Obligate intracellular bacteria are major causes of infectious disease worldwide in terms of incidence and severity. Deciphering mechanisms by which they parasitize host cell metabolites to survive will advance understanding of microbial pathogenesis and may lead to development of novel therapeutics for the infections that they cause. Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium that causes the potentially deadly zoonosis, human granulocytic anaplasmosis. We discovered that Ap hijacks sphingomyelin-rich vesicles from the trans-Golgi network (TGN) to its vacuole in a Rab10- dependent manner to drive conversion from its non-infectious to infectious form. Ap infectious progeny are enriched in the host sphingolipid, ceramide, which is produced by acid sphingomyelinase (ASMase)- mediated hydrolysis of sphingomyelin. Notably, host ASMase is also routed to the Ap vacuole (ApV). Knocking down or inhibiting Rab10 or ASMase arrests the Ap infection cycle, and Ap cannot productively infect ASMase-/- mice. Since Ap parasitizes TGN vesicles, an increase in TGN anterograde traffic would benefit infection. Indeed, we discovered that Ap induces this very phenomenon by upregulating host cell production of the bioactive sphingolipid, CERK-derived ceramide-1-phosphate (C1P). Conspicuously, C1P induces Golgi destabilization and anterograde traffic induction through its interaction with UVRAG. We hypothesize that Ap induces C1P formation at the Golgi, which recruits UVRAG to induce anterograde trafficking of Rab10-positive, sphingomyelin-rich vesicles that the ApV intercepts. We further posit that, at the ApV, hijacked ASMase converts sphingomyelin to ceramide, which drives infectious progeny production.
Aim 1 will determine why Rab10 is critical for Ap virulence.
Aim 2 will define the roles of ASMase and ceramide in Ap pathobiology.
Aim 3 will determine the roles of C1P and UVRAG in hijacking TGN traffic and Ap infection in vivo using novel transgenic mouse models. C1P?s role as a potent regulator of diverse cellular processes including cancer and inflammation has recently emerged. Here, we stand to illuminate for the first time that C1P also plays a critical role in infectious disease. The culmination of our studies will define novel and previously unsurmised mechanisms for ceramide parasitism by intracellular bacteria. If our hypotheses are validated, ASMase and CERK become targets for developing new generations of therapeutics against these types of pathogens. Overall, this work will have a broad and powerful impact.
By studying the emerging human pathogen, Anaplasma phagocytophilum, we discovered novel mechanisms by which it steals nutrients from its host cells to survive and cause disease. This project will determine specifically how A. phagocytophilum executes this task and why it is essential for infection. Our work has broad reaching implications, will advance understanding of microbial pathogenesis, and may foster developmentofnoveltreatmentsformultipleinfectiousdiseases.
