University of Louisville

Poster Title

Salmonella Pathogenesis: A Human Genetics Approach

Institution

University of Louisville

Abstract

Salmonella typhi invades host macrophages upon infection, and can effectively evade detection within a host. Infection is thus a major public health problem with Salmonella causing more than one billion new human infections each year that lead to more than three million deaths. The central question our lab is working to answer is, what host genes does Salmonella use to colonize and exploit its host, and what effectors does Salmonella employ to exploit those host genes. It is our hypothesis that genes regulating autophagy, or self-induced cell death, are required for successful Salmonella infection. To test this hypothesis, we selectively knockdowned the expression of each host gene individually, and then analyzed the resulting effects on the ability of Salmonella to grow within a host. To achieve this, we introduced complementary sequences of small hairpin RNA (shRNA) directly into the nucleus through transfection with the HEK 293T cell line, thus forming a lentiviral particle which is an effective gene delivery vector. Upon binding of the lentiviral particle to the host cell membrane, the RNA interference (RNAi) process will proceed, which results in rapid degradation of the target mRNA, and therefore, decreased protein expression or inhibition of the autophagy pathway. Twenty-four hours post infection, Salmonella colony counts determined if the reduced expression of that specific gene had an impact on growth. In addition, Quantitative Reverse Transcription PCR was used to validate results, and ensure that we removed the specific transcript of the autophagy gene.

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Salmonella Pathogenesis: A Human Genetics Approach

Salmonella typhi invades host macrophages upon infection, and can effectively evade detection within a host. Infection is thus a major public health problem with Salmonella causing more than one billion new human infections each year that lead to more than three million deaths. The central question our lab is working to answer is, what host genes does Salmonella use to colonize and exploit its host, and what effectors does Salmonella employ to exploit those host genes. It is our hypothesis that genes regulating autophagy, or self-induced cell death, are required for successful Salmonella infection. To test this hypothesis, we selectively knockdowned the expression of each host gene individually, and then analyzed the resulting effects on the ability of Salmonella to grow within a host. To achieve this, we introduced complementary sequences of small hairpin RNA (shRNA) directly into the nucleus through transfection with the HEK 293T cell line, thus forming a lentiviral particle which is an effective gene delivery vector. Upon binding of the lentiviral particle to the host cell membrane, the RNA interference (RNAi) process will proceed, which results in rapid degradation of the target mRNA, and therefore, decreased protein expression or inhibition of the autophagy pathway. Twenty-four hours post infection, Salmonella colony counts determined if the reduced expression of that specific gene had an impact on growth. In addition, Quantitative Reverse Transcription PCR was used to validate results, and ensure that we removed the specific transcript of the autophagy gene.