Understanding the adaptive responses of Salmonella Typhimurium during bile stress

Abstract

Salmonella Typhimurium (S. Typhimurium) is an enteric pathogen that causes gastroenteritis. Although gastroenteritis is usually non-life threatening in healthy adults, it can cause fatality in children and immunocompromised individuals. The invasion and colonization of S. Typhimurium comprises uptake and replication in the intestinal mucosa in humans. However, various stress responses such as those involved in acid tolerance, acquisition of nutrients, resistance against reactive oxygen species (ROS), reactive nitrogen species (RNS), antimicrobial peptides and bile tolerance are a pre-requisite for the interactions with the mucosa to become possible. S. Typhimurium encodes several stress response genes which enable the adaption and growth in presence of different physiological stressors. Among the enteric pathogens, S. Typhimurium manifests an extreme example of bile resistance as it resides in the hepatobiliary tract and gall bladder during systemic infection. Bile is a digestive secretion that is involved in the emulsification and solubilisation of lipids. It is also a potent antimicrobial agent that denatures proteins, compromises membrane permeability, damages DNA and chelates essential micronutrients such as iron. Bile can induce secondary structure formation in RNA. Owing to its detergent properties, bile damages phospholipids and proteins in cell membranes and disrupts cellular homeostasis. Furthermore, Immunoglobulin A and mucus are also secreted into bile which act as antimicrobial agent and prevent bacterial adhesion, respectively. Therefore, the ability of pathogens such as S. Typhimurium to tolerate bile affects their survival and subsequent colonisation of the host gastrointestinal tract. One of the proteins essential for survival of S. Typhimurium in presence of bile is the RNA chaperone, Cold shock protein E (CspE). The isogenic strain of S. Typhimurium lacking cspE (ΔcspE) is highly sensitive to bile stress. A previous study from our laboratory has shown that CspE stabilizes the transcripts of stress response genes such as yciF in presence of bile. The uncharacterized gene yciF (STM14_2092) was induced in the wild type (WT) but not in the ΔcspE strain during bile stress. Importantly, overexpression of yciF in the ΔcspE strain significantly enhanced its growth in bile. However, the biological function and the underlying mechanisms of yciF mediated bile tolerance are not known. Besides yciF, CspE can bind and stabilize the transcripts of numerous other genes, many of which are not implicated in bile adaptation, so far. Therefore, the physiological significance of CspE in mediating bile tolerance is not fully deciphered. This study focuses on understanding the adaptive responses of S. Typhimurium during bile stress and identifying novel underlying mechanisms of bile resistance using WT and ΔcspE strains as a model system. In the first part of study, previously uncharacterized protein YciF which is a member of the domain of unknown function (DUF892) family was characterized and its role in bile stress response was delineated. In S. Typhimurium, there are few studies that suggest YciF is a stress response protein. Although, previously YciF has been shown to be involved in bile stress response of S. Typhimurium, the biochemical function as well as the intracellular mechanism of its action is unknown. In this study, the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium in vitro and in vivo was investigated. The DUF892 domain belongs to the ferritin superfamily. Members of this family form higher order oligomeric complexes. However, the crystal structure of both E. coli and S. Typhimurium YciF displays that YciF forms a dimer. Biochemical and biophysical studies with purified wild type YciF demonstrate that YciF forms higher order oligomers. The DUF892 domain is predicted to coordinate iron within the metal ion binding site. However, in the crystal structure Mg2+ occupies the metal ion binding site of YciF. Studies using thermal shift assay, in presence of different metal ions as possible ligands and iron-specific chromogen Ferene-S, display that YciF binds and retains iron. The metal ion coordination sitespecific mutants Q54A, E113Q and E143D were compromised in iron binding. Biochemically, YciF displays ferroxidase activity i.e., it catalyzes the conversion of ferrous ion (Fe2+) into ferric form (Fe3+). The ferric ion can then be stored within the protein. This activity was partially compromised in mutants Q54A and E113Q and completely lost in E143D. To understand the physiological relevance of ferroxidase activity of YciF, intracellular iron homeostasis was investigated. Through transcriptional analysis it was found that the ΔcspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in presence of bile. Several genes involved in iron uptake such as feoB, fepA, fhuC were significantly upregulated while those involved in iron storage such as ftnB and dps were suppressed in ΔcspE strain compared to wild type (WT) during bile stress. Also, the intracellular iron levels of bile treated ΔcspE strain was significantly higher than bile treated WT. Consequently, the possibility of Fenton reaction and iron toxicity was explored. It was observed that the bile mediated iron toxicity in ΔcspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild type YciF, but not the three mutants of the metal ion coordination site, in ΔcspE alleviate ROS in presence of bile. These results establish the role of YciF as a ferroxidase that can sequester excess iron in the cellular milieu to counter ROS-associated cell death. In fact, pre-treatment with an iron chelator mitigates the hypersensitivity of ΔcspE to bile. In addition, overexpression of YciF also increases the survival and growth of peroxide sensitive ΔcspE strain, providing direct evidence on the role of YciF in mitigating oxidative damage. In the second part of study, to better understand the stress and adaptive responses of S. Typhimurium to bile, mRNA-sequencing (RNA-seq) was performed. The transcriptional profiling of S. Typhimurium WT and ΔcspE strains revealed genes differentially expressed upon bile stress. A total of 466 genes were upregulated and 525 genes were downregulated in WT upon bile treatment. This constitutes 9.27% and 10.45% of the total 5,022 genes captured in the normalized expression data of RNASeq. As ΔcspE is sensitive to bile stress, a higher number of genes were differentially expressed compared to WT strain: 729 genes were upregulated whereas 834 genes showed downregulation in bile treated ΔcspE. The differential expression of genes obtained from RNA-seq was validated by q-PCR. To identify the major pathways that are affected in S. Typhimurium challenged with bile, a KEGG pathway analysis was performed with the differentially expressed genes in bile treated WT. The highest number of induced (144) as well as suppressed (59) transcripts were involved in metabolic pathways. Taken together, the expression of 203 genes belonging to various metabolic pathways was significantly altered in presence of bile. Metabolic pathways such as citrate cycle and glycerol metabolism were induced with bile treatment. A transcriptional upregulation of several genes involved in nitrate metabolism, in response to bile stress was observed. These genes were also differentially expressed between the bile-resilient WT and bile-sensitive ΔcspE strain. To understand the roles of nitrate metabolism in bile stress response, a strain lacking fnr (Δfnr) was generated. Fnr is the global regulator of nitrate metabolism in S. Typhimurium. fnr was induced in bile treated WT strain but not in the ΔcspE strain. Notably, the Δfnr strain was susceptible to bile-mediated killing. Therefore, a new role for fnr in mediating the bile stress response was established. Several genes belonging to nitrate-independent anaerobic metabolism such as dmsA, dmsC, eutK, eutL were transcriptionally repressed in bile-sensitive ΔcspE strain. In addition, a strain lacking arcA (ΔarcA), 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 ROS and significantly enhanced the survival and growth of the bile-sensitive ΔcspE strain. Importantly, 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. Overall, this study provides novel mechanistic insights into S. Typhimurium bile stress response. The protective role of a previously uncharacterized protein, YciF in bile stress was deciphered. The ferroxidase activity of YciF combats iron toxicity and oxidative damage caused due to exposure to bile. The biochemical and functional characterization of YciF delineates the significance of the DUF892 domain that has a wide taxonomic distribution encompassing several bacterial pathogens, in bacterial stress responses. This is the first study of characterization of a member of the DUF892 family. In addition, studies on S. Typhimurium bile stress response divulged the importance of comprehensive iron homeostasis in bacteria in presence of bactericidal compounds that tend to generate ROS irrespective of their primary targets. Analysis of the global transcriptome in the presence of bile revealed that bile stress results in major metabolic reprogramming. S. Typhimurium activates genes involved in anaerobic metabolism; specifically nitrate metabolism, that improves survival of bacteria during bile stress. The nitrate metabolism possibly subsides the cytotoxic effects of ROS generated during bile stress which promotes cell survival.