PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni

Abstract

The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile that is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here, we have characterized the roles of C. jejuni PerR in oxidative stress and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30 % H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin was increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-sequencing analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes

A Mutator Phenotype Promoting the Emergence of Spontaneous Oxidative Stress-Resistant Mutants in Campylobacter jejuni

“…Interestingly, C. jejuni, a Gram-negative bacterium, lacks OxyR and instead possesses the metalloregulator PerR, which is normally found in Gram-positive bacteria such as B. subtilis (26), Staphylococcus aureus (27), Enterococcus faecalis (28), and Streptococcus pyogenes (29). Although the insertional inactivation of PerR has been linked to increased aerotolerance and hyperresistance to H 2 O 2 in C. jejuni (10,12,30), spontaneous mutations conferring peroxide resistance in Campylobacter have not been described. In this study, the MutY mutation in the mutator isolate CMT was found to increase the spontaneous mutation frequency in perR, leading to malfunction of PerR, derepression of the PerR regulon, and consequently the increased emergence of OX R mutants in Campylobacter.…”

“…Multiple studies showed that C. jejuni perR mutants displayed significantly increased peroxide resistance and aerotolerance (10,12,30). For example, there were 2 to 3 log more surviving C. jejuni perR insertional mutants than there were wild-type cells after 9 h of incubation under aerobic conditions (30). Recently, aerotolerant C. jejuni and Campylobacter coli have been increasingly isolated from various sources, including chicken or retail meat samples (34-36).…”

“…However, elevated mutations in perR may facilitate Campylobacter survival under certain conditions, such as the food production environment, where Campylobacter is exposed to high-level oxidative stress. Multiple studies showed that C. jejuni perR mutants displayed significantly increased peroxide resistance and aerotolerance (10,12,30). For example, there were 2 to 3 log more surviving C. jejuni perR insertional mutants than there were wild-type cells after 9 h of incubation under aerobic conditions (30).…”

“…PerR plays an important role in defense against oxidative stress in C. jejuni (3,12,30). PerR represses the expression of katA and ahpC, and insertional mutation of perR results in overexpression of the KatA and AhpC proteins, which makes C. jejuni hyperresistant to peroxide stresses such as cumene hydroperoxide and hydrogen peroxide (10).…”

“…In order to investigate the role of the single-nucleotide changes in affecting the binding of PerR to the promoter regions regulated by PerR, EMSAs were performed by a procedure described previously (30,51), with some modifications. Briefly, primers KatAPromF/KatAPromR and DnaEPromF/DnaEPromR (30) were used to amplify the promoter regions of katA and dnaE, respectively.…”