Examining the effects of bile salts on Shigella flexneri pathogenesis
Embargo Date
2025-02-06
OA Version
Citation
Abstract
The Shigella species are Gram-negative facultative anaerobes that are the causative agents of millions of shigellosis infections annually. Shigellosis infections are characterized by watery and/or bloody diarrhea, abdominal cramps, and dehydration. Most cases occur in children under five years of age, especially in low- and middle-income countries (LMICs). The first line of treatment is typically antibiotics, but the rise of multidrug resistant Shigella strains and limited access to newer antibiotics and other treatments in LMICs are causing significant morbidity and mortality. Currently, there is no Shigella vaccine to prevent infection. In order to develop an effective vaccine or other novel therapeutics, the pathogenic mechanisms that Shigella utilizes to cause infection in the colon must be thoroughly understood. As Shigella transits through the small intestine prior to causing infection in the colon, it encounters bile salts. Bile salts are typically bactericidal, but Shigella has developed resistance mechanisms and uses bile salts as host signals to enhance colonic infection by increasing virulence gene expression and protein secretion. The laboratory previously identified two neighboring uncharacterized genes affected by bile salts exposure. This study analyzed both a single gene and double gene deletion constructs to examine growth, virulence protein secretion, and subsequent effects on epithelial cell invasion following exposure to bile salts. Growth curve analyses of the two mutant strains relative to wildtype bacteria, S. flexneri serotype 2a strain 2457T, were performed in various media formulations with different sugars both with and without bile salts to mimic small intestinal conditions. The double mutant demonstrated a significant growth deficiency, more so in the presence of bile salts, relative to 2457T and the other single mutant strain in all conditions, suggesting a role in bile salts resistance. Exposure to bile salts in media with different sugar compositions triggered biofilm formation in all bacterial strains, but the formation was delayed in the double mutant. Additionally, a Congo red (CR) secretion assay was performed to measure virulence protein secretion for all strains following exposure to bile salts. Relative to 2457T, the single mutant displayed enhanced secretion while the double mutant displayed hypersecretion. Lastly, an invasion assay revealed altered infection patterns of epithelial cells by both mutants following bile salts exposure, confirming the CR secretion assay results. Combined with protein homology predictions, the data indicate the two genes facilitate bile salts resistance and virulence. This study contributes to the understanding of Shigella pathogenesis and the genes may serve as novel targets for future therapeutic development.