texas-red and monodansylcadaverine

HEp-2 cells internalize non-pathogenic Escherichia coli bacteria by a low-efficiency internalization mechanism which is upregulated in Pho-derepressed strains (as shown by Sinai and Bavoil in 1993), and is independent of microfilament integrity but requires functional microtubules. Here, we further characterize the microtubule requirement of this pathway using various effectors of microtubule integrity and function. Furthermore, we show that internalization is enhanced upon treatment with monodansylcadaverine, a specific inhibitor of receptor mediated endocytosis, and is insensitive to brefeldin A, which promotes the microtubule-dependent reorganization of the endosome. An assay system is also described to directly evaluate the contribution of pinocytosis to this pathway based on the ability of the bacteria to cointernalize and consequently colocalize with the fluid-phase marker, Texas-red-conjugated dextran (TRD). Using this assay, Hoescht-stained bacteria were observed in TRD-containing vesicles in numbers that are consistent with their observed internalization rate. Overall, these data are strongly supportive of the existence of a low-efficiency macropinocytic mechanism of entry for these non-pathogenic bacteria. Moreover, the observed requirements for host tyrosine kinase and protein kinase C activities suggest that it is inducible.

Topics: Antifungal Agents; Antineoplastic Agents; Brefeldin A; Cadaverine; Cyclopentanes; Endocytosis; Epithelial Cells; Epithelium; Escherichia coli; In Vitro Techniques; Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Pinocytosis; Protein-Tyrosine Kinases; Xanthenes