Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin â, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC).

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of São Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.

Virulence factors of Escherichia coli strains belonging to serogroups O127 and O142.

A total of 102 Escherichia coli strains belonging to serogroups O127 and O142 were examined for genotypic and phenotypic characteristics. The most frequent serotypes found were O127:H21, O127:H40 and O142:H34. The virulence properties were evaluated by adhesion to HeLa cells and hybridization with gene probes for diarrhoeagenic E. coli. Most strains in the two serogroups were categorized as enteropathogenic E. coli, but enteroaggregative E. coli was also detected in both serogroups. All strains that carried the eae sequence presented the LEE region inserted in selC. Five ribotypes were detected in serogroup O127 and four in serogroup O142 and a correlation between serotypes and ribotypes was observed mainly in serogroup O142.

Combinations of putative virulence markers in typical and variant enteroaggregative Escherichia coli strains from children with and without diarrhoea.

Enteroaggregative Escherichia coli (EAEC) is defined by the ability to produce aggregative adherence (AA) to cultured cells. We analysed 128 EAEC strains, isolated from children with and without diarrhoea, regarding the presence of 11 EAEC virulence genes. Seventy strains carried and 58 lacked the EAEC probe sequence; 17 probe positive and 31 probe negative strains showed variations in the AA pattern. All EAEC probe positive strains carried at least one EAEC marker; aspU (94.3%), irp2 (91.4%), and aggR (74.3%) were the most prevalent. Conversely, among the EAEC probe negative strains, 41.4% were devoid of any marker and astA predominated (44.8%). No significant statistical difference in the prevalence of any marker between cases and controls in both EAEC probe groups or AA variants was found. We suggest that the EAEC probe positive strains may have a higher pathogenic potential or alternatively, EAEC probe negative strains may harbour virulence factors as yet undescribed.

Tissue tropism of enteropathogenic Escherichia coli strains belonging to the O55 serogroup.

Four enteropathogenic Escherichia coli (EPEC) strains belonging to the O55 serogroup (G21 and G30 [both O55:H6], G35 [O55:H-], and G58 [O55:H7]) were tested for their tissue tropism by using human intestinal in vitro organ culture. Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyer's patches, with no apparent adhesion to duodenum or colon. G35 and G58 express intimin gamma and show a similar tropism to intimin gamma-expressing enterohemorrhagic E. coli (EHEC) O157:H7. However, strains G21 and G30 were unusual because they expressed intimin alpha and had a restricted tissue tropism of intimin gamma phenotype. The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined. Comparison with the prototype intimin alpha from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins). This mutation was reproduced by site-directed mutagenesis in an intimin alpha plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism. However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture. Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism.

Production of cytolethal distending toxin and other virulence characteristics of Escherichia coli strains of serogroup O86.

Genetic and phenotypic virulence markers of different categories of diarrhoeagenic Escherichia coli were investigated in 106 strains of enteropathogenic E. coli (EPEC) serogroup O86. The most frequent serotype found was O86:H34 (86%). Strains of this serotype and the non motile ones behaved as EPEC i.e., carried eae, bfpA and EAF DNA sequences and presented localised adherence to HeLa cells. Serotypes O86:H2, O86:H6, O86:H10, O86:H18, O86:H27 and O86:H non determined, belonged to other categories. The majority of the strains of serotype O86:H34 and non motile strains produced cytolethal-distending toxin (CDT). The ribotyping analysis showed a correlation among ribotypes, virulence markers and serotypes, thus suggesting that CDT production might be a property associated with a universal clone represented by the O86:H34 serotype.

Inhibition of enteroaggregative Escherichia coli adhesion to HEp-2 cells by secretory immunoglobulin A from human colostrum.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important agent of the persistent diarrhea among low socioeconomic level children in developing countries that may be associated with chronic undernourishment. Breast-feeding is effective in protecting infants against diarrhea and other infectious diseases. The aim of the study is to verify the ability of human colostrum to inhibit aggregative adhesion of EAEC to HEp-2 cells and the presence of antibodies reactive to antigenic fractions of EAEC in colostrum samples. METHODS: Enzyme-linked immunosorbent assay, immunoblotting and adhesion assays of EAEC to HEp-2 cells were done with pooled or individual colostrum samples (n = 35). Assays were performed with a well-known EAEC strain, 044:H18 E. coli (strain 042). Colostral IgA was isolated by affinity chromatography in Sepharose anti-human alpha chain column. RESULTS: Total colostrum and isolated IgA inhibited EAEC adhesion, and this ability was associated with the presence of IgA antibodies against a 15-kDa band, compatible with the subunits of aggregative adherence fimbrial adhesin II, characteristic of the 042 strain, absent in its plasmid-cured isogenic strain, that was used as control. Individual colostrum samples also inhibited adhesion, showed variable antibody titles against EAEC antigens in enzyme-linked immunosorbent assay and recognized many antigenic fractions in immunoblotting assays, including the 15-kDa band. CONCLUSIONS: These results confirm that IgA from human colostrum inhibits adhesion of EAEC to HEp-2 cells and suggest that colostrum IgA antibodies reactive to EAEC antigens may play a role in protection of infants against diarrhea caused by these bacteria.

Intimin and the host cell--is it bound to end in Tir(s)?

Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, intimin and Tir. Some key remaining questions include whether intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate intimin-Tir association. Recent data support the hypothesis that the formation of a stable intimin-Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.

Genetic differences between Escherichia coli O26 strains isolated in Brazil and in other countries.

Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup -- encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains -- was evaluated through random amplified polymorphic DNA (RAPD) analysis. Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters. RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H(-) serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data. The epidemiological significance of these data is discussed.

Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry EAE and lack the EPEC adherence factor and Shiga toxin DNA probe sequences.

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the EAE profile were associated with diarrhea (P=.039).

Inhibition of enteropathogenic Escherichia coli (EPEC) adherence to HEp-2 cells by bovine colostrum and milk.

BACKGROUND: enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhea in Brazil and other developing countries. Human milk IgA protects newborn intestinal mucosa by inhibiting bacterial adhesion to epithelial cells and this effect is shown by in vitro assays of EPEC adhesion to HEp-2 cultured cells. Bovine milk, if effective in promoting this protection, could be an useful tool in the absence of the natural breastfeeding, in high-risk nurseries or in hospital infections. METHODS: the effect of colostrum, milk, and serum from dairy cows on the adherence to EPEC to HEp-2 cells was investigated. Colostrum from immunized and control animals and industrialized milk formulas were fractionated through a membrane device with a molecular weight cut off 10 kDa. The high molecular weight fraction (HMWF) of bovine colostrum was depleted of IgG through an affinity column and absorbed with an EPEC adherent strain. Antibodies were searched by ELISA and immunoblotting (IB). RESULTS: colostrum and milk from EPEC-immunized animals showed and inhibitory activity on adherence similar to that of control non-immunized animals. The inhibitory effect on adhesion was related to the HMWF. IgG-depleted colostrum partially retained the inhibitory effect, whereas IgG-rich eluate lost this property. The EPEC-absorbed fraction retained the inhibitory property. Industrialized milk formulas and respective HMWF also inhibited bacterial adherence. In IB assays, colostrum and milk samples from immunized animals recognized proteins of 30-40 kDa and 94 kDa, a molecular weight consistent with the adhesin intimin, in EPEC extracts. CONCLUSIONS: the inhibitory effect of EPEC adherence may be mediated by HMWF components, and IgG was not the only component responsible for this phenomenon.

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil.

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.

Human colostrum and serum contain antibodies reactive to the intimin-binding region of the enteropathogenic Escherichia coli translocated intimin receptor.

BACKGROUND: In Brazil, enteropathogenic Escherichia coli (EPEC) diarrhoea is endemic in young infants. A characteristic feature of EPEC adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching and effacing" (A/E) lesions on mammalian cells. Two genes directly involved in intimate adhesion, eae and tir, encode the adhesion molecule intimin and its translocated receptor Tir, respectively. The intimin-binding domain of Tir was recently mapped to the middle part of the polypeptide (Tir-M), and the amino (Tir-N) and carboxy (Tir-C) termini were found to be located within infected host cells. Recently, it was shown that colostrum samples from mothers living in Sao Paulo contain IgA-class antibodies reactive with a number of proteins associated with EPEC virulence. It has also been shown that patients infected with verocytotoxin-producing E. coli O157 can produce antibodies to Tir. In the current study antibody responses to the different Tir domains were analyzed in sera and colostrum samples collected in an EPEC-endemic area of Brazil. METHODS: Recombinant Tir, Tir-N, Tir-M, and Tir-C were expressed as His-tagged protein in E. coli BL21a and purified on nickel columns. Western blot analysis was used to investigate colostrum IgA- and serum IgG-class antibodies reactive with the Tir fragments. RESULTS: Anti-Tir IgG antibodies were detected in the serum of children, with (63%) or without (50%) diarrhoea. Anti-Tir IgA-class antibodies were detected in all the colostrum pools tested. With the use of both serum IgG- and colostrum IgA-class antibodies, an immunodominant domain of the Tir-polypeptide, Tir M, was identified. CONCLUSION: The intimin-binding region of Tir (Tir-M) is the immunodominant region of the polypeptide in humans. Both serum IgG-class and colostrum IgA-class antibodies reacted predominantly with the Tir-M domain.

Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon.

Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells. In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca. 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili. An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes. These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.

Diffusely adhering Escherichia coli (DAEC) strains of fecal origin rarely express F1845 adhesin.

A total of 398 diffusely adhering Escherichia coli (DAEC) strains of fecal origin were analyzed for the presence of sequences homologous to the structural subunit gene (daaE) of the F1845 fimbria. For that purpose, a DNA fragment homologous to daaE, obtained by PCR, was used as a probe in colony hybridization assays. Only two strains carried daaE and expressed F1845, suggesting that this fimbria is rare among DAEC strains.

Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups.

The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis. The invasive strains were isolated in different parts of the world and most of them recovered from dysentery. Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages. These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes. The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains.

Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli.

Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

Virulence properties of atypical EPEC strains.

Virulence properties of 31 atypical enteropathogenic Escherichia coli (EPEC) strains isolated from cases of diarrhoea were examined. All except two strains adhered to HEp-2 cells in a localised adherence-like (LAL) pattern. With the exception of two strains, all were fluorescent actin staining (FAS) positive. Gentamicin HEp-2 invasion assay studies showed that all strains were invasive. Transmission electron microscopy of infected HEp-2 cells showed the characteristic attaching and effacing lesion and invasion of the cultured cells. Of the nine strains that hybridised with a DNA probe for alpha-haemolysin, five were haemolytic within 3 h of incubation, while the remaining strains were haemolytic only after incubation for 24 h. Three strains produced enterohaemolysin on blood agar. None of the 31 strains of E. coli induced fluid accumulation in the rabbit intestinal loop assay or displayed cytotoxic effects in HeLa and Vero cells. All the strains belonging to serotypes O26:H11, O26:H- and 0119:H2 expressed intimin beta, whereas all the strains from serotype O55:H7 expressed intimin gamma. The strains belonging to serogroup O111 expressed a non-typable intimin. The participation of intimin in LAL was supported by adhesion inhibition experiments in which antibodies to intimin significantly reduced the level of LAL.