title: Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection.
abstract: Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosine-phosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4-dependent type IV secretion (T4S) system. Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl-interactor 1 (Abi-1), an adaptor protein that interacts with Abl-1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl-1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti-AnkA antibody, Abl-1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl-1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular alpha-proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl-1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA.
authors.forename: Mingqun
authors.initials: M
authors.lastname: Lin
authors.forename: Amke
authors.initials: A
authors.lastname: den Dulk-Ras
authors.forename: Paul J J
authors.initials: PJ
authors.lastname: Hooykaas
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D041081:Anaplasma phagocytophilum / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016873:Ehrlichiosis / Q000382:microbiology; D018922:HL-60 Cells; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014443:Tyrosine; D016315:Proto-Oncogene Proteins c-abl