LEISHMANIA-INDUCED TYROSINE PHOSPHORYLATION IN THE HOST MACROPHAGE AND ITS IMPLICATION TO INFECTION

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a varie ty of pathogens. We have investigated the involvement of protein tyros ine kinase activity in the Leishmania attachment, invasion and surviva l within macrophages, as well as promastigote ability to trigger tyros ine phosphorylation, which could contribute to leishmanicidal activity Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasit e invasion in a dose-dependent manner. Contrary, addition of sodium or thovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot anal ysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa followi ng macrophage challenge with promastigotes, Uninfected macrophages dis played no detectable tyrosine phosphorylated proteins, possibly indica ting an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leish mania lysates. Immunofluorescence and immunogold detection of phosphot yrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-in duced tyrosine phosphorylation is an early, local and short-lived even t. Genistein treatment of Leishmania-infected cells significantly enha nced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activat ed infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regu late the Leishmania promastigote invasion and the macrophage microbici dal activity.