The Effect Of VT2 On The Pathogenesis Of Enterohemorrhagic E.Coli O157:H7

Enterohemorrhagic E. coli (EHEC) is a subgroup of verotoxin-producing E. coli (VTEC), also referred to as Shiga-toxin-producing E. coli (STEC). Serotype O157:H7 is the prototypic EHEC, associated with large outbreaks of disease in North America due to the ingestion of food contaminated with feces from cattle, which are the primary reservoir for EHEC O157:H7. The disease syndromes caused by EHEC infection include mild diarrhea to hemorrhagic colitis (HC) and, in the most severe cases, the hemolytic uremic syndrome (HUS). The key virulence factors for EHEC are a phage-encoded verotoxin (VT) and adherence mediated by the LEE (locus for enterocyte effacement) pathogenicity island (PAI). The LEE encodes proteins responsible for the attaching and effacing lesion (AE), which is characterized by intimate adhesion of the pathogen to the enterocyte surface, formation of an actin-rich pedestal on the host cell surface beneath the bacterium, and destruction of brush border microvilli. The LEE PAI consists of five major operons (LEE1, LEE2, LEE3, tir/LEE5 and LEE4) that encode a typeâ…¢secretion system involved in secretion of translocon and effector proteins and the outer membrane surface adhesin intimin and its receptor Tir (translocated intimin receptor). In addition to binding to its primary receptor Tir, intimin also interacts with host cell intimin receptors (HIR), such asβ1-integrin and nucleolin, on the surface of enterocytes. It is hypothesized that prior to the formation of a stable intimin-Tir interaction, intimin binds to HIRs and brings the bacteria close enough for injection of Tir and other effectors into the host epithelial cells.Verotoxin (VT) has been implicated in promotion of adherence to and colonization of intestinal epithelial cells by enterohemorrhagic Escherichia coli (EHEC) O157:H7. The present study investigated the effect of VT2 on adherence of EHEC O157:H7 strain 86-24 to porcine jejunal (IPEC-J2), human colon (CaCo-2) and human laryngeal carcinoma (HEp-2) cell lines and on expression in IPEC-J2 cells of synthases forβ1-integrin and nucleolin, which are both implicated in bacterial adherence. The effect on expression of Gb3 synthase, the receptor for VT, was also examined. Data were obtained by adherence assays and quantitative reverse transcriptase PCR, using EHEC O157 strain 86-24, a vt2 deletion mutant, a vt2-phage negative strain, and complemented mutants in which the vt2 gene was restored. Compared with the parent and complemented mutant strains, the vt2-negative strains adhered significantly less to all three types of cells. Adherence of the wild type EHEC strain to IPEC-J2 cells was accompanied by increased expression ofβ1-integrin, nucleolin, and Gb3 synthase. IPEC-J2 cells in association with the wild type EHEC O157 or the complemented mutants expressed higher levels ofβ1-integrin than did cells associating with the vt2-negative strains or with no bacteria. Expression of nucleolin was decreased by association with the vt2-negative mutant but complementation failed to restore wild type expression. The data indicate that VT2 plays a role in adherence of EHEC O157:H7 to intestinal epithelial cells, possibly by increasing the expression of the host receptorβ1-integrin.Nucleolin is a multifunctional nuclear protein that can be expressed at the surface of many cell types and serves as a receptor for some viruses and intimin. Integrins are a large family of heterodimeric receptors that are associated with a wide range of cell to cell interactions. Intimin of enteropathogenic E. coli (EPEC) specifically binds toβ1 integrin. Immunostainedβ1 integrin clusters at the sites of bacterial adherence to porcine and bovine tissues, suggesting thatβ1 integrin potentially serves as a receptor for intimin during EHEC O157:H7 infection. VT, an AB₅ toxin that inhibits protein synthesis in eukaryotic cells, is responsible for the severe clinical manifestations of EHEC infection, such as HC and HUS. The toxins are internalized by receptor-mediated endocytosis after binding of the B pentamer to the target cell glycolipid receptor globotriaosylceramide (Gb3). Recent studies have indicated that VT2 also promotes EHEC colonization of the intestine by stimulating expression of nucleolin. However, this role is still controversial.In a previous study, we showed that a Nal^R vt2-insertion mutant caused a reduction in adherence of EHEC O157:H7 strain 86-24 to IPEC-J2 and HEp-2 cells. In the present study, new vt2-deletion and VT2-phage cured mutants were generated in a Nal-sensitive strain 86-24 and the effect of the mutants on adherence of EHEC O157:H7 to IPEC-J2 cells was assessed and compared to effects on adherence to HEp-2 and CaCo-2 cells. To further characterize the new in-vitro model of porcine jejunal IPEC-J2 cells for use in combination with pig gut-loops for the study of EHEC O157:H7 pathogenesis, we analyzed the effect of EHEC O157:H7 strain 86-24 and its isogenic vt2^- mutant on expression of nucleolin,β1-intergrin, and Gb3 synthase in IPEC-J2 cells.