CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is an intestinal attaching and effacing pathogen that utilizes a type III secretion system (T3SS) for the delivery of effectors into host cells. The chaperone CesT has been shown to bind and stabilize the type III translocated effectors Tir and Map in the bacterial cytoplasm prior to their delivery into host cells. In this study we demonstrate a role for CesT in effector recruitment to the membrane embedded T3SS. CesT-mediated effector recruitment was dependent on the presence of the T3SS membrane-associated ATPase EscN. EPEC DeltacesT carrying a C-terminal CesT variant, CesT(E142G), exhibited normal cytoplasmic Tir stability function, but was less efficient in secreting Tir, further implicating CesT in type III secretion. In vivo co-immunoprecipitation studies using CesT-FLAG containing EPEC lysates demonstrated that CesT interacts with Tir and EscN, consistent with the notion of CesT recruiting Tir to the T3SS. CesT was also shown to be required for the efficient secretion of several type III effectors encoded within and outside the locus of enterocyte effacement (LEE) in addition to Tir and Map. Furthermore, a CesT affinity column was shown to specifically retain multiple effector proteins from EPEC culture supernatants. These findings indicate that CesT is centrally involved in recruiting multiple type III effectors to the T3SS via EscN for efficient secretion, and functionally redefine the role of CesT in multiple type III effector interactions.     

Atypical Shigella boydii 13 encodes virulence factors seen in attaching and effacing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes watery diarrhea and hemorrhagic colitis. In this study, we identified StcE, a secreted zinc metalloprotease that contributes to intimate adherence of EHEC to host cells, in culture supernatants of atypical Shigella boydii 13 (Shigella B13) strains. Further examination of the Shigella B13 strains revealed that this cluster of pathogens does not invade but forms pedestals on HEp-2 cells similar to EHEC and enteropathogenic E.?coli. This study also demonstrates that atypical Shigella B13 strains are more closely related to attaching and effacing E.?coli and that their evolution recapitulates the progression from ancestral E.?coli to EHEC.     

Prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria

AIMS: Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria. METHODS AND RESULTS: Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs cm(-2). For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analysed harboured at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2.9%) positive by colony hybridization were isolated. The majority (60.6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype (eae+ stx-). Only three enterohaemorrhagic E. coli (EHEC)-like (eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. CONCLUSIONS: In this study, the global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of this type in Algeria, which indicates that the general hygiene of the slaughterhouse must be improved.     

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

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 NleA₄₇₉₅-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 nleA₄₇₉₅ in the genome of an Stx1 prophage.     

Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium . We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.     

Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes.

Attaching and effacing Escherichia coli is a new causal agent of diarrhea in calves. Its major virulence factors are the intimin protein, encoded by the eaeA gene, and the Shiga-like toxins, encoded by slt genes. Because the sequences of these genes are available, we selected specific primers to amplify each virulence gene so as to develop a new identification test based on multiplex amplification of virulence-associated genes. Of 30 tested strains, 14 were eaeA+, 15 were eaeA+ slt-I+, 1 was eaeA+ slt-I+ slt-II+, and 1 was eaeA+ slt-II+. The method proved in our hands to be fast and specific and in perfect correlation with the hybridization method.     

Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221

We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221. When comparing the predicted amino acid sequence of the eae_DEPEC to that of the Eae proteins from enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E. coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion. Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein. We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221. Binding of MBP-Eae_DEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy. In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-Eae_DEPEC fusion protein.     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .     

Development of an enzyme-labeled oligonucleotide probe for detecting the Escherichia coli attaching and effacing A gene.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) can produce attaching and effacing (AE) lesions on intestinal epithelium in vitro and in vivo. A gene necessary to cause the AE lesion has been identified and designated Escherichia coli attaching and effacing A (eaeA) gene. In this study, an alkaline phosphatase (ALP)-conjugated oligonucleotide probe for the eaeA gene was developed and used to detect the eaeA gene among 163 strains of classical EPEC and 25 strains of EHEC O157. The prevalence rates of eaeA gene in the strains of classical EPEC and EHEC O157 were 51.5 and 100%, respectively. The eaeA-positive rate (60.0%) in strains of class I EPEC serogroups (O26, O55, O86, O111, O119, O125, O126, O127, O128ab, and O142) was significantly higher than that (22.9%) in strains of the class II EPEC serogroups (O18, O44, O114) (P<0.01). A total of 109 eaeA-positive classical EPEC and EHEC O157 were positive for fluorescent actin staining (FAS) assay, whereas 79 eaeA-negative classical EPEC were negative. Both the sensitivity and specificity of the eaeA probe versus the FAS assay positivity were 100%. Thus, use of the ALP-conjugated oligonucleotide probe for the eaeA gene would be specific and reliable in identifying the adherence capability of EPEC and EHEC.     

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:H7 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7.     

Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.     

Fibronectin binding and cell-surface hydrophobicity of attaching effacing enteropathogenic Escherichia coli strains isolated from newborn and weanling rabbits with diarrhoea

Abstract 32 different strains of Escherichia coli isolated from rabbits with diarrhoea were studied for cell-surface properties which may be involved in intestinal colonisation. Strains isolated from diarrhoeic suckling (6 strains) and weaning (26 strains) rabbits which were shown to attach to brush borders in vivo, showed high relative cell-surface hydrophobicity as determined by the Salt Aggregation Test (SAT) when grown on Colonisation Factor Antigen (CFA) agar at 33°C. Cells of these strains grown to express surface hydrophobicity were also defined as high, moderate or low binders of ¹²⁵I-fibronectin or its ¹²⁵I-29-kDa fragment in a standard binding assay. Based on these findings, we propose that binding to intestinal cell surface (mucus)-associated fibronectin may be an early important step in intestinal colonisation of the small bowel in enteropathogenic E. coli (EPEC) diarrhoea in rabbits and other animal species.     

Shiga toxin-producing and attaching and effacing Escherichia coli in cats and dogs in a high hemolytic uremic syndrome incidence region in Argentina

Shiga toxin-producing Escherichia coli (STEC), responsible for the hemolytic uremic syndrome, is an endemic pathogen in Argentina. We studied the prevalence of STEC in fecal samples from cats and dogs of Buenos Aires city and suburbs. Cultures were used for screening stx1/stx2 and rfbO157 by multiplex PCR. All E. coli-positive colonies for these genes were further characterized for the eae gene and for serotypes. In dogs, 17 (3.7%), 19 (4.2%) and 34 (7.5%) of samples were positive for stx2, stx1 and rfb, respectively. In cats, six (4.0%) of the samples were positive for stx2, three (2.0%) for stx1 and four (2.7%) for rfbO157. In 18 (4.0%) of the dog samples, a bacteriological diagnosis was obtained by isolation. The percentage of positive isolates corresponding to the rfbO157 and to the stx2 genotypes were 2.9% and 1.1%, respectively. In four of the cat samples, the bacteriological diagnosis for stx2 (2.6% prevalence of STEC) was confirmed. Although these data suggest that the high infection index of STEC in children in Argentina does not seem to be due mainly to the role of cats and dogs, there are some strains with virulence genes in common for humans and their domestic animals.     

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.     

Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacing Escherichia coli infections

Using a porcine ileal in vitro organ culture model, we have demonstrated that egg yolk-derived antibodies specific for the attaching and effacing Escherichia coli (AEEC) virulence factors intimin and translocated intimin receptor (Tir), but not those specific for the AEEC-secreted proteins EspA, EspB and EspD, significantly reduced the bacterial adherence of the porcine enteropathogenic E. coli strain ECL1001, formerly 86-1390. Moreover, antibodies specific for intimin and Tir also significantly reduced bacterial adherence of heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli strains, as well as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we demonstrated that the oral administration of these anti-intimin antibodies significantly reduced the extent of attaching and effacing lesions found in the small intestine of weaned pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Overall, our results underline the potential use of specific egg yolk-derived antibodies as a novel approach for the prevention of AEEC infections.     

Terminal reassortment drives the quantum evolution of type III effectors in bacterial pathogens

Many bacterial pathogens employ a type III secretion system to deliver type III secreted effectors (T3SEs) into host cells, where they interact directly with host substrates to modulate defense pathways and promote disease. This interaction creates intense selective pressures on these secreted effectors, necessitating rapid evolution to overcome host surveillance systems and defenses. Using computational and evolutionary approaches, we have identified numerous mosaic and truncated T3SEs among animal and plant pathogens. We propose that these secreted virulence genes have evolved through a shuffling process we have called "terminal reassortment." In terminal reassortment, existing T3SE termini are mobilized within the genome, creating random genetic fusions that result in chimeric genes. Up to 32% of T3SE families in species with relatively large and well-characterized T3SE repertoires show evidence of terminal reassortment, as compared to only 7% of non-T3SE families. Terminal reassortment may permit the near instantaneous evolution of new T3SEs and appears responsible for major modifications to effector activity and function. Because this process plays a more significant role in the evolution of T3SEs than non-effectors, it provides insight into the evolutionary origins of T3SEs and may also help explain the rapid emergence of new infectious agents.