Project Grant F32AI183749

ANAPLASMA PHAGOCYTOPHILUM EXPLOITATION OF REGULATED EXOCYTOSIS. - SUMMARY REGULATED EXOCYTOSIS IS A CONSERVED EUKARYOTIC PROCESS THAT IS ESSENTIAL FOR NUMEROUS CELLULAR FUNCTIONS, MAKING IT A PRIME TARGET FOR EXPLOITATION BY BOTH INFECTIOUS AND NON-INFECTIOUS DISEASES. INDEED, VARIOUS MALIGNANCIES, VIRUSES, BACTERIA, AND PROTOZOA COMMANDEER THIS PATHWAY TO PROMOTE DISEASE PROGRESSION. NUMEROUS STUDIES HAVE INTERROGATED THE MOLECULAR PLAYERS OF REGULATED EXOCYTOSIS THAT ARE MODULATED IN CANCER FORMATION, INCLUDING THE CONSERVED SECOND MESSENGERS, CALCIUM (CA2+) AND SPHINGOSINE-1-PHOSPHATE (S1P). HOWEVER, FAR LESS IS KNOWN ABOUT THE MECHANISMS THAT INTRACELLULAR BACTERIAL PATHOGENS EMPLOY TO EXPLOIT THIS PROCESS. HUMAN GRANULOCYTIC ANAPLASMOSIS IS AN EMERGENT DISEASE CAUSED BY THE OBLIGATE INTRACELLULAR VACUOLAR-ADAPTED BACTERIUM, ANAPLASMA PHAGOCYTOPHILUM (AP). AP IS A TICK-TRANSMITTED PATHOGEN THAT EXHIBITS AN UNUSUAL TROPISM FOR NEUTROPHILS. A PRIMARY FUNCTION OF NEUTROPHILS IS TO DESTROY MICROORGANISMS THROUGH DEGRANULATION, A FORM OF REGULATED EXOCYTOSIS. INTRIGUINGLY, BOTH AP-INFECTED NEUTROPHILS AND TICKS EXHIBIT PROLONGED DEGRANULATION AND INCREASED EXOCYTOSIS, RESPECTIVELY. WE RECENTLY PUBLISHED THAT THE AP- CONTAINING VACUOLE (APV) RESEMBLES A HOST MULTIVESICULAR BODY (AN ORGANELLE THAT UNDERGOES REGULATED EXOCYTOSIS) AND THAT LATE-STAGE APVS MOBILIZE TO AND FUSE WITH THE PLASMA MEMBRANE TO RELEASE AP INTO EXTRACELLULAR MILIEU FOR REINFECTION. OUR PRELIMINARY RESULTS INDICATE THAT INTRACELLULAR CA2+ CONCENTRATION ([CA2+]I), SPHINGOSINE KINASE 1 (SK1 [PRODUCES S1P]), AND S1P ARE INCREASED LATE IN AP INFECTION AND THAT INHIBITION OF AP PROTEIN SYNTHESIS PREVENTS THESE PHENOMENA AS WELL AS APV EXOCYTOSIS. THESE DATA SUGGEST THAT AP MANIPULATES REGULATED EXOCYTOSIS FOR EGRESS. WE ALSO UNCOVERED A POTENTIAL MECHANISTIC ROLE FOR THE AP EFFECTOR, P130, IN THIS PROCESS. P130 IS UPREGULATED DURING LATE AP INFECTION AND LOCALIZES TO THE CYTOSOLIC FACE OF THE APV MEMBRANE. WE DISCOVERED THAT P130 INTERACTS WITH HOST CALCIUM AND INTEGRIN-BINDING PROTEIN 1 (CIB1), WHICH, TO DATE, HAS BEEN INDIRECTLY IMPLICATED IN REGULATED EXOCYTOSIS. CIB1 ALSO LOCALIZES WITH P130 ON EXOCYTOSING APVS, FURTHER SUPPORTING ITS ROLE IN AP DISSEMINATION. IN AIM 1, WE WILL INTERROGATE THE RELEVANCE OF [CA2+]I AND S1P TO APV EXOCYTOSIS AND INFECTIOUS PROGENY RELEASE USING IN VITRO AND IN VIVO APPROACHES. IN AIM 2, WE WILL IDENTIFY THE P130 DOMAINS NECESSARY FOR ITS INTERACTION WITH CIB1 AND ITS PROMOTION OF APV EXOCYTOSIS AS WELL AS DETERMINE THE RELEVANCE OF CIB1 TO APV EXOCYTOSIS IN VITRO AND IN VIVO. THIS WORK WILL ADVANCE OUR FUNDAMENTAL UNDERSTANDING OF THE MECHANISMS THAT INTRACELLULAR BACTERIAL PATHOGENS UTILIZE TO FACILITATE THEIR RELEASE AND POTENTIALLY DEFINE NOVEL ROLES FOR CIB1 IN HOST CELL BIOLOGY AND INFECTIOUS DISEASE.