Upon invasion of a host cell, intracellular pathogens must actively ensure their survival in an immediately hostile environment. One such survival tactic of some pathogenic bacteria is through the subversion of host membrane fusion machinery, thereby inhibiting phagolysosomal fusion and subsequent delivery of the bacterium to the host degradative lysosome. The foodborne pathogen, Salmonella enterica, and the causative agent of Legionnaire’s disease, Legionella pneumophila, are examples of such bacterial pathogens that utilize this particular survival tactic. While evading host cell defenses in this manner is key to the organism’s ability to cause infection and disease, the mechanisms underlying these evasion pathways remain poorly understood. Many studies have tentatively identified bacterial factors thought to be important for the disruption of normal host membrane dynamics, but the biochemical analysis of these factors remains lacking. By employing a powerful in vivo and in vitro model system of eukaryotic membrane fusion, my laboratory will investigate the biochemistry of eukaryotic membrane fusion, identify and biochemically characterize bacterial effectors capable of modulating membrane fusion, and finally analyze these activities within the context of pathogenesis.
Vacuoles of the budding yeast Saccharomyces cerevisiae serve the equivalent physiological function of the mammalian lysosome, and undergo constant rounds of fission and homotypic (self) fusion in response to cellular growth conditions. Isolation of these fusogenic organelles from yeast is now a straightforward task, and robust colorimetric assays have been developed to assay the multi-stage process of their fusion in vitro. As an excellent model of general eukaryotic SNARE-, Rab GTPase-, and SM protein-dependent intracellular membrane fusion, the yeast homotypic vacuole fusion system will comprise the backbone of our genetic, molecular, and biochemical approaches. Initial studies in the lab will characterize factors that allow an organism to drive a given membrane fusion event with a specific set of fusion machinery. The recent discovery that a yeast protein complex (the so-called HOPS complex) provides a proofreading activity to ensure proper homotypic vacuole fusion will be further studied. In addition, we will conduct genetic and biochemical screens of the intracellular pathogens Salmonella and Legionella to identify bacterially-produced inhibitors of vacuole fusion in vivo and in vitro. Mechanistic information gleaned from these studies will open new avenues towards the detailed study of basic bacterial pathogenesis.