Functional characterization ofSalmonellaTyphimurium encoded YciF, a domain of unknown function (DUF892) family protein, and its role in protection during bile and oxidative stress

Abstract

YciF (STM14_2092) is a member of domain of unknown function (DUF892) family. It is an uncharacterized protein, involved in stress responses in Salmonella Typhimurium. In this study, we investigated the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium. Purified wild type YciF forms higher order oligomers, binds to iron and displays ferroxidase activity. Studies on the site-specific mutants revealed that the ferroxidase activity of YciF is dependent on the two metal binding sites present within the DUF892 domain. Transcriptional analysis displayed that the delta cspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in presence of bile. Utilizing this observation, we demonstrate that the bile mediated iron toxicity in deltacspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild type YciF, but not the three mutants of the DUF892 domain, in delta cspE alleviate ROS in presence of bile. Our 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 deltacspE to bile.

Global transcriptome analysis reveals Salmonella Typhimurium employs nitrate metabolism to combat bile stress

“…To understand the mechanism by which fnr rescued bile-sensitive DcspE strain, we quantitated the level of intracellular reactive oxygen species (ROS) as fnr has been reported to protect the cells against oxidative stress [45]. Consistent with the previous studies [7,[10][11][12], bile treatment led to an increase in intracellular ROS in the WT strain (Fig. 5B).…”

“…Bile also causes generation of reactive oxygen species, thereby causing oxidative damage to the cells Abbreviations cspC, gene encoding Cold shock protein C; cspE, gene encoding Cold shock protein E; FDR, false discovery rate; fnr, gene encoding fumarate and nitrate reductase protein; glpA/B/C/D, genes encoding glycerol-3-phosphate dehydrogenase subunits; glpK, gene encoding glycerol kinase; narG/H/I/K, genes encoding components of cytoplasmic nitrate reductase; narL, gene encoding two-component system response regulator for nitrate reductase; nirD, gene encoding nitrite reductase; nrfB, gene encoding periplasmic nitrite reductase; qRT-PCR, quantitative real-time polymerase chain reaction; RNA-Seq, RNA sequencing; ROS, reactive oxygen species; S. Typhimuirum, Salmonella Typhimurium; TCA cycle, Tricarboxylic Acid Cycle; WT, wild type strain; DcspE, mutant strain of Salmonella with deletion in cspE; Dfnr, mutant strain of Salmonella with deletion in fnr; DnarL, mutant strain of Salmonella with deletion in narL. [7,[10][11][12]. As a pathogen, S. Typhimuirum displays an extreme example of bile tolerance as it is able to colonize the hepatobiliary tract and gall bladder during infection [9,13].…”