IDENTIFICATION OF PUTATIVE CYTOSKELETAL PROTEIN HOMOLOGS IN THE PROTOZOAN HOST HARTMANNELLA-VERMIFORMIS AS SUBSTRATES FOR INDUCED TYROSINE PHOSPHATASE-ACTIVITY UPON ATTACHMENT TO THE LEGIONNAIRES-DISEASE BACTERIUM, LEGIONELLA-PNEUMOPHILA

The Legionnaires' disease bacterium, Legionella pneumophila, is a facu ltative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells . Invasion and intracellular replication within protozoa are thought t o be major factors in the transmission of Legionnaires' disease. We ha ve recently reported the identification of a galactose/N-acetyl-D-gala ctosamine (Gal/GalNAc) lectin in the protozoan host Hart-mannella verm iformis as a receptor for attachment and invasion by L. pneumophila (V enkataraman, C., B.J. Haack, S. Bondada, and Y.A. Kwaik. 1997.J. Exp. Med. 186:537-547). In this report, we extended our studies to the effe cts of bacterial attachment and invasion on the cytoskeletal proteins of H. vermiformis. We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125(FAK), paxillin, and vinculin, al l of which were basally tyrosine phosphorylated in resting H. vermifor mis. In addition to L. pneumophila-induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated. with tyrosine dephosphorylation of paxillin, pp125(FAK), and vinculin, wher eas actin was minimally affected. Inhibition of bacterial attachment t o H. vermiformis by Gal or GalNAc monomers blocked bacteria-induced ty rosine dephosphorylation of detergent-insoluble proteins. In contrast, inhibition of bacterial invasion but not attachment failed to block b acteria-induced tyrosine dephosphorylation of H. vermiformis proteins. This was further supported by the observation that 10 mutants of L. p neumophila that were defective in invasion of H. vermiformis were capa ble of inducing tyrosine dephosphorylation of H. vermiformis proteins. Entry of L. pneumophila into H. vermiformis was predominantly medial- ed by noncoated receptor-mediated endocytosis (93%) but coiling phagoc ytosis was infrequently observed (7%). We conclude that attachment but not invasion by L. pneumophila into H. vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H. vermifor mis. These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis. A model for attachment and entry of L. pneumophila into H. vermiformis is proposed.