Kyle Perry

Previous studies have shown that the mammalian cell cytosol is generally non-permissive for the growth of bacteria, which suggests that intracellular bacterial pathogens such as L. monocytogenes have evolved specific mechanisms to allow for growth in this environment. However, relatively little is known about the mechanisms facilitating intracellular survival, growth, and spread of bacterial pathogens. To identify bacterial factors that allow intracellular infection by L. monocytogenes, I am employing two fluorescence-activated cell sorting (FACS)-based genetic screens.

To identify bacterial factors specifically involved in cell-to-cell spread, murine bone marrow-derived macrophages (BMM) are infected with a transposon library of GFP-expressing L. monocytogenes. After several hours of infection, host cells are processed via FACS to enrich for BMM with high fluorescence, which contain transposon mutants with cell-to-cell spread defects. To identify bacterial factors involved in intracellular replication, BMM are infected with a transposon library of ActA-deficient L. monocytogenes expressing GFP. ActA-deficient bacteria are defective for intracellular actin-based motility and cell-to-cell spread. Infected host cells are similarly processed via FACS to enrich for BMM with low fluorescence, which contain transposon mutants with intracellular replication defects. Cell-to-cell spread and intracellular replication defects of mutants identified in both screens are being characterized by genetic analyses and intracellular infection models.

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Figure. Flow cytometry analysis of infected bone marrow-derived macrophages (BMM). Exponential phase bacterial cultures were used to infect BMM at an MOI of 1.  After 6 hr, the infected macrophages were analyzed by flow cytometry. The black line represents the fluorescence intensity of macrophages infected with an ActA-deficient strain of L. monocytogenes expressing GFP. The blue line represents the fluorescence intensity of macrophages infected with a transposon mutant of the GFP-expressing ActA-deficient strain that results in a defect in intracellular replication.