The Shigella species cause millions of cases of watery or bloody diarrhea each year in developing countries, with children under the age of five years most vulnerable to infection. Emerging strains of multidrug resistant Shigella emphasize the need for a comprehensive and cost-efficient vaccine; however, an effective vaccine has yet to be produced despite years of research. Several studies have demonstrated that Shigella utilizes host physiology, specifically bile salts as signals for invasion and virulence gene expression. This study aimed to build upon previous research analyzing the bile salts transcriptional profile of Shigella flexneri 2457T, in which an induction of the uncharacterized gene was demonstrated during bile salts exposure. Here, a mutant and wild-type 2457T strains were used in infection of HT-29 colonic epithelial cells to compare invasion ability and intracellular replication. The Congo red (CR) secretion assay was also used as a measure of virulence protein secretion from the type-III secretion system (T3SS), while interleukin-8 (IL-8) secretion from infected HT-29 cells was measured as a marker for the epithelial cell response to infection. Infection analyses included subculturing the strains in media with 0.4% bile salts to mimic small intestine physiology and gastrointestinal transit of S. flexneri prior to infection. The mutant strain displayed both increased invasion of and intracellular replication in HT-29 cells compared to 2457T. The presence of bile salts enhanced both invasion and intracellular replication in both strains when compared to wild-type without bile salts exposure during subculture. The CR assays revealed increased protein secretion from the mutant compared to 2457T, and that bile salts increased T3 secretion in both strains. Increased IL-8 secretion from infected HT-29 cells was detected when both strains were subcultured in bile salts; however, a decrease in IL-8 secretion was observed following infection with the mutant subcultured without bile salts. Overall, the data suggest that this bile salt-induced gene encodes a negative regulator of virulence, and that the gene product likely prevents a hypervirulence phenotype that would compromise the ability of S. flexneri to control infection and regulate the host immune response. This work has provided insights into the function of this uncharacterized gene, which could serve as a novel target for future therapeutic development. / 2027-06-30T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41125 |
| Date | 04 June 2020 |
| Creators | Carey, James |
| Contributors | Offner, Gwynneth, Faherty, Christina |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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