INTERNALIN-B IS ESSENTIAL FOR ADHESION AND MEDIATES THE INVASION OF LISTERIA-MONOCYTOGENES INTO HUMAN ENDOTHELIAL-CELLS

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In b oth instances, the infection occurs by the crossing of endothelial cel ls lining a physiological barrier, the blood-brain barrier or the tran splacental barrier. In this study, the ability of L. monocytogenes wil d-type EGD to invade human umbilical vein endothelial cells (HUVECs) w as evaluated using wild-type bacteria and isogenic Listeria mutants. H ere, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin a gene product. This was demonstra ted in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Third ly, entry of wild-type EGD could be blocked effectively with antibodie s to InlB. Fourthly, cell binding assays and flow cytometry with HUVEC s showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically incr eased their ability to invade HUVECs. In laser-scanning confocal micro scopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry proce ss by scanning electron microscopy revealed that both wild-type EGD an d a recombinant strain overexpressing only InlB enter HUVECs in a simi lar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.