Nitric oxide reactivities of the two globins of the foodborne pathogen Campylobacter jejuni: Roles in protection from nitrosative stress and analysis of potential reductants

作者：Tinajero Trejo Mariana; Vreugdenhil Angie; Sedelnikova Svetlana E; Davidge Kelly S; Poole Robert K^*

来源：Nitric Oxide-Biology and Chemistry, 2013, 34: 65-75.

DOI：10.1016/j.niox.2013.06.002

• Campylobacter jejuni
• Bacterial haemoglobin
• Nitrosative stress
• Reductase
• Heterologous expression
• Escherichia coli

Background: During infection and pathogenesis, Campylobacter, the leading cause of gastroenteritis, encounters NO and reactive nitrogen species (RNS) derived from the host. To combat these species, Campylobacter jejuni expresses two haemoglobins: the single domain haemoglobin (Cgb) detoxifies NO but the role of the truncated globin (Ctb) is unclear. Confirmation of Cgb activity and more extensive exploration of Ctb s) in vivo are restricted due to difficulties in expressing proteins in Campylobacter and our lack of understanding of how the globin haems are re-reduced after ligand reactions. %26lt;br%26gt;Methods: The cgb and ctb genes were cloned under the control of arabinose-inducible promoters and the globins expressed in an Escherichia cob mutant lacking the main NO detoxification mechanisms (Hmp and the Nor system comprising the transcription regulator NorR, the flavorubredoxin and its reductase (NorVW)); cellular responses under oxidative and nitrosative stress conditions were assessed. Spectroscopic changes of the Cgb and Ctb haems in soluble fractions after oxidation by NO were evaluated. Construction of E. cob nor mutants and a ubiquinone-defective strain allowed the exploration of the flavorubredoxin reductase and the aerobic respiratory chain as candidates for Cgb electron donors in E. coli mutants. %26lt;br%26gt;Results: Cgb, but not Ctb, complements the NO- and RNS-sensitive phenotype of an E. coli hmp mutant in aerobic conditions; however, Cgb fails to protect an hmp norR mutant in the absence of oxygen. Reduction of Cgb and Ctb in E. coli and C jejuni soluble extracts and turnover after NO oxidation is demonstrated. Finally, we report a minor role for NorW as a Cgb reductase partner in E. coli but no role for respiratory electron flux in globin redox cycling. %26lt;br%26gt;Conclusions: The NO detoxification capacity of Cgb is confirmed by heterologous expression in E. coli. The reducibility of Cgb and Ctb in E. coli and C.jejuni extracts and the lack of dependence of reduction upon flavorubredoxin reductase and the respiratory chain in E. cob argue in favor of a non-specific reductase system. %26lt;br%26gt;General significance: We present the most persuasive evidence to date that Cgb, but not Ctb, confers tolerance to NO and RNS by reaction with NO. Since certain hypotheses for the mechanism of haem re-reduction in E. cob following the reaction with NO are not proven, the mechanisms of reduction in C jejuni now require challenging experimental evaluation.

• 出版日期2013-11-1