Global transcriptome analysis reveals Salmonella Typhimurium employs the nitrate-dependent anaerobic pathway to combat bile stress

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyse the stress and adaptive responses of S. Typhimurium to bile. We identified the cellular pathways affected during bile stress in wild type (WT) and a mutant lacking cspE ({Delta}cspE), which plays an essential role in protection from bile stress. We observed transcriptional upregulation of several genes involved in nitrate metabolism, in response to bile stress. These genes were also differentially expressed between the bile-resilient WT and the bile-sensitive {Delta}cspE strain. To understand the role of nitrate metabolism in bile stress response, we generated a strain lacking fnr ({Delta}fnr), which is the global regulator of nitrate metabolism in S. Typhimurium. fnr was highly induced in the bile treated WT strain but not in the {Delta}cspE strain. Notably, the {Delta}fnr strain was susceptible to bile-mediated killing. Our studies revealed a new role for fnr in mediating the bile stress response. In addition, a strain lacking arcA ({Delta}arcA), a two-component system response regulator involved in anaerobic metabolism, also showed a marked reduction in growth in presence of bile. This corroborated the significance of anaerobic metabolism in S. Typhimurium bile tolerance. Importantly, overexpression of fnr and arcA lowered reactive oxygen species and significantly enhanced the survival of the bile-sensitive {Delta}cspE strain. We also observed that S. Typhimurium pre-treated with nitrate displayed better growth in the presence of bile. Together, these results demonstrate that nitrate-dependent anaerobic metabolism promotes adaptation of S. Typhimurium to bile. ImportanceSalmonella Typhimurium, as an enteric pathogen, manifests an extreme example of bile tolerance. This study describes the diverse metabolic changes at the level of transcriptome in S. Typhimurium exposed to bile. We identified the differential expression of several genes involved in anaerobic metabolism between bile-tolerant WT and bile-sensitive {Delta}cspE strains. Two major regulators of anaerobic metabolism, fnr and arcA, support the growth of S. Typhimurium in bile. Our results highlight that, in presence of bile, S. Typhimurium activates genes involved in anaerobic metabolism, specifically nitrate metabolism, that improves survival of bacteria during bile stress.