Toxoplasma gondii and Listeria monocytogenes are intracellular pathogens classified as NIAID category B priority agents. Both organisms can lead to significant disease and mortality in immunodeficient or immunocompetent hosts. Our long-term goal is to elucidate the steps involved in microbial invasion to identify potential therapeutic targets for treatment of toxoplasmosis and listeriosis. We hypothesize that the host cell plays an active role during invasion, and a large-scale siRNA screen will identify components important for this process. Specific Aim 1 will utilize a human whole genome siRNA library (Dharmacon) in conjunction with high throughput luciferase assays to identify host molecules that contribute to Toxoplasma or Listeria invasion of epithelial cells. Targeted genes that result in defects in attachment or entry will be examined in greater detail in Specific Aim 2 to confirm a reduction of corresponding mRNA and protein levels, in addition to determining subcellular localization of host targets during invasion. Host genes that are identified as being important for entry of these disparate microbial pathogens may provide some insight into processes common to invasion by many pathogens. This proposal offers a unique opportunity to study two distinct host-pathogen interactions and gain a broader understanding of microbial invasion strategies. PUBLIC HEALTH RELEVANCE: T. gondii is a human parasite that infects an estimated 23% of the population in the USA. While generally asymptomatic in healthy individuals, it can be deadly under certain circumstances: 1) maternal infection can lead to serious birth defects or loss of pregnancy, and 2) immuno-compromised individuals (either as a result of AIDS, or through organ transplant immuno-suppression) can succumb to toxoplasmosis. Listeria similarly poses a threat to immunodeficient individuals and pregnant women by foodborne ingestion of meat or dairy products contaminated during processing.