Deoxynivalenol (DON) is a common mycotoxin that contaminates cereals and their by-products. The gastrointestinal tract is the first physical barrier against ingested food contaminants. DON contributes to the loss of barrier function of the intestine through the decreased expression of claudin-4 protein, a tight junction protein. The mechanism by which DON alters the intestinal barrier function remains poorly characterized. Therefore, we investigated the involvement of mitogen-activated protein kinases (MAPK) in the DON-induced loss of barrier function. We first verified that 30 μmol/L of DON activated MAPK in a highly sensitive porcine intestinal epithelial cell line (IPEC-1). Inhibition of p44/42 extracellular signal-regulated kinase (ERK) phosphorylation, with 0.5 μmol/L of the specific MAPK pharmacological inhibitor U0126 for 2 h, restored the barrier function of the differentiated intestinal epithelial cell monolayers. The restoration of barrier function was evaluated by trans-epithelial electrical resistance measurements and tracer flux paracellular permeability experiments. The U0126 also restored the intestinal expression of claudin-4 protein, thereby demonstrating that MAPK activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function. Further experiments indicated that p44/42 ERK is not involved in the transcriptional regulation of claudin-4. In conclusion, we demonstrated that DON-induced activation of the p44/42 ERK signaling pathway inhibits the expression of claudin-4 protein, which leads to impaired intestinal barrier function. Given the high levels of DON in cereal grains, these observations of impaired barrier function have implications for human and animal health.

Epidemiological and phylogenetic associations of the pks genomic island of extraintestinal pathogenic Escherichia coli (ExPEC), which encodes the genotoxin colibactin, are incompletely defined. clbB and clbN (as markers for the 5 and 3 regions of the pks island, respectively), clbA and clbQ (as supplemental pks island markers), and 12 other putative ExPEC virulence genes were newly sought by PCR among 131 published E. coli isolates from hospitalized veterans (62 blood isolates and 69 fecal isolates). Blood and fecal isolates and clbB-positive and -negative isolates were compared for 66 newly and previously assessed traits. Among the 14 newly sought traits, clbB and clbN (colibactin polyketide synthesis system), hra (heat-resistant agglutinin), and vat (vacuolating toxin) were significantly associated with bacteremia. clbB and clbN identified a subset within phylogenetic group B2 with extremely high virulence scores and a high proportion of blood isolates. However, by multivariable analysis, other traits were more predictive of blood source than clbB and clbN were; indeed, among the newly sought traits, only pic significantly predicted bacteremia (negative association). By correspondence analysis, clbB and clbN were closely associated with group B2 and multiple B2-associated traits; by principal coordinate analysis, clbB and clbN partitioned the data set better than did blood versus fecal source. Thus, the pks island was significantly associated with bacteremia, multiple ExPEC-associated virulence genes, and group B2, and within group B2, it identified an especially high-virulence subset. This extends previous work regarding the pks island and supports investigation of the colibactin system as a potential therapeutic target.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.Although Escherichia coli belongs to the normal microflora present in the gastrointestinal tracts of most mammals and birds, certain E. coli strains have been associated with intestinal or extraintestinal infections. Among these pathogenic E. coli strains, enteropathogenic E. coli (EPEC) is a major cause of infant diarrhea in developing countries (for a recent review, see reference 51) and is a significant category of diarrheagenic E. coli in different animal species. In addition, EPEC is an important cause of morbidity and mortality in weaned rabbits (5,54,56). EPEC is also pathogenic in neonatal calves (20,53) and seems to be isolated more frequently in farms with recurrent diarrhea (7). In swine, EPEC is involved in cases of postweaning diarrhea (67). There is also increasing evidence for a diarrheagenic role of EPEC in dogs (16,64). Finally, EPEC has been isolated ...

SummaryEscherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.

Genomic diversity of enterohemorrhagic Escherichia coli strains

Comparing the genomes of O157 and non-O157 enterohemorrhagic Escherichia coli (EHEC) strains reveals the selective conservation of a large number of virulence determinants.

Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli.In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G 2 -M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential "cargo" genes or "morons" and therefore play a crucial role in the generation of genetic diversity within ExPEC.Cytolethal distending toxins (CDTs) were the first bacterial toxins that block the eukaryotic cell cycle that were described, and they suppress cell proliferation and eventually lead to cell death. CDTs represent an emerging toxin family, which was initially described for Escherichia coli but is actually widely distributed among a variety of pathogenic bacteria, including Shigella dysenteriae, Salmonella enterica serovar Typhi, Campylobacter spp., Helicobacter spp., Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, and Haemophilus ducreyi (31,34).A CDT is a tripartite holotoxin (28) in which CdtB is the active subunit and CdtA and CdtC form a heterodimeric subunit apparatus required for delivery of CdtB into the cell (8,23,24). The toxin of S. enterica serovar Typhi is an exception. This organism does not encode CdtA or CdtC; instead, the enzymatic CdtB is delivered directly into host cells by a bacterial internalization pathway (14). Nuclear entry of CdtB, which relies on an atypical nuclear localization signal, is crucial for the cytotoxic activity (25,27,29). CdtB has DNase I-like activity (10,15,23). Once in the host cell nucleus, CdtB induces DNA double-strand breaks in both proliferating and nonproliferating cells (13,15,26). Although pathogenic roles of CDT have been shown for chronic infection in mouse models (12, 39), CDT has not played a significant role in acute infection models tested to date (25,40,48,49). Nevertheless, CDT appears to be a virulence factor; accordingly, studying its spread is important.So far, five different CDTs have been reported for E. coli, and they were designated in order of publication. CDT-I (44) and CDT-II (38) were identified in enteropathogenic E. coli (EPEC) serotype O86:H34 and O128:NM strains, respectively. CDT-III was cloned from an...

SummaryAttaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non-polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, -B, -D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE-negative E2348/ 69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Espdependent but Eae (intimin)-independent alteration of the host cell cytoskeleton by certain EPEC strains.