Coregulation by arcA and fnr protects Salmonella Typhimurium from bile stress by maintaining redox homeostasis and membrane integrity

Bile salts constitute a major antimicrobial barrier encountered by Salmonella Typhimurium during infections. Here, we identified the interplay of two global regulators, Fnr (fumarate and nitrate reductase) and ArcA (aerobic respiration control protein A), in determining adaptive responses to bile. We utilized WT, {Delta}fnr, {Delta}arcA and {Delta}arcA{Delta}fnr strains and compared the expression and functional responses of S. Typhimurium to bile. The highlights of this study are: First, the {Delta}arcA and {Delta}arcA{Delta}fnr strain form smaller colonies on LB agar plates and the {Delta}arcA{Delta}fnr strain displays lower motility. Second, qRT-PCR expression analysis demonstrates that fnr is induced early with bile, followed by arcA. Also, arcA transcripts are lower in the {Delta}fnr strain and fnr expression is also partially lower in the {Delta}arcA strain. Third, the {Delta}arcA and {Delta}fnr strains display partial sensitivity to bile, whereas the {Delta}arcA{Delta}fnr strain exhibits hypersensitivity to bile. Upon bile exposure, the {Delta}arcA{Delta}fnr strain displays elevated transcripts of the major antioxidant genes (sodA and katG) and outer membrane protein (ompC), higher induction of reactive oxygen species (ROS), and greater membrane damage. Fourth, intracellular nitrite is induced earlier than ROS with bile. Fifth, arcA and fnr protects S. Typhimurium from bile induced stress by activating the nitrate metabolism pathway, which lowers ROS. Functionally, pretreatment of the deletion strains with sodium nitrate reduces ROS, improves membrane integrity and survival with bile. Overall, these findings demonstrate that arcA and fnr function cooperatively to utilize alternate electron acceptors, reduce dependence on aerobic respiration and lower ROS to improve survival of S. Typhimurium during bile stress.