Characterization of HEp-2 cell projection formation induced by diffusely adherent Escherichia coli

Diffusely adherent Escherichia coli (DAEC) are diarrheagenic E. coli whose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Preincubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be "embedded' within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon TnphoA mutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.