Functional characterization of Salmonella Typhimurium 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 {Delta}cspE 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 {Delta}cspE to bile. ImportanceThe DUF892 domain has a wide taxonomic distribution encompassing several bacterial pathogens. This domain belongs to the ferritin-like superfamily; however, it has not been biochemically and functionally characterized. This is the first report of characterization of a member of this family. In this study, we demonstrate that S. Typhimurium YciF is an iron binding protein with ferroxidase activity, which is dependent on the metal binding sites present within the DUF892 domain. It combats iron toxicity and oxidative damage caused due to exposure to bile. The functional characterization of YciF delineates the significance of the DUF892 domain in bacteria. In addition, our 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.