Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

Abstract

There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA 15 (gene from OI-15) and aidA 48 (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA 48 had no effect on adherence, whereas deletion of aidA 15 resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.

Involvement of Quorum Sensing and Heat-Stable Enterotoxin a in Cell Damage Caused by a Porcine Enterotoxigenic Escherichia coli Strain

“…For each RNA sample, genomic DNA contamination was examined by a PCR assay with primers specific to a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapA) (8), after DNase I (Ambion, TX) digestion. RNA integrity was verified by visualization in an agarose gel (38). The concentration of total RNA was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).…”

Verotoxin 2 Enhances Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Intestinal Epithelial Cells and Expression of β1-Integrin by IPEC-J2 Cells

“…Both media were supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 g/ml). The adherence assays and the quantification of adherence were conducted as previously detailed, with approximately 10 7 bacteria of an overnight culture of each strain cultured in 3 ml BHIN for 16 to 18 h without shaking (30). Briefly, the cells were infected for 6 h at 37°C under 5% CO 2 with a medium change at 3 h, washed with phosphatebuffered saline (PBS) to remove unbound bacteria, fixed with 70% methanol for 10 min, and stained with 1:40 Giemsa (Sigma) for 30 min.…”

“…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 (22,30). However, this role is still controversial.…”

“…In a previous study, we showed that a nalidixic acid-resistant (Nal r ) vt2 insertion mutant caused a reduction in the level of adherence of EHEC O157:H7 strain 86-24 to IPEC-J2 and HEp-2 cells (30). In the present study, new vt2 deletion and vt2 phage-cured mutants were generated in Nal-sensitive strain 86-24, and the effects of the mutations on the adherence of EHEC O157:H7 to IPEC-J2 cells were assessed and compared to their effects on adherence to HEp-2 and CaCo-2 cells.…”

The IPEC-J2 Cell Line

“…However, various EHEC mutants adhere differently to IPEC-J2 cells and porcine ileal loops. This indicates that different model systems may behave differently (Yin et al 2009 ). Nonetheless, the ability of some EHEC mutants to adhere to IPEC-J2 cells and ileal loops was highly correlated.…”