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A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells.

ABSTRACT: The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% of E. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.

SUBMITTER: Jerse AE

PROVIDER: S-EPMC54845 | biostudies-other | 1990 Oct

REPOSITORIES: biostudies-other

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A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells.

Jerse A E AE Yu J J Tall B D BD Kaper J B JB

Proceedings of the National Academy of Sciences of the United States of America 19901001 20

The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% of E. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produc ...[more]

PMID: 2172966

Similar Datasets

Enteropathogenic Escherichia coli O125:H6 triggers attaching and effacing lesions on human intestinal biopsy specimens independently of Nck and TccP/TccP2.

Project description:Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.

| S-EPMC2223649 | biostudies-literature

Atypical enteropathogenic Escherichia coli that contains functional locus of enterocyte effacement genes can be attaching-and-effacing negative in cultured epithelial cells.

Project description:Enteropathogenic Escherichia coli (EPEC) induces a characteristic histopathology on enterocytes known as the attaching-and-effacing (A/E) lesion, which is triggered by proteins encoded by the locus of enterocyte effacement (LEE). EPEC is currently classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid, respectively. Here we analyzed the LEE regions of three aEPEC strains displaying the localized adherence-like (LAL), aggregative adherence (AA), and diffuse adherence (DA) patterns on HEp-2 cells as well as one nonadherent (NA) strain. The adherence characteristics and the ability to induce A/E lesions were investigated with HeLa, Caco-2, T84, and HT29 cells. The adherence patterns and fluorescent actin staining (FAS) assay results were reproducible with all cell lines. The LEE region was structurally intact and functional in all strains regardless of their inability to cause A/E lesions. An EspF(U)-expressing plasmid (pKC471) was introduced into all strains, demonstrating no influence of this protein on either the adherence patterns or the capacity to cause A/E of the adherent strains. However, the NA strain harboring pKC471 expressed the LAL pattern and was able to induce A/E lesions on HeLa cells. Our data indicate that FAS-negative aEPEC strains are potentially able to induce A/E in vivo, emphasizing the concern about this test for the determination of aEPEC virulence. Also, the presence of EspF(U) was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain via the direct or indirect activation of the LEE4 and LEE5 operons.

| S-EPMC3088124 | biostudies-literature

Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains.

Project description:In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa). The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta. Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.

| S-EPMC154638 | biostudies-literature

Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation.

Project description:Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.

| S-EPMC41273 | biostudies-other

Quantitative proteomic analysis of type III secretome of enteropathogenic Escherichia coli reveals an expanded effector repertoire for attaching/effacing bacterial pathogens.

Project description:Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.

| S-EPMC3434768 | biostudies-literature

Late establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli depends on protein expression regulated by Per.

Project description:Enteropathogenic Escherichia coli (EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC, a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC (perABC) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains.

| S-EPMC4288892 | biostudies-literature

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains.

Project description:Streptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Strr) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Strr mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Strr restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Strr should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.

| S-EPMC3754346 | biostudies-literature

Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models.

Project description:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3-4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

| S-EPMC3014348 | biostudies-literature

Genetic background and mobility of variants of the gene nleA in attaching and effacing Escherichia coli.

Project description:In this study, we characterized the genetic background of various nleA variants in 106 Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains. The flanking regions of eight nleA variants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzed nleA variants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genes avrA, ospB, nleH, and nleG and IS elements were detected in the neighborhood of nleA. The structure of the eight analyzed regions flanking nleA primarily resembled the corresponding region of the NleA????-encoding prophage BP-4795. Using PCR, the gene order flanking 13 nleA variants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particular nleA variant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilize nleA by transduction in 20% of strains determined, which comprised in particular EPEC strains harboring an nleA variant, the gene encoding the protein known as "EspI-like." Plaque hybridization was used to identify phages that harbor the genes stx and nleA. However, only two strains harbored variant nleA???? in the genome of an Stx1 prophage.

| S-EPMC3233075 | biostudies-literature

Prevalence of the paa gene (porcine attaching and effacing associated) in porcine enteropathogenic Escherichia coli (PEPEC) associated with postweaning diarrhea in south Brazil.

Project description:Paa (porcine attaching and effacing associated) may be an important virulence factor E. coli of piglets with diarrhea. This study showed for the first time in Brazil the prevalence of the paa gene (22%) in E. coli strains isolated from piglets and these isolates also harboured genes for other adhesins and toxins LT II, STa and STb.

| S-EPMC3833155 | biostudies-literature

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