Listeria monocytogenes is an intracellular parasite which causes bacteremia and meningitis in immunocompromised patients. The incidence of listeriosis in AIDS is 100-250 times higher than the normal population. Listeria invades the host cell cytoplasm and induces host cell actin to assemble into a rocket tail which propels the bacterium rapidly through the cytoplasm so it can spread from cell to cell. To address the question, """"""""How do Listeria interact with the host cells to accomplish this remarkable task?"""""""", we plan to: I. Determine which actin-binding proteins are critical for Listeria intracellular movement and cell-to-cell spread. PtK2 cell (epithelial cells) and J774 cells (macrophage-like cells) will be infected with Listeria and immunofluorescence used to examine the localization of actin and actin-binding proteins at early and late stages of infection. In addition to normal wild type strains, the MACK mutant strain which constitutively overproduces surface proteins will be studied. To better synchronize the infection bacteria will be microinjected into cells. Listeria motility in Xenopus extracts will be studied by microscopy. A living demonstration of how the actin severing protein, gelsolin, affects Listeria motility will be provided by infecting 3T3 fibroblasts permanently transfected with human gelsolin cDNA. Bodipy phalloidin (an actin filament stain) will be used to measure bacteria rocket tail lengths (an indirect measure of bacterial speed). Cells will be microinjected with fluorescein labeled actin so that actin assembly and disassembly rates as well as migration speed can be measured using time lapse video image analysis. II. Purify ActA protein and investigate its mechanism of action. A bacterial surface protein, ActA, which is necessary for Listeria induction of actin assembly, will be purified from the Listeria MACK strain, using high salt extraction, ion exchange and gel filtration chromatography. Alternatively the pET12 protein expression vector can be used to produce recombinant protein. Purified ActA will be microinjected into living cells. The protein will be added to macrophage extracts and actin assembly monitored using the pyrenyl actin. ActA will be coated on polystyrene beads and motility examined in cell extracts. An ActA affinity column will be used to purify host cell ActA-binding proteins. III. Search for additional genes important for Listeria intracellular movement and cell-to-cell spread. The actA gene will be transduced into Bacillus subtilis (hly) to determine if the ActA gene alone can mediate actin assembly. Other genes important for actin assembly will be sought by screening Tn9l6 Listeria mutants using phase contrast microscopy of infected PtK2 cells. The genetic basis for the immotile Listeria mutant M117 will be determined using Tn9l6 probes and nucleic acid sequencing. Understanding how Listeria utilizes the host cell's contractile system to infect new host cells should be applicable to other intracellular pathogens such as Shigella, Trypanosoma Cruzi, and Rickettsia. These discoveries may lead to the development new methods for treating intracellular infections in AIDS patients.
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