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Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni.

ABSTRACT: Copper is both a required micronutrient and a source of toxicity in most organisms, including Campylobacter jejuni Two proteins expressed in C. jejuni (termed CopA and CueO) have been shown to be a copper transporter and multicopper oxidase, respectively. We have isolated strains with mutations in these genes, and here we report that they were more susceptible to both the addition of copper in the growth media and to induced oxidative stress. Both mutant strains were defective in colonization of an avian host, and copper in the feed exacerbated the colonization deficiency. Overexpression of a cytoplasmic peptide derived from the normally periplasmic copper-binding region of CueO also caused copper intolerance compared to nonexpressing strains or strains expressing the non-copper-binding versions of the peptide. Taken together, the results indicate that copper toxicity in C. jejuni is due to a failure to effectively sequester cytoplasmic copper, resulting in an increase in copper-mediated oxidative damage.IMPORTANCE Copper is a required micronutrient for most aerobic organisms, but it is universally toxic at elevated levels. These organisms use homeostatic mechanisms that allow for cells to acquire enough of the element to sustain metabolic requirements while ensuring that lethal levels cannot build up in the cell. Campylobacter jejuni is an important foodborne pathogen that typically makes its way into the food chain through contaminated poultry. C. jejuni has a metabolic requirement for copper and encodes a copper detoxification system. In the course of studying this system, we have learned that it is important for avian colonization. We have also gained insight into how copper exerts its toxic effects in C. jejuni by promoting oxidative stress.

SUBMITTER: Gardner SP

PROVIDER: S-EPMC6182239 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni.

Gardner Susan P SP Olson Jonathan W JW

Journal of bacteriology 20181010 21

Copper is both a required micronutrient and a source of toxicity in most organisms, including <i>Campylobacter jejuni</i> Two proteins expressed in <i>C. jejuni</i> (termed CopA and CueO) have been shown to be a copper transporter and multicopper oxidase, respectively. We have isolated strains with mutations in these genes, and here we report that they were more susceptible to both the addition of copper in the growth media and to induced oxidative stress. Both mutant strains were defective in c ...[more]

PMID: 30150230

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Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.

Project description:Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OXR) in C. jejuni We further demonstrated that MutY malfunction did not directly contribute to the OXR phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OXR phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OXR mutants in bacterial organisms.IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OXR mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OXR mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, the same mechanism may operate in multiple bacterial species. Additionally, we developed a novel method that allows for rapid quantification of spontaneous OXR mutants in a bacterial population. This method represents a technical innovation and may also be applied to other bacterial species. These findings significantly advance our understanding of bacterial mechanisms for survival under oxidative stress.

Dataset Information

Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni.

Publications

Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni.

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