Phenylpropionic acid metabolism: a marker for enteropathogenic Escherichia coli clonal group 2 strains.
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Biomarkers↗
Biomedical subjects
L R Trabulsi
Publications and source records attributed to L R Trabulsi.
Molecular and ultrastructural characterisation of EspA from different enteropathogenic Escherichia coli serotypes.
Enteropathogenic Escherichia coli (EPEC) encode a type III secretion system located on a pathogenicity island known as the locus for enterocyte effacement. Four proteins are known to be exported by this type III secretion system--EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor protein (Tir) required for intimin-mediated intimate attachment and attaching and effacing lesion formation. The espA gene is located within the locus for enterocyte effacement and the EspA polypeptide from the prototype EPEC strain E2348/69 (O127:H6) has recently been shown to be a component of a filamentous structure involved in bacteria-host cell interaction and locus for enterocyte effacement-encoded protein translocation involved in attaching and effacing lesion formation. In this study we have extended our investigation of EspA to strains belonging to other classical EPEC serotypes. DNA sequencing demonstrated that the espA gene from the different EPEC strains share at least 65% DNA identity. In addition, we detected morphologically and antigenically similar EspA filaments in all but one of the bacterial strains examined including recombinant, non-pathogenic E. coli expressing espA from a cloned locus for enterocyte effacement region (HB101(pCVD462)).
Amino Acid Sequence↗
Penicillin-binding proteins of pathogenic Escherichia coli strains.
The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [3H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by beta-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.
Anti-Bacterial Agents↗
Characterization of enteroinvasive Escherichia coli and Shigella strains by RAPD analysis.
Genetic variation of 33 enteroinvasive Escherichia coli (EIEC), 12 non-EIEC and 39 Shigella strains (representing the 4 species of this genus) was analyzed using the random amplified polymorphic DNA (RAPD) technique. Reproducible polymorphisms were generated and the combined data allowed us to construct a dendrogram using Jaccard's distance. Two main groups were obtained: one for Shigella and the other for EIEC and non-EIEC strains. The first group contained four clusters, one for each Shigella species. The second group contained one cluster for EIEC and another for non-EIEC strains. The main clusters encompassed many small clusters corresponding to different serotypes. It was possible to characterize each one of the 84 strains under study as well as the boundaries among Shigella species and between this genus and EIEC strains.
Bacterial Typing Techniques↗
Characterization of the locus of enterocyte effacement (LEE) in different enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing Escherichia coli (STEC) serotypes.
All proteins involved in the attachment and effacement lesion produced by enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing E. coli (STEC) are encoded by the locus of enterocyte effacement (LEE). We studied the presence and insertion site of the LEE in different EPEC and STEC strains. In serotypes O119:H6/H-, O55:H6, O55:H7, O142:H6, O111ac:H9/H-, O111ab:H9/H- LEE is inserted downstream of selC as previously described for EPEC O127:H6 and STEC O157:H7. In serotypes O111ac:H8/H- and O26:H11/H- the LEE is inserted in pheU as previously described for STEC O26:H-. However in EPEC from serotype O111ab:H25 the LEE is not inserted in either site suggesting a third insertion site in the K12 chromosome. We also cloned fragments of 2.3 kb and 1.0 kb from the right and left hand sides of the LEE of a O111ac:H- strain and identified additional insertion sequences on these LEE fragments, suggesting that the LEE may be larger and may have undergone more recombination events in these serotypes.
Bacterial Adhesion↗
Edwardsiella tarda induces plasma membrane ruffles on infection of HEp-2 cells.
Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated. By electron microscopy we observed at 1 h post infection that E. tarda induced formation of extensive plasma membrane projections resembling membrane ruffles. The ruffles did not coincide with adhering bacteria. Only few invading bacteria were seen. Vacuolated nuclear membrane was occasionally observed. Three hours post infection, E. tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus. Only one of the E. tarda strains was seen residing within the host cell remains. The results indicate that E. tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis.
Adult↗
Human colostrum contains IgA antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins: intimin, BfpA, EspA, and EspB.
BACKGROUND: In Brazil, enteropathogenic Escherichia coli diarrhoea is endemic among infants born into low economic levels, and it is one of the main causes of morbidity and mortality in this group. Binding of enteropathogenic E. coli to the brush border mucosa triggers a cascade of transmembrane and intracellular signals, causing cytoskeletal reorganization and formation of a specific lesion, termed the attaching and effacing lesion. Several enteropathogenic E. coli gene products have been implicated in formation of attaching and effacing lesions. Evaluation of pathogen-specific protective factors shows that breast feeding is effective against enteropathogenic E. coli infection. To investigate the nature of the protection, defatted colostrum and secretory immunoglobulin A obtained from mothers living in Sao Paulo were investigated for the ability to recognise selected enteropathogenic E. coli-associated virulence factors. METHODS: Western blot analysis was used to investigate the IgA repertoire in pooled colostrum that is reactive with specific enteropathogenic E. coli proteins. Whole enteropathogenic E. coli bacterial cell extracts, nonpathogenic E. coli strains overexpressing specific virulence factors, and purified polypeptides were used as antigen sources in this study. RESULTS: Reaction of the colostrum samples in Western blots of whole bacterial cell extracts and selected purified enteropathogenic E. coli proteins showed that they contained a secretory immunoglobulin A reactive with all the virulence-associated proteins studied. CONCLUSION: These results suggest that maternal antibodies may protect infants from enteropathogenic E. coli infection by interfering with adherence processes (anti-intimin and anti-bundle-forming pili antibodies) and cell signaling (anti-enteropathogenic Escherichia coli-secreted protein A and B antibodies.
Adhesins, Bacterial↗
Characterisation of diarrhoeagenic Escherichia coli clones by ribotyping and ERIC-PCR.
The ability of ribotyping and enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) to discriminate diarrhoeagenic Escherichia coli clones of 122 strains belonging to 26 distinct serotypes was evaluated. The 26 serotypes corresponded to 24 ribotypes and 25 ERIC-types. Correlation between multilocus enzyme electrophoresis, ERIC-PCR and ribotyping was c. 90% for the dominant ribotypes. Related clones such as O55:H7 and O157:H7 presented similar ribotypes and clustered together in a dendrogram, and the two divergent clonal groups of enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) were included in distinct branches. The results suggest the possibility of applying these two simpler techniques as tools to identify clones of diarrhoeagenic E. coli.
Bacterial Typing Techniques↗
Identification of immunodominant regions within the C-terminal cell binding domain of intimin alpha and intimin beta from enteropathogenic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) strains are a common cause of infantile diarrhea in developing countries. EPEC strains induce a characteristic attaching and effacing (A/E) lesion on epithelial cells. A/E lesion formation requires intimin, an outer membrane adhesin protein. The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of the polypeptide (Int280). So far, four distinct Int280 types (alpha, beta, gamma, and delta) have been identified. The aim of this study was to identify immunodominant regions within the Int280alpha and Int280beta domains. Recombinant DNA was used to construct and express overlapping polypeptides spanning these domains. Rabbit anti-Int280 antisera and human colostral immunoglobulin A were reacted with these polypeptides in Western blots and enzyme-linked immunosorbent assays. The results obtained with the rabbit antisera showed the presence of two separate immunodominant regions which are common to both Int280alpha and Int280beta. The first localized within the N-terminal region of Int280, and the second localized between amino acids 80 and 130. The results with the human colostra revealed one reactivity pattern against the Int280alpha fragments but two different reactivity patterns against the Int280beta domain.
Adhesins, Bacterial↗
Increased levels of intracellular calcium are not required for the formation of attaching and effacing lesions by enteropathogenic and enterohemorrhagic Escherichia coli.
Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]i are not required for A/E lesion formation by EPEC and EHEC.
Calcium↗
Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens.
Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.
Adhesins, Bacterial↗
The traditional enteropathogenic Escherichia coli (EPEC) serogroup O125 comprises serotypes which are mainly associated with the category of enteroaggregative E. coli.
Genotypic and phenotypic virulence markers of the different categories of diarrheagenic Escherichia coli were investigated in 76 strains of the enteropathogenic E. coli (EPEC) serogroup O125. The most frequent serotype found was O125ac:H21. None of the serotypes behaved as EPEC, i.e. carried the eaeA, bfpA, and EAF DNA sequences simultaneously and presented localized adherence to HeLa cells. All strains of O125ac:H6 were atypical EPEC since they carried eaeA only, and presented an indefinite pattern of adherence. All strains of O125ab:H9, O125ac:H9, O125?:H16, and O125ab:H21 and 79% of the O125ac:H21 strains were enteroaggregative E. coli, since they carried a specific DNA sequence and presented the typical aggregative adherence pattern.
Diarrhea, Infantile↗
Virulence properties of Escherichia coli O111:H12 strains.
Seventeen Escherichia coli O111:H12 strains isolated from the feces of children with acute diarrhea were studied with regard to their adherence properties and other virulence characteristics. All strains showed an aggregative adherence pattern to HEp-2 cells and agglutinated bovine and sheep red cells in the presence of mannose. These strains did not have gene sequences homologous to the aggregative adherence fimbria I gene and did not react with any of the DNA probes used to detect other virulence genes in enteropathogens. With one exception, the O111:H12 strains did not induce fluid accumulation in the rabbit ileal loop assay, although 16 of the strains had the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST) gene sequences. A 60-70 MDa plasmid was present in 16 of the strains studied. We conclude that the O111:H12 serotype, one of the first E. coli identified in infantile diarrhea, belongs to the enteroaggregative E. coli category but the genes encoding its adherence phenotype are distinct from those previously described.
Animals↗
Colostral mononuclear phagocytes are able to kill enteropathogenic Escherichia coli opsonized with colostral IgA.
Enteropathogenic Escherichia coli (EPEC) is the main aetiological agent of acute diarrhoea among low socioeconomic level infants in developing countries. Breast-feeding provides infant protection against acute gastrointestinal and respiratory infections; however, little is known about the protective role of colostral phagocytes in the gut of newborn infants. In the present investigation we studied the ability of human colostral MN phagocytes to kill EPEC as well as the interactions between these cells and colostral and serum opsonins. The authors observed that the microbicidal activity of colostrum MN phagocytes was dependent on previous EPEC opsonization with colostral supernatant or blood serum. A defatted colostrum supernatant pool presented opsonic activity for EPEC killing at levels equivalent to those of normal serum. IgA-depleted colostrum supernatant showed significantly lower opsonic activity, whereas purified IgA from the same colostrum pool was a potent opsonin which induced EPEC killing at levels equivalent to those of untreated colostrum. Colostral MN phagocytes are able to release superoxide anion when incubated with both EPEC opsonized with untreated colostrum and purified IgA. Purified IgA was also able to restore opsonic activity of IgA-depleted colostrum. A colostrum pool without C3 and IgG induced EPEC killing by colostral MN phagocytes at rates equivalent to those of untreated colostrum supernatant. Addition of an IgM MoAb (My43) anti-human Fc alpha receptor resulted in a significant inhibition of EPEC killing when bacteria were opsonized with purified IgA, suggesting an interaction between IgA and Fc alpha R. With respect to serum opsonins, we observed that IgG plus complement component C3 were necessary to induce EPEC killing by the colostrum MN phagocytes. Colostral phagocyte killing of enteropathogenic bacteria may represent an additional mechanism of breast-feeding protein against intestinal infections during the first week of life.
Adolescent↗
Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes.
A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E. coli (EPEC). Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA). The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains. Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period. The second pattern was represented by strains which hybridized with the eaeA gene only. Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype). The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe. Most (80%) of these strains exhibited the LA-like phenotype. Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not. The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs). ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H-. The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype. The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype. Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.
Bacterial Adhesion↗
Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55.
Virulence properties and genetic variation as determined by multilocus enzyme electrophoresis were studied in 70 strains of Escherichia coli 055, a common serogroup of enteropathogenic E. coli (EPEC), a major cause of infantile diarrhea in developing countries. Nearly 40% of the strains were originally isolated in Brazil and represented serotypes 055:H6, 055:H7, and 055:H51 and nonmotile (055:H-) strains. The analysis of electrophoretic variants of 20 enzymes defined seven distinct electrophoretic types (ETs). ET 1 was represented by 41% of the strains, including strains which usually hybridized with DNA probes for the intimin gene (eaeA), the EPEC adherence plasmid (EAF), and the gene for the pilin subunit of the bundle-forming pilus (bfpA). The ET 1 strains were also typically serotype 055:H6, displayed localized adherence (LA) in tissue culture assays, and were positive in the fluorescent-actin staining test for intimate cell adherence. These same characteristics were observed in the closely related ETs 2 to 4, which clustered in the same branch as ET 1. No known virulence marker could be identified in ET 6. ET 5 included 23 strains, all of which carried the eaeA gene but otherwise displayed a striking array of distinct virulence traits. This ET was represented by 055:H7 strains with phenotypes as diverse as the simultaneous expression of LA and diffuse adherence and the ability to form a newly described adherence pattern, called LA-like adherence. The results suggest that ET 5 marks a special pathogenic clone with a propensity to acquire virulence factors which may facilitate the emergence of new pathogenic strains.
Brazil↗
Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA.
This paper demonstrates that human colostrum can inhibit the invasion of HEp-2 cells by enteroinvasive Escherichia coli (EIEC) of serotypes O28:H- and O29:H-, and that IgA antibodies mediate the inhibitory process. Seventy three of 77 (95.9%) colostrum samples prevented invasion of HEp-2 cells by E. coli O28:H-. Most of these samples contained high levels of IgA reactive to EIEC in immunoenzymatic assays. IgA eluted from an affinity chromatography column strongly inhibited HEp-2 invasion by EIEC, whereas IgA-depleted colostrum had no inhibitory effect on bacterial invasion. Immunoblots of colostrum samples with high (> 60%) invasion-inhibiting levels were performed with water extracts of invasive and noninvasive strains. Bacterial antigens from the invasive strain were recognized and the size of some was consistent with the invasion plasmid antigens (Ipas) A, B, C, and D, with stronger reactions with Ipas A and C. Colostrum samples with high inhibitory levels showed a strong reaction in Western blot assays, in contrast to the faint bands observed with poor-inhibitory samples. The results obtained in the present study suggest that colostrum IgA may protect infants against invasive E. coli infections.
Antibodies, Bacterial↗
Inhibition of enteropathogenic Escherichia coli adhesion to HeLa cells by serum of infants with diarrhea and by cord serum.
We have studied the effect of serum from infants with diarrhea and of cord serum on the localized adherence of enteropathogenic Escherichia coli (EPEC) to HeLa cells. Serum samples from 16 infants with diarrhea due to EPEC of serotypes O55:H6, O111: H-, O111:H2, O119:H6 and O142:H6 were used. The adherence ability of EPEC strains belonging to serotypes identical to (homologous) or different from (heterologous) those isolated from the infants' feces was highly inhibited by samples of infant serum collected both during the acute phase of the illness and upon discharge from the hospital. These data confirm the development of antibodies against EPEC adhesins and the cross-reaction between different EPEC serotypes. Cord serum inhibited the localized adherence of EPEC strains at different levels according to the serotype of the strain studied. These results suggest that the placental transfer of adhesin-related antibodies does not protect the newborn against EPEC infections, since half of our patients were less than 30 days old.
Bacterial Adhesion↗