LOW-EFFICIENCY (MACRO-)PINOCYTIC INTERNALIZATION OF NONPATHOGENIC ESCHERICHIA-COLI INTO HEP-2 CELLS

HEp-2 cells internalize non-pathogenic Escherichia coli bacteria by a low-efficiency internalization mechanism which is upregulated in Pho-d erepressed strains (as shown by Sinai and Bavoil in 1993), and is inde pendent of microfilament integrity but requires functional microtubule s. Here, we further characterize the microtubule requirement of this p athway using various effecters of microtubule integrity and function. Furthermore, we show that internalization is enhanced upon treatment w ith monodansylcadaverine, a specific inhibitor of receptor mediated en docytosis, and is insensitive to brefeldin A,which promotes the microt ubule-dependent reorganization of the endosome. An assay system is als o described to directly evaluate the contribution of pinocytosis to th is pathway based on the ability of the bacteria to cointernalize and c onsequently colocalize with the fluid-phase marker, Texas-red-conjugat ed dextran (TRD). Using this assay, Hoescht-stained bacteria were obse rved in TRD-containing vesicles in numbers that are consistent with th eir observed internalization rate. Overall, these data are strongly su pportive of the existence of a low-efficiency macropinocytic mechanism of entry for these non-pathogenic bacteria. Moreover, the observed re quirements for host tyrosine kinase and protein kinase C activities su ggest that it is inducible.