Legionella pneumophila invasion of MRC-5 cells induces tyrosine protein phosphorylation

After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like ac tin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton, Dysregulation of the cellular phosphorylatio n and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins during intracellular growt h of L. pneumophila. By using an anti-tyrosine phosphoprotein antibody we s howed that proteins phosphorylated on tyrosine residues accumulated progres sively during late infection exclusively around or in phagosomes filled wit h bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cel ls were treated with cycloheximide, an inhibitor of eukaryotic protein synt hesis. The newly synthesized proteins were labeled metabolically with [S-35 ]methionine-cysteine and immunoprecipitated with a phospho tyrosine-specifi c antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for sy nthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a t yrosine kinase inhibitor, revealed that tyrosine protein phosphorylation wa s not important for bacterial uptake but contributed to intracellular growt h of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed in tracellular structural changes may be important to understanding the proces s involved in intracellular growth of L. pneumophila.