Defense Date


Document Type


Degree Name

Doctor of Philosophy


Microbiology & Immunology

First Advisor

Jason A. Carlyon


Anaplasma phagocytophilum is the etiologic agent of the emerging infection, granulocytic anaplasmosis. This obligate intracellular bacterium lives in a host cell-derived vacuole that receives membrane traffic from multiple organelles to fuel its proliferation and from which it must ultimately exit to disseminate infection. Understanding of these essential pathogenic mechanisms has remained poor. A. phagocytophilum infection induces fragmentation of the Golgi apparatus to enhance parasitism of nutrient rich vesicles from this organelle. Multivesicular bodies (MVBs) are late endosomal compartments that receive biomolecules from organelles, such as the Golgi, and encapsulate them into intralumenal vesicles (ILVs) using endosomal sorting complexes required for transport (ESCRT)-dependent and ESCRT-independent machinery. Association of the ESCRT-independent protein, ALIX, directs MVBs to the plasma membrane where they release ILVs as exosomes. We report that A. phagocytophilum induces Golgi fragmentation to facilitate bacterial replication mediated by increased levels of ceramide-1-phosphate (C1P), a previously uncharacterized regulator of Golgi stabilization. We establish that the A. phagocytophilum vacuole (ApV) is acidified and enriched in lysobisphosphatidic acid, a lipid that is abundant in MVBs. ESCRT-0 and ESCRT-III components along with ALIX localize to the ApV membrane. siRNA-mediated inactivation of ESCRT-0 and ALIX together impairs A. phagocytophilum proliferation and infectious progeny production. RNA silencing of ESCRT-III, which regulates ILV scission, pronouncedly reduces ILV formation in ApVs and halts infection by arresting bacterial growth. Rab27a and its effector Munc13-4, which drive MVB trafficking to the plasma membrane and subsequent exosome release, localize to the ApV. Treatment with Nexinhib20, a small molecule inhibitor that specifically targets Rab27a to block MVB exocytosis, abrogates A. phagocytophilum infectious progeny release. Finally, we show that the ILVs within ApVs of infected cells are released as modified exosomes that preemptively trigger Golgi fragmentation in naïve cells. Thus, A. phagocytophilum modulates intracellular lipid homeostasis that induces Golgi fragmentation to drive delivery of nutrient rich vesicles to the ApV. We show that MVB biogenesis machinery is exploited by A. phagocytophilum for nutrient internalization and exocytosis. These strategies benefit each major stage of the A. phagocytophilum intracellular infection cycle: intravacuolar growth, conversion to the infectious form, and exit from the host cell.


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