INTIMIN-DEPENDENT BINDING OF ENTEROPATHOGENIC ESCHERICHIA-COLI TO HOST-CELLS TRIGGERS NOVEL SIGNALING EVENTS, INCLUDING TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA-1

Enteropathogenic Escherichia coli (EPEC) interactions with HeLa epithe lial cells induced the tyrosine phosphorylation of a host protein of a pproximately 150 kDa, Hp150. Phosphorylation of this protein hand was dependent on the interaction of the EPEC protein intimin with epitheli al cell surfaces and was correlated with pedestal formation, Hp150 pho sphorylation was specifically inhibited by the addition of cytochalasi n D, an inhibitor of actin polymerization, although this appeared to b e an indirect effect preventing interaction of intimin with its recept or, tyrosine-phosphorylated Hp90, and thus triggering Hp150 phosphoryl ation. This suggests the involvement of an actin-based movement of mem brane-bound tyrosine-phosphorylated Hp90 to allow its interaction with intimin. Analysis of the tyrosine-phosphorylated Hp150 protein demons trated that it is heterogeneous in composition, with phospholipase C-g amma 1 (PLC-gamma 1) being a minor component. Activation of PLC-gamma 1 by tyrosine phosphorylation leads to inositol triphosphate and Ca2fluxes, events defected following EPEC infection. EPEC also induced ty rosine dephosphorylation of host proteins, including a 240-kDa host pr otein (Hp240), following EPEC infection, Protein dephosphorylation app ears to be a signaling event which occurs independently of intimin. In hibition of host tyrosine dephosphorylation events by the addition of the tyrosine phosphatase inhibitor sodium vanadate did not pl:event ac tin accumulation beneath the adherent bacteria. We conclude that EPEC induces two sets of signaling events following infection, One set is d ependent on EPEC proteins secreted by the type III secretion pathway ( EspA and EspB) which induces Hp90 tyrosine phosphorylation and dephosp horylation of host phosphotyrosine proteins. The second set, which is also dependent on the first signaling events, requires intimin interac tion with its receptor, tyrosine-phosphorylated Hp90, to trigger Hp150 and PLC-gamma 1 tyrosine phosphorylation as well as pedestal formatio n. Inhibition of pedestal formation by tyrosine kinase inhibitors indi cates an important role for tyrosine phosphorylation events during EPE C subversion of host processes.