Anaplasma phagocytophilum is an intracellular pathogen that infects and survives in neutrophilic granulocytes. The A. phagocytophilum genome encodes a type four
secretion system (T4SS) that may facilitate intracellular survival by translocation of
virulence factors, but to date, no such factors have been identified. Because T4SS-translocated
proteins of several intracellular organisms undergo
tyrosine phosphorylation by host cell
kinases, we investigated
tyrosine phosphorylation of A. phagocytophilum
proteins during
infection. Within minutes after incubation of A. phagocytophilum with HL-60 cells or PMN, a 190 kDa
bacterial protein, AnkA, was increasingly
tyrosine-phosphorylated. A. phagocytophilum binding to host cells without entry was sufficient for AnkA
tyrosine phosphorylation. An in vitro
Src kinase assay demonstrated that purified AnkA expressed in Escherichia coli was phosphorylated at tyrosines located at the C-terminal portion of AnkA. Similarly, AnkA expressed in COS-7 cells underwent
tyrosine phosphorylation by Src at the C-terminus. The phosphorylated tyrosines were located in EPIYA motifs that display the consensus sequence for binding to SH2 domains. Immunoprecipitation studies demonstrated AnkA binding to the host cell
phosphatase SHP-1 during early
infection. Phosphorylation of the EPIYA motifs and the presence of the SH2 domains were necessary for AnkA-SHP-1 interaction. We conclude that AnkA is a translocated
virulence factor that is
tyrosine-phosphorylated by host cell
kinases upon translocation into the host cell early during
infection. A. phagocytophilum may manipulate the host cell through SHP-1 recruitment.