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Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates
Published on: May 9, 2015
Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress
Annika Flint1, Yi-Qian Sun, James Butcher
1Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
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.
Insights
Campylobacter jejuni survival during host colonization depends on its oxidative stress defenses. This study identified 22 key genes involved in resisting reactive oxygen species (ROS) and found motility plays an indirect role in this defense.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Oxidative Stress Response
Background:
- Campylobacter jejuni encounters reactive oxygen species (ROS) during host colonization, necessitating robust defense mechanisms.
- Understanding these defenses is crucial for controlling C. jejuni infections in the gastrointestinal tract.
- Previous transcriptomic studies identified potential oxidative stress response genes in C. jejuni.
Purpose of the Study:
- To construct and screen a library of C. jejuni isogenic deletion mutants to identify genes involved in oxidative stress survival.
- To assess the role of these genes in host colonization using an in vivo chick model.
- To elucidate the mechanisms underlying oxidative stress resistance in C. jejuni.
Main Methods:
- Construction of 109 isogenic deletion mutants for identified oxidative stress response genes.
- Phenotypic screening of mutants for sensitivity/resistance to oxidants.
- In vivo colonization assays in a chick model.
- Analysis of motility mutants and the impact of fumarate supplementation and gene deletions on oxidant sensitivity.
Main Results:
- 22 C. jejuni genes were identified as critical for oxidant defense, showing hypersensitivity or hyposensitivity upon deletion.
- A nonmotile flagellum mutant (ΔmotAB) exhibited increased sensitivity to oxidants, suggesting an indirect role for motility.
- Superoxide sensitivity in the ΔmotAB mutant was restored by fumarate or deletion of ccoQ, implicating proton gradient disruption.
- Genes involved in cation transport, detoxification, and energy metabolism were confirmed as important for oxidant defense.
Conclusions:
- This study provides the first comprehensive analysis of C. jejuni oxidative stress defense genes using an isogenic deletion mutant library.
- Motility, cation transport, detoxification, and energy metabolism are significant factors in C. jejuni's resistance to oxidative stress.
- Understanding these defenses offers potential targets for controlling C. jejuni colonization and infection.
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