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The study of intracellular microbial pathogens allows a multidisciplinary approach to fundamental questions related to host cell/microbe interactions. In my lab, we use Listeria monocytogenes as a model system to study these host/microbe interactions. It is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. L. monocytogenes multiplies in the cytosol of mammalian cells, and subverts host humoral immune system by spreading from cell-to-cell without leaving the intracellular milieu. The efficiency of spreading is directly related to the virulence of the bacterium. The long-term goal of our research is to define the cellular and molecular mechanisms leading to L. monocytogenes cell-to-cell spread. Among the bacterial factors involved in cell-cell-spread are a broad-range phospholipase C (PC-PLC) and a metalloprotease (Mpl). PC-PLC is secreted as an inactive precursor (proPC-PLC), and proteolytic cleavage at its N-terminus generates the active form of the enzyme. We have shown that the intracellular activation of proPC-PLC is mediated by a bacterial Mpl, which is also active in broth culture, and a cysteine protease, whose activity can only be detected during intracellular infection. Furthermore, proPC-PLC activation by either pathway is dependent on bacterial localization to a vacuole, and on vacuolar acidification. Recently, we discovered that PC-PLC is sequestered within cytosolic bacteria and acid pH triggers its activation and release. We hypothesize that PC-PLC and Mpl are sorted to a bacterial compartment, and shortly after bacterial entrapment in a vacuole, a drop in pH triggers Mpl-mediated activation and release of active PC-PLC into the lumen of the inner vacuole. This cascade of events influences the kinetics of action of other bacterial factors, vacuolar maturation and bacterial escape from the vacuole. Future studies aim at (i) defining the bacterial compartment in which PC-PLC is sorted, (ii) the sorting mechanism of PC-PLC, and (iii) the mechanism controlling release of PC-PLC by the bacterial cell. In addition, we aim at (iv) defining the signaling pathways that are involved in bacterial cell-to-cell spread and the role of bacterial factors on vacuolar maturation. These studies will contribute to our understanding of the mechanisms regulating L. monocytogenes cell-to-cell spread.
NON-TECHNICAL SUMMARY: The study of intracellular microbial pathogens allows a multidisciplinary approach to fundamental questions related to host/microbe interactions. Listeria monocytogenes is a good model system to study these interactions. As a foodborne pathogen, the bacterium has emerged as a significant public health problem. L. monocytogenes multiplies in the cytosol of mammalian cells and spreads directly from cell-to-cell. Our research aims at defining how Listeria spreads.
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APPROACH: The approaches we use are multidisciplinary. We will use biochemistry and immunochemistry to localize bacteria-associated PC-PLC and Mpl, and to define the efficiency of PC-PLC activation and bacterial release of active PC-PLC in response to a change in pH and redox potential. We will use molecular genetics and biochemistry to define the structural features of PC-PLC and Mpl that regulate their sorting and activation. We will use cell biology, molecular biology and biochemistry to define the signaling pathways that are involved in bacterial cell-to-cell spread and the role of bacterial factors on vacuolar maturation.