Listeria rnonocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised adults. The bacterium grows directly in the cytoplasm of infected host cells and moves rapidly throughout and between infected cells using a form of actin-based motility. An L. rnonocytogenes protein, ActA, induces polymerization of host cell actin to form a """"""""comet tail"""""""" structure that pushes the bacterium through the host cell cytoplasm. The overall goal of this proposal is to understand the mechanism of the actin-based motility of L. monocytogenes. Three complementary approaches will be used to study this problem: biochemical, biophysical, and cell biological. A major goal is the establishment of a simplified biochemical system that can support L. monocytogenes motility, and development of quantitative assays so that the precise role of each component in actin-based motility can be assessed. The amount of force generated by moving bacteria will be measured directly using a laser force trap (optical tweezers) and compliant microneedles. The mechanism of coupling between actin filament polymerization and production of a motile force will be examined. Finally, videomicroscopy techniques will be used to observe the process of bacterial spread from one host cell to another, and a combination of genetic and pharmacological perturbations will be used to define the contributions of the bacterium, the host cell, and the actin-rich comet tail associated with the moving bacterium, to the process of intercellular spread. Successful completion of our research goals would give significant insight into the mechanisms by which pathogenic bacteria such as L. monocytogenes communicate specifically with the cells of their human hosts. This understanding might pave the way for the development of new ways to prevent and cure bacterial infections. In addition, the results of our research would contribute to our understanding of a wide variety of basic biological process involving actin-based cell movement, including wound healing, immune system responses, and embryonic development. Furthermore, since most malignant tumors do not become lethal until the cancer cells move away from the tumor site and invade other tissues, a detailed understanding of the basic mechanisms that regulate actin-based motility may also be important in the development of therapeutic strategies for combating metastatic cancers.
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