PASTEURELLA-MULTOCIDA ENTERS POLARIZED EPITHELIAL-CELLS BY INTERACTING WITH HOST F-ACTIN

We investigated the interaction of an avian strain of Pasteurella mult ocida with the cytoskeleton of MDCK cells, which formed a polarized ep ithelium when grown on type I collagen coated filters. Bacteria were i ncubated with MDCK cells for 30 min, 2, 4 and 6 hours and their locati on and association with the cell cytoskeleton determined by double-lab el immunofluorescence confocal microscopy. Cells were stained with a p olyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) ac tin. Confocal microscopy revealed that bacteria entered the cells by 3 0 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Ele ctron microscopy demonstrated that by 2 hours, each bacterium was asso ciated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection di srupted the actin cytoskeleton and inhibited entry of P. multocida.