Mutations in the Histone-Like Nucleoid Structuring Regulatory Gene (hns) Decrease the Adherence of Shiga Toxin-Producing Escherichia coli 091:H21 Strain B2F1 to Human Colonic Epithelial Cells and Increase the Production of Hemolysin

摘要

Non-0 157 serotype Shiga toxin-producing E. coli (STEC) cause a significant amount of food-borne hemorrhagic colitis (HC) and life-threatening hemolytic uremic syndrome (HUS) worldwide and yet these strains do not encode intimin a major adherence factor encoded by the most common serotype 0157:117 isolated from documented cases of HC and HUS. In this investigation. we attempted to isolate specific adhesins critical for attachment of intimin- negative STEC 091:H21 to human colonic epithelial cells. Transposon mutagenesis of B2F1 was accomplished with the mini-Tn5phoACm mobile element and a mutant bank of B2F1 colonies that carried putative in-frame PhoA-positive transposon insertions was isolated. The bank was screened for mutants unable to adhere to the human colonic epithelial T84 cell line in both its nonpolarized and polarized states. An adherence mutant designated as 34.7 was identified that did not bind to these cells. Subsequently, mutant 34.7 was found to contain an out-of-frame insertion of the transposon into hns. The hns gene encodes the H- NS protein that has been reported to modulate the expression of various housekeeping as well as virulence-associated genes encoded by E. coli and other gram-negative organisms. Disruption of hns in 34.7 not only caused reduced adherence to T84 cells but resulted in over- production of STEC-hemolysin encoded on the large 90 kb plasmid carried by this strain. When H-NS was supplied to 34.7 in trans on a moderate copy number plasmid, both the reduced adherence phenotype and the hyper-hemolytic phenotype reverted to wild-type. Because hemolysin can destroy the T84 monolayer over the course of the adherence assay, it was difficult to assess the direct effect of the hns mutation on adherence of B2F1 to T84 cells. Hence, we sought to separate the reduced adherence phenotype from the hyper-hemolytic phenotype exhibited by mutant 34.7.