The roles of ceramide and its derivatives in A. phagocytophilum pathogenesis

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Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium that causes the emerging and potentially fatal infection, human granulocytic anaplasmosis (HGA). The microbial-host interactions that facilitate Ap intracellular proliferation and dissemination have remained poorly characterized. We uncovered a novel role for the host bioactive sphingolipid, ceramide-1-phosphate (C1P), as a regulator of Golgi fragmentation. Specifically, C1P activates a PKC/Cdc42/JNK signaling axis that phosphorylates GRASP55 (Golgi reassembly stacking protein 55 kDa) to destabilize the trans-Golgi network (TGN) and amplify TGN anterograde traffic. We determined that Ap infection upregulates C1P synthesis and induces Golgi fragmentation in a C1P signaling-dependent manner. Multivesicular bodies (MVBs) are endosomal sorting stations that receive Golgi traffic. MVB limiting membrane invagination and scission deliver cargo into the MVB in intraluminal vesicles (ILVs). ILV membranes are enriched in cholesterol, sphingolipids, and sphingolipid metabolic enzymes that promote cholesterol release from ILVs. MVB fusion with the plasma membrane releases ILVs as exosomes, which induce a battery of responses in recipient cells including signaling, immunity, and inflammation. The importance of exosome- mediated cell-to-cell communication in infectious disease is just beginning to be appreciated. We discovered that Ap resides in a pathogen-modified MVB that receives sphingomyelin-rich TGN cargo as ILVs. The bacterium coopts MVB lipid metabolic machinery to parasitize sphingolipids and cholesterol, which is essential for its growth and conversion from its replicative to infectious form. Consistent with MVB exosome release, the Ap vacuole fuses with the plasma membrane in a Rab27a-dependent manner to disperse infectious progeny to naïve cells. We also found that Ap alters exosome content. In this competitive renewal, we will interrogate our hypothesis that Ap induces C1P to destabilize the Golgi and promote TGN-to-ApV trafficking of SM-rich vesicles that enables Ap to parasitize host lipids for proliferation and dissemination. We will also test our hypothesis that Ap modulates the proteomic and lipidomic content of ILVs that, when released as exosomes together with progeny bacteria, synergistically benefit infection of naïve cells and/or contributes to immunopathology associated with HGA. Completing the Aims herein will yield one of the most refined models for intracellular bacterial pathogenesis, further illuminate our newly discovered roles of C1P as both a regulator of Golgi stability and microbial target for host modulation, and further understanding of how exosomes contribute to pathogenic processes.