Conditioned medium from enterohemorrhagic Escherichia coli-infected T84 cells inhibits signal transducer and activator of transcription 1 activation by gamma interferon.

Gamma interferon (IFN-gamma) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-gamma-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-gamma and protein extracts were analyzed by immunoblotting. The data showed that IFN-gamma-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-gamma receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-gamma signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.

Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function.

Intestinal bacteria play an etiologic role in triggering and perpetuating chronic inflammatory bowel disorders. However, the precise mechanisms whereby the gut microflora influences intestinal cell function remain undefined. Therefore, the effects of the non-pathogenic prototype translocating Escherichia coli, strain C25 on the barrier properties of human T84 and Madine-Darby canine kidney type 1 epithelial cells were examined. T-84 cells were also infected with commensal E. coil, strains F18 and HB101, and enterohaemorrhagic E. coli, serotype O157:H7. Strains F18 and HB101 had no effect on transepithelial electrical resistance (TER) of T84 monolayers. By contrast, epithelial cells infected with strain C25 displayed a time-dependent decrease in TER, preceded by an altered distribution of the cytoskeletal protein alpha-actinin, comparable to infection with E. coli O157:H7. E. coli C25 infection also led to activation of nuclear factor kappaB (NF-kappaB), interleukin-8 secretion and alterations in localization of claudin-1, but not zona occludens-1 or claudin-4, in T84 cells. There were adherent C25 bacteria on the intact apical surface of infected T84 cells, while mitochondria appeared swollen and vacuolated. These novel findings demonstrate the ability of a translocating commensal bacterium to adhere to and modulate intestinal epithelial barrier function and to induce morphological changes in a manner distinct from the known enteric pathogen, E. coli O157:H7.

Cholesterol-enriched membrane microdomains are required for inducing host cell cytoskeleton rearrangements in response to attaching-effacing Escherichia coli.

The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and enteropathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced alpha-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection.

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products--for example, adhesins and toxins--and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10 percent of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products - for example, adhesins and toxins - and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.