Contribution of the stereospecific methionine sulphoxide reductases MsrA and MsrB to oxidative and nitrosative stress resistance in the food-borne pathogen Campylobacter jejuni

Abstract

The microaerophilic food-borne pathogen Campylobacter jejuni is exposed to highly variable oxygen concentrations during its life cycle and employs a variety of protection mechanisms to resist oxidative stress. However, not all of the enzymes that mediate such protection have yet been identified. Two genes in strain NCTC 11168, Cj0637c and Cj1112c, are predicted to encode unrelated methionine sulphoxide reductases, which may repair oxidized methionine residues in proteins and thus contribute to oxidative stress defence. Cj0637 and Cj1112 were overexpressed, purified and shown by a coupled thioredoxin-thioredoxin reductase-NADPH assay to catalyse the stereospecific reduction of the S and R diastereoisomers, respectively, of the model compound methyl p-tolyl sulphoxide. Cj0637 is thus identified as MsrA and Cj1112 as MsrB. The contribution of these enzymes to oxidative and nitrosative stress resistance in C. jejuni was assessed by phenotypic analysis of a set of isogenic msrA, msrB and msrA/B insertion mutants. As RT-PCR data suggested a polar effect on Cj1111c in the msrB mutant, an msrB/ msrB + merodiploid complementation strain was also constructed. The msrA/B strain was severely growth inhibited under standard microaerobic conditions, whereas the msrA and msrB strains grew normally. Agar plate disc diffusion assays showed that all mutants displayed increased sensitivity to hydrogen peroxide, organic peroxide, superoxide, and nitrosative and disulphide stress, but quantitative cell viability assays showed that the msrA/B double mutant was markedly more sensitive to both oxidative and nitrosative stress. All of the stress-sensitivity phenotypes observed for the msrB mutant were restored to wild-type in the msrB/msrB + merodiploid. It is concluded that MsrA and MsrB make a significant contribution to the protection of C. jejuni against oxidative and nitrosative stress.

Gene Expression and Physiological Role of Pseudomonas aeruginosa Methionine Sulfoxide Reductases during Oxidative Stress

“…Superoxide anion-mediated oxidation of Met residues in proteins and free Met has been reported (53,54). In other bacteria, inactivation of msr genes has been shown to increase sensitivity to the superoxide generator paraquat (53); therefore, the physiological roles of P. aeruginosa msrA and msrB in superoxide stress protection were evaluated.…”

The Campylobacter jejuni Transcriptional Regulator Cj1556 Plays a Role in the Oxidative and Aerobic Stress Response and Is Important for Bacterial SurvivalIn Vivo

“…The incomplete reduction of oxygen to water creates reactive oxygen species (ROS) molecules, such as hydrogen peroxide (H 2 O 2 ), that are used by the host against invading pathogens (20). ROS are also released by the immune system to combat invading microorganisms (4). An example of ROS release is the deposition of various oxygen species generated by the respiratory burst oxidase as the bacterium remains bound within an endosome (41).…”

“…C. jejuni is a microaerophilic organism optimally suited to low levels of atmospheric oxygen; however, the bacterium is able to survive oxidative stresses in vivo (4). The incomplete reduction of oxygen to water creates reactive oxygen species (ROS) molecules, such as hydrogen peroxide (H 2 O 2 ), that are used by the host against invading pathogens (20).…”

“…C. jejuni infection has also been associated with postinfectious sequelae, including septicemia and neuropathies, such as Guillain-Barré syndrome (GBS) (52). C. jejuni is a commensal in avian species, and the consumption and handling of poultry products is a major source of human infection (4,39). However, a diverse range of environmental sources, such as untreated water, raw or unpasteurized milk, vegetables, and transmission from pets, are also all recognized sources of infection (55).…”

“…Oxidative, nitrosative, and aerobic (O 2 ) stresses are major factors that pathogens must counteract in order to survive within the host (4,25,57,87). C. jejuni is a microaerophilic organism optimally suited to low levels of atmospheric oxygen; however, the bacterium is able to survive oxidative stresses in vivo (4).…”