Project Grant F31AI203139

STRUCTURAL BASIS OF PHOSPHOINOSITIDE RECOGNITION IN BACTERIAL PATHOGENS - PROJECT SUMMARY ANTIMICROBIAL RESISTANCE POSES A CRITICAL PUBLIC HEALTH THREAT, WITH OVER 2.8 MILLION ANTIBIOTIC-RESISTANT INFECTIONS OCCURRING ANNUALLY IN THE UNITED STATES. INTRACELLULAR BACTERIAL PATHOGENS ARE ESPECIALLY DIFFICULT TO TREAT BECAUSE THEY PROLIFERATE WITHIN PROTECTIVE MEMBRANE COMPARTMENTS THAT SHIELD THEM FROM IMMUNE DEFENSES AND ANTIBIOTICS. OF THE CDC'S LIST OF HIGH PRIORITY BIOLOGICAL AGENTS, MANY ARE INTRACELLULAR BACTERIAL PATHOGENS. THESE PATHOGENS ESTABLISH THEIR REPLICATIVE NICHE BY SECRETING EFFECTOR PROTEINS THAT MANIPULATE HOST CELL MEMBRANES. RECENT DISCOVERIES REVEAL THAT MANY BACTERIAL EFFECTORS TARGET SPECIFIC HOST MEMBRANES BY RECOGNIZING PHOSPHOINOSITIDE (PIP) LIPIDS, WHICH ARE CRITICAL REGULATORS OF MEMBRANE TRAFFICKING AND IDENTITY. WHILE EUKARYOTIC PIP-BINDING DOMAINS ARE WELL CHARACTERIZED, THE MECHANISMS BY WHICH BACTERIAL EFFECTORS SELECTIVELY ENGAGE PIPS REMAIN POORLY UNDERSTOOD. THIS KNOWLEDGE GAP POSITIONS BACTERIAL PIP-BINDING EFFECTORS AS PROMISING TARGETS FOR ANTI-VIRULENCE THERAPIES. MY LONG-TERM GOAL IS TO ELUCIDATE HOW BACTERIAL EFFECTORS EXPLOIT PIP LIPIDS TO SUBVERT HOST MEMBRANE BIOLOGY AND REVEAL DRUGGABLE TARGETS FOR TREATING INTRACELLULAR INFECTIONS. I HYPOTHESIZE THAT INTRACELLULAR BACTERIAL PATHOGENS HAVE EVOLVED STRUCTURALLY DISTINCT PIP-BINDING DOMAINS THAT ENABLE SELECTIVE HOST MEMBRANE TARGETING. THE INTRACELLULAR BACTERIAL PATHOGEN, LEGIONELLA PNEUMOPHILA, INFECTS AND REPLICATES WITHIN HUMAN LUNG MACROPHAGES, CAUSING A SEVERE PNEUMONIA, LEGIONNAIRES' DISEASE. L. PNEUMOPHILA PROVIDES AN IDEAL MODEL SYSTEM AS IT DEPLOYS OVER 350 EFFECTORS, INCLUDING MORE THAN 50 PIP-BINDING EFFECTORS, 30 OF WHICH I HELPED VALIDATE IN MY PRELIMINARY DATA. STRUCTURAL MAPPING REVEALED THAT DESPITE LACKING CONSERVED SEQUENCE MOTIFS, THESE EFFECTORS SHARE STRUCTURAL HOMOLOGY, SUGGESTING BACTERIA-SPECIFIC PIP-BINDING FOLDS. MY PRELIMINARY WORK IDENTIFIED A RECURRING FOUR-A-HELIX MOTIF, TERMED THE LIPID-BINDING 4-HELIX DOMAIN OR "L4H", THE FOCUS OF THIS PROPOSAL. IN AIM 1, I WILL DETERMINE THE STRUCTURAL BASIS AND SPECIFICITY OF PIP RECOGNITION BY SOLVING CO-CRYSTAL STRUCTURES OF L4H BOUND TO PIP HEADGROUPS USING X-RAY CRYSTALLOGRAPHY AND PERFORMING STRUCTURE-GUIDED MUTAGENESIS COUPLED WITH BIOPHYSICAL BINDING ASSAYS. IN AIM 2, I WILL DEFINE L4H MEMBRANE SPECIFICITY AND HOST PROTEIN INTERACTIONS USING FLUORESCENCE MICROSCOPY AND CO-IMMUNOPRECIPITATION IN MAMMALIAN CELLS, ESTABLISHING PIP-DEPENDENCE THROUGH BIOSYNTHETIC PATHWAY DISRUPTION. IN AIM 3, I WILL VALIDATE L4H CONSERVATION ACROSS MULTIPLE INTRACELLULAR PATHOGENS USING PROTEIN-LIPID OVERLAY ASSAYS AND CELLULAR LOCALIZATION STUDIES. THIS WORK WILL DEFINE A BACTERIAL-SPECIFIC LIPID-BINDING DOMAIN, PROVIDING FUNDAMENTAL INSIGHT INTO HOW INTRACELLULAR PATHOGENS MANIPULATE HOST MEMBRANES. SUCCESSFUL COMPLETION OF THESE AIMS WILL REVEAL NOVEL PRINCIPLES OF HOST-PATHOGEN INTERACTIONS AND ESTABLISH BACTERIAL PIP-BINDING DOMAINS AS CANDIDATE TARGETS FOR ANTI-VIRULENCE THERAPIES, DIRECTLY ADDRESSING THE URGENT NEED FOR IMPROVED STRATEGIES TO COMBAT L. PNEUMOPHILA AND RELATED PATHOGENS.