The Small Protein CydX Is Required for Cytochrome Quinol Oxidase Stability and Function in Salmonella Enterica Serovar Typhimurium: a Phenotypic Study

Cytochrome quinol oxidases, which have a greater affinity for oxygen than heme-copper cytochrome oxidases (HCOs), promote bacterial respiration and fitness in low-oxygen environments, such as host tissues. Here, we show that, in addition to the CydA and CydB subunits, the small protein CydX is required for the assembly and function of the cytochrome complex in the enteric pathogen serovar Typhimurium. Mutant Typhimurium lacking CydX showed a loss of proper heme arrangement and impaired oxidase activity comparable to that of a Δ mutant lacking all cytochrome subunits. Moreover, both the Δ mutant and the Δ mutant showed increased sensitivity to β-mercaptoethanol and nitric oxide (NO). Cytochrome -mediated protection from β-mercaptoethanol was not a result of resistance to reducing damage but, rather, was due to cytochrome oxidase managing respiration, while β-mercaptoethanol interacted with the copper ions necessary for the HCO activity of the cytochrome -type quinol oxidase. Interactions between NO and hemes in cytochrome and cytochrome -dependent respiration during nitrosative stress indicated a direct role for cytochrome in mediating resistance to NO. Additionally, CydX was required for Typhimurium proliferation inside macrophages. Mutants deficient in cytochrome , however, showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. The essential role of CydX in cytochrome assembly and function suggests that targeting this small protein could be a useful antimicrobial strategy, but potential drug tolerance responses should also be considered. Cytochrome quinol oxidases, which are found only in bacteria, govern the fitness of many facultative anaerobic pathogens by promoting respiration in low-oxygen environments and by conferring resistance to antimicrobial radicals. Thus, cytochrome complex assembly and activity are considered potential therapeutic targets. Here we report that the small protein CydX is required for the assembly and function of the cytochrome complex in Typhimurium under stress conditions, including exposure to β-mercaptoethanol, nitric oxide, or the phagocytic intracellular environment, demonstrating its crucial function for fitness. However, cytochrome inactivation also leads to increased resistance to some antibiotics, so considerable caution should be taken when developing therapeutic strategies targeting the CydX-dependent cytochrome .