Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection

Jacob W. Ijdo, Adam C. Carlson, Elizabeth L. Kennedy

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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.

Original languageEnglish (US)
Pages (from-to)1284-1296
Number of pages13
JournalCellular Microbiology
Issue number5
StatePublished - May 2007
Externally publishedYes


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