Faculty Sponsor

Dr. Lon Chubiz

Final Abstract for URS Program

Salmonella is a relatively abundant, virulent species of bacteria that is most known for spreading gastrointestinal diseases through food. These illnesses result in approximately 1.35 million infections, including over 25,000 hospitalizations each year, in the U.S. alone (CDC.gov). As antibiotic resistance becomes an increasingly urgent public health problem, the importance of developing alternative treatment methods is only becoming more crucial. One of the genes responsible for this virulence is known as hilA. HilA is the main transcriptional regulator of Salmonella Pathogenicity Island-1 gene (UniProt). SPI-1 plays an important role in the invasion of Salmonella into epithelial cells. The proteins encoded for by this gene assemble into a “needle complex,” which delivers bacterial proteins into host cells (Lou et al, 2019). Based off preliminary evidence that another protein, MarA (“Multiple Antibiotic Resistance Protein” A) indirectly represses the hilA promoter, the purpose of this experiment was to attempt to further resolve the repression process and identify additional genes that are required for MarA to repress hilA. Are there entirely separate proteins involved in a pathway started off by MarA’s gene product, and if so, what genes can we attempt to begin to implicate in this pathway? Transposon mutagenesis was conducted in order to create mutants of a strain of Salmonella that both express hilA alongside a lacZ reporter, and overexpress the MarA protein. The purpose of this was to introduce a DNA sequence into the wild-type genome. Wherever the transposon was inserted would be disrupted. These electroporated mutants were then plated on X-gal media, and any that had a gene involved in hilA repression disrupted, would be able to express hilA and grow blue colonies. The hilA activity of these mutants was then measured via B-galactosidase kinetic assays, and mutations with high hilA expression were reinserted into the wild-type salmonella strain via transduction. HilA activity was then screened for once more, and those whose mutations had been confirmed to disrupt the repression pathway of MarA will then have their genomes sequenced for further study. Though this project is still in progress, we have identified several mutants that exhibit increased amounts of B-gal activity compared to the wild-type strain. Furthermore, variable amounts of B-gal activity across different cell samples implies that there are different routes to disrupt the repression of hilA, and that a multi-gene/gene product pathway in involved in this repression process. Future sequencing will allow us to resolve potential genes and gene products important for this process.

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