Rab27a via its effector JFC1 localizes to Anaplasma inclusions and promotes Anaplasma proliferation in leukocytes

Abstract

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes tick-borne zoonosis called human granulocytic anaplasmosis. Mechanisms by which Anaplasma replicates inside of the membrane-bound compartment called “inclusion” in neutrophils are incompletely understood. A small GTPase Rab27a is found in the secretory granules and multivesicular endosomes. In this study we found Rab27a-containing granules were localized to Anaplasma inclusions in guanine nucleotide-dependent manner, and constitutively active Rab27a enhanced Anaplasma infection and dominant-negative Rab27a inhibited Anaplasma infection. Rab27a effector, JFC1 is known to mediate docking/fusion of Rab27a-bearing granules for exocytosis in leukocytes. shRNA stable knockdown of Rab27a or JFC1 inhibited Anaplasma infection in HL-60 cells. Similar to Rab27a, both endogenous and transfected JFC1 were localized to Anaplasma inclusions by immunostaining or live cell imaging. The JFC1 C2A domain that binds 3′-phosphoinositides, was sufficient and required for JFC1 and Rab27a localization to Anaplasma inclusions which were enriched with phosphatidylinositol 3-phosphate. Nexinhib20, the small molecule inhibitor specific to Rab27a and JFC1 binding, inhibited Anaplasma infection. Taken together, these results imply elevated phosphatidylinositol 3-phosphate in the inclusion membrane recruits JFC1 to mediate Rab27a-bearing granules/vesicles to dock/fuse with Anaplasma inclusions, the lumen of which is topologically equivalent to the exterior of the cell to benefit Anaplasma proliferation.