Angela Smith, Texas A&M University – Corpus Christi

Increased Salmonella enterica serovar Typhimurium-host cell invasion in vitro through induction of SPI1 Type Three Secretion System

Abstract: Salmonella Typhimurium is a facultative intracellular pathogen that causes gastroenteritis in humans and a systemic typhoid-like disease in susceptible mice.  Expression of the Salmonella Pathogenicity Island 1 (SPI1)-encoded Type Three Secretion System 1 (T3SS1) is crucial for invasion of non-phagocytic cells, such as intestinal epithelial cells.  This process is regulated by environmental cues and must be induced prior to invasion of host cells.  The goal of this project was to develop a protocol to optimize growth parameters of S. Typhimurium in lysogeny broth (LB) to maximally induce SPI1 expression and subsequent invasiveness.  We monitored SPI1 induction using a transcriptional prgH fusion to a destabilized variant of GFP, through whole population and single cell analysis.  Invasiveness was monitored by measuring internalization into HeLa cells using a gentamicin protection assay.  Our results indicate that culture vessel and culture surface-to-volume ratio are important for optimal SPI1 induction.  Sub-cultures grown in 5 ml LB-Miller in 14-ml tubes with aeration resulted in SPI1 reporter expression in nearly 76% of the population, compared with the 31% seen when using previously described growth conditions with a larger surface-to-volume ratio.  Bacteria grown under the new conditions resulted in increased invasion efficiency of HeLa cells by 2.64% of the input inoculum.  We conclude that at least two-fold higher levels of SPI1 induction occur with Salmonella Typhimurium when grown aerobically with a low surface-to-volume ratio.

Presentation Author(s):
Angela Smith*, Kendal Cooper, Olivia Steele-Mortimer, and Gregory Buck

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