title: Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress.
abstract: During host colonization, Campylobacter jejuni is exposed to harmful reactive oxygen species (ROS) produced from the host immune system and from the gut microbiota. Consequently, identification and characterization of oxidative stress defenses are important for understanding how C. jejuni survives ROS stress during colonization of the gastrointestinal tract. Previous transcriptomic studies have defined the genes belonging to oxidant stimulons within C. jejuni. We have constructed isogenic deletion mutants of these identified genes to assess their role in oxidative stress survival. Phenotypic screening of 109 isogenic deletion mutants identified 22 genes which were either hypersensitive or hyposensitive to oxidants, demonstrating important roles for these genes in oxidant defense. The significance of these genes in host colonization was also assessed in an in vivo chick model of C. jejuni colonization. Overall, our findings identify an indirect role for motility in resistance to oxidative stress. We found that a nonmotile flagellum mutant, the ΔmotAB mutant, displayed increased sensitivity to oxidants. Restoration of sensitivity to superoxide in the ΔmotAB mutant was achieved by fumarate supplementation or tandem deletion of motAB with ccoQ, suggesting that disruption of the proton gradient across the inner membrane resulted in increased superoxide production in this strain. Furthermore, we have identified genes involved in cation transport and binding, detoxification, and energy metabolism that are also important factors in oxidant defense. This report describes the first isogenic deletion mutant library construction for screening of relevant oxidative stress defense genes within C. jejuni, thus providing a comprehensive analysis of the total set of oxidative stress defenses.
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Annika
authors.initials: A
authors.lastname: Flint
authors.forename: Yi-Qian
authors.initials: YQ
authors.lastname: Sun
authors.forename: James
authors.initials: J
authors.lastname: Butcher
authors.forename: Martin
authors.initials: M
authors.lastname: Stahl
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Huang
authors.forename: Alain
authors.initials: A
authors.lastname: Stintzi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002169:Campylobacter Infections; D016123:Campylobacter jejuni / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002645:Chickens; D004195:Disease Models, Animal; D005407:Flagella / Q000502:physiology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species