Pathobiological roles of Anaplasma phagocytophilum inclusion membrane proteins

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DESCRIPTION (provided by applicant): Pathogen-occupied vacuoles (PVs) are host cell-derived endosomes/phagosomes that are converted by obligate vacuolar pathogens into unique organelles that serve as safe-havens sequestered from the normal endocytic continuum. Pathogen derived proteins that localize to the PV membrane (PVM) are essential for the construction and altered fusogenicity of PVs. Furthermore, pathogen-encoded PVM proteins serve as interfaces for PV interactions with host membrane traffic and signaling pathways. Thus, understanding the pathobiological roles of bacterial PVM proteins is key to understanding the survival mechanisms of intravacuolar pathogens. The functions of the vast majority of identified/predicted bacterial PVM proteins remain undefined, which represents a considerable gap in our knowledge of how obligate vacuolar pathogens manipulate their host cells to escape destruction. Anaplasma phagocytophilum is an obligate vacuolar bacterium that invades neutrophils to cause the emerging and potentially deadly disease, human granulocytic anaplasmosis (HGA). It is a tick-transmitted pathogen that protects itself from neutrophil killing by residing within the A. phagocytophilum-occupied vacuole (APV), which does not mature along the endosomal pathway, avoids lysosomal fusion, and intercepts recycling endosome- and Golgi-derived traffic. How this bacterium manipulates vesicular traffic is unknown. We have identified the 1st 2 bacterial-encoded APV membrane (AVM) proteins - APH0032 and APH1387. Both carry large sections of tandem direct repeats, eukaryotic-like endocytic sorting motifs, and decorate the cytoplasmic face of the AVM. Both carry eukaryotic-like Ser/Thr phosphorylation motifs and migrate as multiple isoforms of larger than expected sizes upon SDS-PAGE. We hypothesize that APH0032 and APH1387 contribute to the ApV's selective fusogenicity and/or A. phagocytophilum pathogenesis by interacting with host membrane traffic regulators and/or signaling pathways. The goals of this proposal are (1) to determine which membrane traffic pathways APH0032 and APH1387 intercept and whether host Ser/Thr kinases phosphorylate them; and (2) to identify the host proteins with which APH0032 and APH1387 interact. These exploratory aims will yield a high payoff by addressing a paradigm of intracellular pathogenesis - host cell manipulation by pathogen-derived PVM proteins; furthering our understanding of how A. phagocytophilum survives within neutrophils; and will establish a foundation for building long-term studies of the pathobiological roles of APH0032 and APH1387. PUBLIC HEALTH RELEVANCE: Human granulocytic anaplasmosis is an emerging, debilitating, and potentially fatal infectious disease. Our research seeks to identify bacterial proteins that enable the bacterial pathogen that causes HGA to survive inside a particular white blood cell. Doing so may reveal novel therapeutic or vaccine targets for treating or preventing HGA.