| dc.description.abstract | Salmonella enterica is a Gram-negative bacterium of the Enterobacteriaceae family and a major causative agent of foodborne illnesses, ranging from self-limiting gastroenteritis to systemic infections. As a versatile pathogen, Salmonella can withstand diverse environmental and host-associated stresses, including antibiotics, temperature fluctuations, pH changes, osmotic stress, nutrient limitation, short-chain fatty acids, and bile salts. In this study, we characterised the role of gene yqhD, an aldehyde reductase that detoxifies reactive aldehydes to alcohols. Previous transcriptomic data revealed that yqhD is differentially regulated under various stress conditions, including sodium chloride, iron limitation, and bile salts. The presence of the yqhD gene reduced Salmonella survival in the presence of bile salts, suggesting its role in modulating bile-stress responses. We further investigated the molecular mechanisms by which yqhD influences Salmonella's interaction with bile salts. Secondly, nutrient limitation can arise within the host due to competition with commensal microbiota or the host's dietary preferences. Dietary polysaccharides in food are broken down into disaccharides, such as maltose, sucrose, and lactose, whose absorption is significantly reduced following Salmonella infection in mice due to mucosal damage. Among these, maltose shows the highest residual absorption, suggesting a potential role in supporting Salmonella growth and pathogenesis. Notably, maltose modulates growth and virulence in various bacterial species. This led us to investigate how maltose influences Salmonella's lifecycle and pathogenesis.
Objective 1: To determine the role of yqhD in the bile salt susceptibility of Salmonella
Bile salts play a crucial role in the interaction between Salmonella and the host gut. They possess antimicrobial properties that can disrupt the bacterial membranes, causing DNA damage and producing reactive oxygen species. To counteract bile's effects, Salmonella regulates the expression of several genes, including porins (ompF and ompC), efflux pumps (acrAB), DNA damage repair genes, and membrane remodelling genes. In our study, we explored the role of yqhD in Salmonella Typhimurium in response to bile. Deletion of yqhD conferred a growth advantage to Salmonella Typhimurium both in vitro and in vivo. The yqhD mutant exhibited better survival when exposed to primary bile salts (chenodeoxycholic acid and cholic acid) and secondary bile salts (deoxycholic acid). Similar results were observed in the liver cell line (HepG2). In C57BL/6 mice, upon supplementation with primary bile salt-sodium cholate, there was an increased organ burden of STM ΔyqhD in the cecum one day post-infection. Physiologically, bile salts are produced for the digestion of fat. The yqhD mutant showed reduced organ burden compared to wild-type on a standard chow diet 3 days post-infection; a high-fat diet (HFD) significantly enhanced its colonisation in secondary sites such as the liver and spleen.
STM ΔyqhD exhibits increased oxidative stress compared to the STM WT on bile salt treatment. We investigated whether increased oxidative stress was essential for the increased growth of STM ΔyqhD under bile salt exposure. Treatment with the antioxidant glutathione leads to similar growth of STM WT and STM ΔyqhD. In the gp91 phox -/- mice, there was a similar organ burden in STM WT and STM ΔyqhD on chow diet and high-fat diet, but there was more severe pathology in the liver and spleen of STM WT with HFD. These results show that oxidative stress was essential for the increased survival of STM ΔyqhD on bile salt exposure. Furthermore, in response to bile salt treatment, the mutant showed upregulation of the efflux pump (acrAB). The increased efflux activity mediated by the AcrAB pump was found to be mediated by the RamA/R operon, and deletion of the RamA/R or AcrAB led to a loss of the growth advantage of STM ΔyqhD.
Bile resistance in Salmonella is a key factor in establishing the carrier state and prolonging bacterial shedding in infected individuals. While several genes have been implicated in mediating bile salt susceptibility, our study identifies the antioxidant gene yqhD as a novel regulator of the same. We show that yqhD increases Salmonella susceptibility to bile salts, whereas its deletion confers a growth advantage under bile stress by modulating efflux pump activity via the RamA/R regulon. Interestingly, although loss of yqhD enhances bile resistance, it compromises the bacterium's ability to invade HepG2 cells, indicating a trade-off between stress adaptation and virulence. The underlying mechanism by which yqhD influences host cell invasion remains to be elucidated.
Objective 2: To elucidate the role of maltose metabolism in Salmonella growth in nitrogen-deficient conditions and pathogenesis
Maltose and maltodextrins play different roles in different bacteria. Reports in the literature have shown that in the in-vitro studies, maltose supports growth in nitrogen-deficient conditions. To determine the role of maltose in Salmonella growth, we used a nitrogen-deficient medium and observed that maltose supplementation enhanced Salmonella growth and length. The growth was dependent on the NtrBC, a two-component system responsible for sensing and utilising nitrogen and membrane stress response sigma factor (rpoE); their deletion led to reduced Salmonella growth in Burk's medium at the early time points, but the growth became similar to that of STM WT at the late time point of 12 hours. At the same time, the global stress response regulator (rpoS) showed growth similar to that of STM WT. Salmonella grown in nitrogen-deficient conditions was less pathogenic in the Caco-2 cell line and the C57BL/6 mice model than LB-grown bacteria.
Maltose has been reported to increase infection with Salmonella and E.coli in a mouse model. Meanwhile, maltose derivatives reduce the pathogenesis of Vibrio cholerae and Vibrio alginolyticus, reducing motility and biofilm formation in Pseudomonas aeruginosa. To further understand the pathogenesis, we deleted the malQ gene, which converts the disaccharide maltose into two glucose molecules. We observed increased invasion and adhesion of STM ΔmalQ compared to STM WT in the Caco-2 cell line. This increased adhesion was mediated by enhanced expression of type 1 fimbriae in STM ΔmalQ. Type 1 fimbriae bind to mannose residues on the host cells. Blocking the type 1 fimbriae with mannose reversed the advantage of increased adhesion of STM ΔmalQ. STM ΔmalQ also shows high expression of sigma factor rpoE, and the increased adhesion was lost upon rpoE deletion in STM ΔmalQ.
In summary, our study demonstrates that maltose enhances Salmonella survival under nitrogen-limited conditions while concurrently suppressing the expression of type 1 fimbriae, key adhesins involved in host attachment. Further investigations are warranted to identify the genetic determinants mediating Salmonella survival during nitrogen deficiency at late time points. | en_US |
