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RESEARCH INTERESTS

The overall goal of our research is to understand the molecular and cellular basis of microbial pathogenesis and the mechanisms used by the host to defend against infection. Specifically, the lab is focused on the interaction of the facultative intracellular bacterial pathogen Listeria monocytogenes and mammalian cells. This fascinating microorganism is able to enter cells, escape from a phagosome and grow rapidly in the cytosol. By exploiting a host system of actin-based motility, the bacteria move through the cytosol to the cell membrane and into pseudopod-like projections (listeriopods) that are ingested by neighboring cells. This mechanism allows pathogens to spread from one cell to another without ever leaving the host cytoplasm thereby avoiding the immune response.

Escape from a vacuole and cell to cell spread of L. monocytogenes. The stippled material depicts F-actin.

 

CURRENT PROJECTS

Cell biology of infection. The primary L. monocytogenes determinant responsible for lysis of host cell vacuoles is the pore-forming cytolysin, listeriolysin O (LLO). We will continue to focus on the control of LLO synthesis and secretion, and its mechanism of action. The ultimate goal is to relate structural and biochemical information to its precise mechanism of action in both tissue culture and in mice. We are also characterizing a number of fail-safe mechanisms that prevent LLO toxicity in the host cytosol and thereby compartmentalize its activity to acidic vacuoles. Interestingly, mutants that fail to properly compartmentalize LLO activity are cytotoxic to infected host cells and attenuated for virulence in mice.

The L. monocytogenes ActA protein is necessary and sufficient to mediate actin-based motility in cells and cell-free extracts. Actin polymerization is controlled by interaction of ActA with the host Arp2/3 complex and members of the Ena/VASP family. During the next few years we will focus on the mechanism of ActA function and evaluate its role during infection.

We have recently developed a model to examine the interaction of L. monocytogenes with cultured Drosophila cells and are using RNA interference to identify and characterize the role of host proteins during infection.

Mechanisms of secretion. We are interested in how bacteria regulate secretion. We have recently found that unlike gram-negative bacteria, L. monocytogenes and many other gram-positive bacteria have two secA genes. SecA1 is essential for viability while SecA2 mediates secretion of a subset of bacterial proteins including at least two peptidoglycan hydrolases. We are currently characterizing the role of the SecA2-dependent hydrolases during infection.

Immunity to infection. Murine listeriosis is an outstanding model to study basic aspects of innate and acquired cell-mediated immunity. Using bacterial mutants blocked at various stages in the infection process, we are elucidating pathways of host cell gene expression in response to microbial infection. Our studies clearly document the presence of a vacuolar and cytosolic pathway of innate immune recognition. Microarrary analysis has revealed dozens of genes expressed in response to the cytosolic pathway including beta-interferon. We are now investigating the in vivo roles of the cytosolic pathway using a combination of knockout mice and bacterial mutants.

 

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