes.     

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae ). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae -defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.     

Molecular epidemiological study of a mass outbreak caused by enteropathogenic Escherichia coli O157:H45.

We made a molecular analysis of O157:H45 Escherichia coli isolated from a mass outbreak that occurred in Obihiro City. Using DNA analysis, we confirmed this infection case as a mass outbreak. Although the isolates expressed O157 antigen, they did not produce Vero toxin. We concluded they were enteropathogenic E. coli (EPEC) because they had a bfp gene and an EAF plasmid, and further they exhibited local adherence to HEp-2 cells. We believe this is the first report of a mass outbreak by O157 EPEC, and we suggest that PCR using eae- and bfp-specific primers and HEp-2 adherence assay are useful to identify EPEC.     

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.     

Magnesium ions are required for HEp-2 cell adhesion by enteroaggregative strains of Escherichia coli O126:H27 and O44:H18

Enteroaggregative strains of Escherichia coli, belonging to serotypes O44:H18 and O126:H27, were used to show that magnesium ions were essential for the adhesion of these enteroaggregative strains to HEp-2 cells. The removal of Mg²⁺ ions from culture media was correlated with the inability of strains to produce an outer membrane-associated protein of 18 kDa and a pellicle. It was concluded that magnesium ions were directly involved with the expression of an 18 kDa outer membrane-associated protein by strains of E. coli O126:H27 and O44:H18, and that the outer membrane-associated protein was involved in both HEp-2 adhesion and pellicle formation.     

Interleukin-8 secretion by epithelial cells infected with diffusely adherent Escherichia coli possessing Afa adhesin-coding genes

Escherichia coli that adhere sparsely to human epithelial (HEp-2) cells are known as diffusely adherent E. coli(DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)-identified originally as a uropathogenic factor-in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL-8 in cultures of human carcinoma epithelial cells (e.g., HEp-2, Caco-2, and T84). Nineteen afa-positive DAEC strains were examined for their ability to induce IL-8 secretion, and only 7 strains (37%; 7/19) induced IL-8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL-8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.     

Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Relationship between O-serogroup and presence of pathogenic factor genes in Escherichia coli

A total of 383 isolates of serogroup-based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes. The serogroup-based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers. The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%). The difference was not significant. The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA-positive EPEC strains from patients but was not detected in the 9 strains from carriers. In serogroup-based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers. The 4 ipaH-positive strains possessed the invasive plasmid. These results suggested that the serogroup-based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene.     

An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCalpha

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea. Despite the advances made in understanding EPEC-host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully. Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process. Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with beta-catenin, both of which constitute the adherens junctions. Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC. In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers. We also observed significant phosphorylation of protein kinase Calpha (PKCalpha) in cells treated with either whole EPEC or its OMP. Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCalpha monoclonal antibodies and anti-beta-catenins revealed that in these cells, phosphorylated PKCalpha is associated with cadherins, leading to the dissociation of the cadherin/beta-catenin complex. Immunofluorescence showed beta-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm. Expression of dominant negative PKCalpha reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability. It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.     

Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene

Abstract Invasive Escherichia coli strains of certain serotypes invade by the same mechanism as the Shigella sp. It has been proposed that invasion of epithelial cells by EPEC strains may also occur; this is a previously overlooked property. In the present study E. coli strains isolated from patients with diarrhoea or ulcerative colitis, lacking the inv plasmid mediating classical invasion, but hybridizing with probes for different adhesins, were analyzed for their ability to invade HeLa and Caco-2 cells. The majority of strains invaded Caco-2 cells to a higher extent than HeLa cells. Adhesion to Caco-2 cells was a prerequisite for subsequent invasion of the cells but EAF, eae , EAgg and other known virulence factors were not sufficient to mediate invasion. In 8/9 E. coli strains invasion was enhanced after growth under iron restriction. Growth during anaerobic conditions did not influence subsequent invasion by E. coli strains whereas 6/9 strains had their invasive ability significantly decreased after growth in the presence of 1% glucose. The invasive process was inhibited by mannose but not by lactose, fucose or galactose. Our data indicate that strains of E. coli may invade Caco-2 cells by novel mechanisms which require adhesion to the cells but which differ from those of Salmonella sp., Yersinia sp., Shigella sp. and classical enteroinvasive E. coli .     

HEp-2 cell adherence and Vero cell cytotoxin production by EPEC strains isolated from children with diarrhoea in New Zealand

Abstract A total of 112 EPEC strains isolated from children with diarrhoea in New Zealand were examined for mannose-resistant HEp-2 cell adherence and production of exotoxins. Enterotoxin production was not detected in any of the strains examined. Verotoxin production was detected in 13 (11.6%) strains and of these 4 were also found to adhere to HEp-2 cells. HEp-2 cell adherence was displayed by a total of 29 (25.8%) strains of which 22 were diffusely adherent. Only 3 (2.7%) strains were shown to belong to the new virulence phenotype, entero-aggregative adherence, when examined in the adherence assay. We identified one strain with the novel characteristics of causing detachment of HEp-2 cells from glass coverslips and are further investigating this possible virulence mechanism. These results suggest that if EPEC strains are to be considered as a cause of diarrhoea, the search for new virulence factors must be extended.     

Comparison of adherence to and penetration of a human laryngeal epithelial cell line by group A streptococci of various M protein types

Clinically isolated group A streptococci (GAS) of different M protein types were studied using aminoglycoside exclusion and [2,8-3H]adenine radiolabeled GAS assays to compare the abilities of different strains to adhere to and internalize within human laryngeal epithelial (HEp-2) cells. GAS isolated from patients with pharyngitis and GAS isolated from patients with more severe disease, such as necrotizing fasciitis, adhered to and penetrated HEp-2 cells equally well. M3, M4, M6, and M12 strains adhered to and were internalized within HEp-2 cells more than M1 strains. M18 GAS producing hyaluronic acid capsules were less adherent and less invasive than the M3, M4, M6, and M12 strains. An M3-producing GAS strain and its M protein-deficient isogenic strain adhered similarly to HEp-2 cells, but the M protein-deficient strain exhibited greater penetration. Preincubation of HEp-2 cells with an N-terminal synthetic M3 peptide did not alter the adherence or penetration by an M3 strain. In summary, this study demonstrates that GAS from invasive and non-invasive disease adhere to and penetrate HEp-2 cells equally well and that multiple strains of GAS with various M protein types have the ability to adhere to and penetrate HEp-2 cells.     

Expression of a new porin 'OmpE' by strains of Salmonella enteritidis

Abstract Strains of Salmonella enteritidis expressed a novel porin with a subunit size of 35.5 kDa as shown by SDS-PAGE. This protein was expressed as a major outer membrane protein (MOMP) when grown on nutrient agar, McConkey agar or blood agar, or in Tris-succinate medium; but was only produced in trace amounts when strains were grown in nutrient broth. This OMP was produced by all strains of S. enteritidis examined, regardless of phage type, and expression was not related to the possession of a 38-MDa mouse-virulence plasmid or the ability of strains to make long-chain lipopolysaccharide. This new porin has been tentatively called OmpE.     

Cloning and characterization of the bundle-forming pilin gene of enteropathogenic Escherichia coli and its distribution in Salmonella serotypes

bfp, the structural gene of the major repeating bundle-forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (0111:NM). The bfp open reading frame encoded a 193-amlno-acid protein; comparison of this sequence with the biochemically determined N-terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13-amino-acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21000 dalton protein that co-migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonelia serotypes. Fifty per cent of these bfp probe-sensitive salmonellae exhibited the localized-adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid-containing EPEC strains. Scanning electron micrographs of a bfp probe-positive, LA-positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP-like fibres form inter-bacterial linkages. For EAF plasmid-containing EPEC strains and for severai Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.