Project Grant R21AI178389

CONNECTING THE BACTERIAL METALLOME TO CELLULAR METABOLISM AT SINGLE CELL RESOLUTION - PROJECT SUMMARY PATHOGENIC BACTERIA ENGAGE IN COMPETITION WITH THEIR HOSTS FOR RESOURCES. HOSTS ACTIVELY DENY RESOURCES - ESPECIALLY TRANSITION METALS - TO INVADING BACTERIA TO SUPPRESS THEIR GROWTH. THIS IS A STRATEGY CALLED NUTRITIONAL IMMUNITY. UNDERSTANDING HOW BACTERIAL POPULATIONS USE METALS AND HOW THEIR METAL ACQUISITION RELATES TO THEIR CENTRAL METABOLISM IS CRITICAL TO UNDERSTANDING THIS HOST-MICROBE COMPETITION AND DEVELOPING NEW THERAPEUTIC STRATEGIES. WE CURRENTLY HAVE A POOR UNDERSTANDING BOTH OF BACTERIAL METAL REQUIREMENTS, AND OF HOW BACTERIAL METAL CONCENTRATIONS RELATE TO THEIR CENTRAL METABOLIC STRATEGIES. THE LONG-TERM AIM OF THIS PROJECT IS TO BETTER UNDERSTAND THE FUNDAMENTAL NATURE OF THE RELATIONSHIP BETWEEN THE METAL CONTENT OF INDIVIDUAL BACTERIAL CELLS WITHIN POPULATIONS OF GENETICALLY HOMOGENOUS INDIVIDUALS, AND HOW THIS RELATES TO VARIATIONS IN THEIR EXPRESSION OF CENTRAL METABOLISM. THE FIRST AIM OF THIS PROJECT IS TO TEST THE HYPOTHESIS THAT THE METAL CONCENTRATIONS OF INDIVIDUAL BACTERIAL CELLS CORRELATE WITH THEIR CENTRAL METABOLIC STRATEGY. THIS IS LIKELY TO BE TRUE, SINCE ENZYMES INVOLVED IN CENTRAL METABOLISM REQUIRE METAL COFACTORS, BUT IT HAS NEVER BEEN DEMONSTRATED FOR INDIVIDUAL CELLS. TO APPROACH THIS QUESTION, WE WILL DEVELOP A SECONDARY-ION MASS SPECTROMETRY (SIMS) IMAGING METHOD CAPABLE OF SIMULTANEOUSLY TRACKING METABOLISM AND METAL CONTENT IN HUNDREDS OF INDIVIDUAL BACTERIAL CELLS. THIS IS POSSIBLE DUE TO RECENT DEVELOPMENTS IN SIMS THAT HAVE IMPROVED SENSITIVITY AND THROUGHPUT TO LEVELS CAPABLE OF INVESTIGATING BACTERIAL POPULATIONS. THIS TECHNIQUE IS CAPABLE OF MEASURING SINGLE-CELL BACTERIAL METALLOMES, WHILE SIMULTANEOUSLY MEASURING ABUNDANCES OF ISOTOPE LABELS THAT WERE EXPERIMENTALLY PROVIDED TO TRACE CENTRAL METABOLISM (STABLE ISOTOPE PROBING). THE SECOND AIM OF THIS PROJECT IS TO TEST THE HYPOTHESIS THAT THE PHENOTYPIC TRAITS EXAMINED IN AIM 1 ARE CORRELATED WITH PATTERNS OF GENE EXPRESSION. CURRENTLY AVAILABLE METHODS ARE CAPABLE OF REVEALING PATTERNS OF GENE EXPRESSION IN SINGLE BACTERIAL CELLS USING AMPLIFIED HYBRIDIZATION TECHNIQUES COUPLED TO FLUORESCENT REPORTERS. WE WILL MODIFY THIS TECHNIQUE TO USE A HALOGEN REPORTER THAT IS CAPABLE OF BEING VISUALIZED BY SIMS. THE ABUNDANCE OF THE HALOGEN TAG IN SINGLE CELLS CAN BE MEASURED, CORRELATING TARGETED PATTERNS OF GENE EXPRESSION WITH SINGLE-CELL BACTERIAL METALLOMES AND BACTERIAL METABOLISM. THIS IS SIGNIFICANT BECAUSE IT WILL HELP US UNDERSTAND FUNDAMENTAL RELATIONSHIPS BETWEEN RESOURCE ABUNDANCE AND CENTRAL METABOLISM, AND POPULATION STRATEGIES FOR ALLOCATING RESOURCES AND METABOLISM AMONG INDIVIDUAL BACTERIAL CELLS.