Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors.
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