Project Grant F31AI183650

INFLUENCE OF ARGININE ON KLEBSIELLA PNEUMONIAE MUCOIDY AND PATHOGENESIS - PROJECT SUMMARY HYPERVIRULENT KLEBSIELLA PNEUMONIAE (HVKP) IS A GROWING GLOBAL THREAT WITHIN COMMUNITY SETTINGS. HVKP CAUSES SEVERE LIVER ABSCESSES THAT CAN SPREAD TO THE EYES AND CENTRAL NERVOUS SYSTEM. ONE DISTINCT FEATURE OF MOST HVKP STRAINS IS THE PRESENCE OF VISUALLY DISTINGUISHABLE MUCOID COLONIES, THAT ADHERE TO LOOPS AND EXHIBIT A MEASURABLE STRETCH FROM AGAR PLATES. THESE COLONIES HAVE LONGER CAPSULAR POLYSACCHARIDE CHAINS, RESULTING IN A MUCOID APPEARANCE AND INCREASED DISEASE SEVERITY. HOWEVER, THE REGULATION OF MUCOIDY BY EXTRACELLULAR SIGNALS AND ITS ADAPTATION TO HUMAN HOSTS IS POORLY UNDERSTOOD. THIS PROPOSAL AIMS TO PROVIDE HIGH-IMPACT TRAINING TO A DOCTORAL STUDENT, IN PART THROUGH MENTORED RESEARCH TO IDENTIFY EXTERNAL NUTRIENT SIGNALS RESPONSIBLE FOR REGULATING MUCOIDY IN K. PNEUMONIAE. INITIAL OBSERVATIONS SHOWED INCREASED MUCOIDY WHEN K. PNEUMONIAE WAS GROWN IN A MINIMAL MEDIUM SUPPLEMENTED WITH CASAMINO ACIDS. IMPORTANTLY, THIS INCREASE IN MUCOIDY WAS INDEPENDENT OF CAPSULE ABUNDANCE, THUS PROMPTING OUR FOCUS ON AMINO ACIDS. SUBSEQUENT EXPERIMENTS ELIMINATING ONE AMINO ACID AT A TIME SUGGESTED THAT ARGININE IS A KEY SIGNAL FOR MUCOIDY VIA EFFECTS ON CAPSULE CHAIN LENGTH. THE CENTRAL HYPOTHESIS OF THE RESEARCH COMPONENT OF THIS TRAINING PROPOSAL IS THAT GENES OR METABOLITES IN ARGININE CATABOLIC PATHWAYS REGULATE MUCOIDY AND CONTRIBUTE TO PATHOGENESIS. TO ADDRESS THIS HYPOTHESIS AND ACHIEVE THE PROPOSAL'S GOALS, WE WILL FIRST IDENTIFY THE ARGININE SIGNALING PATHWAYS THAT ARE ALTERING MUCOIDY. WE WILL EMPLOY ESTABLISHED SEDIMENTATION ASSAYS TO ASSESS MUCOIDY IN VARIOUS ARGININE MUTANTS. WE WILL ALSO DETERMINE THE EFFECTS OF ARGININE ON TRANSCRIPT LEVELS OF MUCOID-RELATED GENES, USING QPCR. FURTHERMORE, WE WILL EVALUATE THE INTRACELLULAR SURVIVAL, ESCAPE ABILITY, AND MUCOIDY OF AN ARGININE IMPORT MUTANT WITHIN CULTURED MACROPHAGES INFECTED BY K. PNEUMONIAE. THIS INVESTIGATION WILL SHED LIGHT ON THE ROLES OF ARGININE AVAILABILITY, AND MUCOIDY IN THE INTRACELLULAR SURVIVAL OF K. PNEUMONIAE WITHIN MACROPHAGES. ADDITIONALLY, USING A MURINE BACTEREMIA MODEL, WE WILL ASSESS DISSEMINATION AND RMPD EXPRESSION IN AN ARGININE IMPORT MUTANT TO EVALUATE THE CONTRIBUTION OF HOST ARGININE IN HVKP DISSEMINATION. COLLECTIVELY, THESE DATA WILL IDENTIFY HOW ARGININE REGULATES MUCOIDY AND ALTERS THE INTRACELLULAR FITNESS OF K. PNEUMONIAE. THESE FINDINGS LAY THE GROUNDWORK FOR FUTURE RESEARCH ENDEAVORS INTO THE PATHOGENICITY OF K. PNEUMONIAE IN DISEASE STATES THAT CAUSE FLUCTUATIONS IN ARGININE LEVELS, ENHANCING OUR ABILITY TO COMBAT INFECTIONS CAUSED BY HYPERVIRULENT STRAINS. THIS RESEARCH WILL BE CARRIED OUT IN THE CONTEXT OF A RIGOROUS AND ACTIVELY MENTORED TRAINING PROGRAM.