Genetic Mapping and Functional Characterization of Zebrafish Mutants that are Hypersusceptible to Mycobacterium marinum

Abstract

University of Washington Abstract Genetic Mapping and Functional Characterization of Zebrafish Mutants That are Hypersusceptible to Mycobacterium marinum Russell David Berg Chair of the Supervisory Committee: Lalita Ramakrishnan, M.B.B.S., Ph.D. Professor, Microbiology and Adjunct Professor, Immunology Tuberculosis (TB) has historically killed more humans than any other single cause of mortality, and continues to haunt us in the form of resistant strains of Mycobacterium tuberculosis (Mtb) that are untreatable with current antibiotics. To address the need for new therapeutic approaches to TB, we have conducted a forward genetic screen in the zebrafish (Danio rerio) to identify genes required for host immunity to Mycobacterium marinum (Mm), the closest genetic relative of the Mtb complex. Forward genetics approaches have illuminated much of our understanding of biology and immunity, a summary of which is presented. In this work, we have described the identification of a hypersusceptible zebrafish mutant, snapc1, and characterization of the cellular and molecular defects responsible for failed immunity to Mm infection. We showed that the basal transcription factor snapc1 is required for adequate production of the lysosomal protease cathepsin L1, a defect in which leads to lysosomal storage of engulfed dead cells in mutant macrophages. Engorged macrophages exhibited an aberrant "vacuous" morphology and were defective in migration towards bacteria. This lead to premature extracellular escape of Mm, as vacuous macrophages failed to participate in immunity. The snapc1 phenotype was mimicked by ablation of lysosomal hydrolases associated with lysosomal storage disorders. Together, these findings highlight the phagocytic burden placed on macrophages during physiological processes and their potential to alter macrophages' ability to combat infection. They reveal that homeostatic dead cell clearance and effective phagocytic killing place competing demands on macrophages. Finally, in addition to snapc1, I discuss some new and ongoing searches for host determinants of immunity.