Functional and Regulatory Characteristics of Vibrio vulnificus Alkyl Hydroperoxide Reductase (AhpC1) and Catalase (KatG)

Abstract

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpC1 and katG, homologues of E. coli ahpC encoding a peroxiredoxin and katG encoding a bifunctional catalase-peroxidase, respectively, were identified among the Vibrio vulnificus genes specifically induced by exposure to H2O2. The ahpC1 mutant was susceptible to various oxidative stress induced by H2O2, tert-butyl hydroperoxide (t-BOOH), and cumene hydroperoxide (CHP). In contrast, the katG mutant was more sensitive to H2O2 than the ahpC1 mutant and was not sensitive to oxidative stress induced by t-BOOH and CHP. The purified AhpC1 reduced H2O2 and t-BOOH in the presence of AhpF and NADH as a hydrogen donor. It revealed that V. vulnificus AhpC1 is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. In addition, the catalase activity of KatG was determined using native polyacrylamide gel electrophoresis, indicating that V. vulnificus KatG has an antioxidant function against H2O2. Compared to wild type, the ahpC1 and katG mutants exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. To define the promoters of ahpC1 and katG, PahpC1 and PkatG, primer extensions were performed, showing that the transcriptional start sites of ahpC1 and katG were determined and putative -10 and -35 regions of the promoters were predicted. In addition, there were OxyR binding consensus sequences located in the upstream of -35 regions of PahpC1 and PkatG. In order to verify whether the redox sensing transcriptional regulator, OxyR, regulates the expression levels of ahpC1 and katG in V. vulnificus, qRT-PCR was performed using the RNA derived from wild type and the oxyR mutant treated with H2O2. The results showed that the expression levels of both ahpC1 and katG in the oxyR mutant were significantly reduced as compared to those in the wild type. It indicated that the expression of ahpC1 and katG is positive regulated by OxyR under oxidative stress. Both gel mobility shift assay and DNase I footprinting experiment using either oxidized or reduced OxyR revealed that the two forms of OxyR binds to the OxyR binding consensus sequences located in the PahpC1 and PkatG. Interestingly, reduced OxyR bound to the -35 region of PahpC1 and -10 region of PkatG, indicating that the reduced OxyR may block RNA polymerase binding to the PahpC1 and PkatG. In order to confirm that the expression of ahpC1 and katG was repressed by reduced OxyR, C199S-OxyR mutant, mimicking reduced OxyR, was constructed and then qRT-PCR was performed using the RNA isolated from wild type and C199S-OxyR mutant treated with H2O2. Compared to the expression levels of ahpC1 and katG in wild type, those in C199S-OxyR mutant were significantly reduced, indicating that reduced OxyR repressed the expression of ahpC1 and katG. In some bacteria, it has been known that ahpC and ahpF are an operon. In order to confirm whether the V. vulnificus ahpC1 and ahpF genes are an operon, northern blot analysis was performed, indicating that transcription of ahpC1F results in two different transcripts, ahpC1 transcript and ahpC1F transcript. Mutational analysis of the regulatory region suggested that the ahpC1F operon has a single promoter, PahpC1. These results indicated that the ahpC1 transcript results from attenuation of the ahpC1F transcript at the ahpC1 and ahpF intergenic region. The 3end of ahpC1 transcript was determined by 3RACE and a potential stem-loop structure responsible for the transcriptional attenuation was predicted in the ahpC1 and ahpF intergenic region. To confirm whether the transcription of ahpC1F can be attenuated by the stem-loop structure, a reporter containing the PahpC1 fused to promoterless lux genes was constructed. Several DNA fragments of the ahpC1 and ahpF intergenic region were inserted downstream of PahpC1, and only the DNA fragment containing the predicted stem-loop structure had the reporter RLU reduced. These results indicated that the ahpC1F operon is regulated by a single promoter and the ahpC1 and ahpF intergenic region attenuator.