The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InIB protein is sufficient for entry into mammalian cells, stimulation ofPI 3-kinase and membrane ruffling

The Listeria monocytogenes InIB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, in cluding hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 a nd HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton, We previ ously showed that InIB is sufficient for entry of InIB-coated latex beads i nto cells and recent results indicate that purified InIB can stimulate PI 3 -kinase activity and is thus the first bacterial agonist of this lipid kina se, In this study, we identified the region of InIB responsible for entry a nd stimulation of signal transduction events. Eight monoclonal antibodies d irected against InIB were raised and, of those, five inhibited bacterial en try. These five antibodies recognized epitopes within the leucine-rich repe at (LRR) region and/or the inter-repeat (IR) region. InIB-staphylococcal pr otein A (SPA) fusion proteins and recombinant InIB derivatives were generat ed and tested for their capacity to mediate entry into cultured mammalian c ells. All the InIB derivatives that carried the amino-terminal 213-amino-ac id LRR region conferred invasiveness to the normally non-invasive bacterium L. innocoa or to inert latex beads and the corresponding purified polypept ides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoske leton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRR s similar to those of InIB, Taken together, these results show that the fir st 213 amino acids of InIB are critical for its specific properties.