Roles of Alkyl Hydroperoxide Reductase Subunit C (AhpC) in Viable but Nonculturable Vibrio parahaemolyticus

Abstract

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state in Vibrio parahaemolyticus. In this investigation, ahpC1 (VPA1683) and ahpC2 (VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these two ahpC genes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar-3% NaCl in the presence of the extrinsic peroxides H 2 O 2 and tert-butyl hydroperoxide (t-BOOH) at different incubation temperatures. The results revealed that both ahpC genes were protective against t-BOOH, while ahpC1 was protective against H 2 O 2 . The protective function of ahpC2 at 4°C was higher than that of ahpC1. The times required to induce the VBNC state (4.7 weeks) at 4°C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in an ahpC2 mutant and an ahpC1 ahpC2 double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and the ahpC1 mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (P < 0.03). Complementation with an ahpC2 gene reversed the effects of the ahpC2 mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the two ahpC genes and confirmed the particular role of ahpC2 in the VBNC state of V. parahaemolyticus.

Influence of oxyR on Growth, Biofilm Formation, and Mobility of Vibrio parahaemolyticus

“…This study demonstrated the antioxidative role of oxyR in V. parahaemolyticus (Table 4 and Fig. 1) in regulating the expression of the major functional ahpC1 gene and katE-homologous VPA1418 gene (Table 5) (9,10). Phenotypic defects associated with the oxyR mutant in this study may be associated with the decreased antioxidative activity.…”

“…5), but the swarming of this mutant was not significantly affected according to an assay with a 0.6% agar medium at 37°C (31; data not shown). In another study, we revealed decreased swarming mobility in the ahpC mutant of V. parahaemolyticus in agar medium that contained 1.5% agar and that was incubated at 30°C (10), and this result suggests that 0.6% agar medium at 37°C was not suitable for assaying the swarming mobility of this pathogen.…”

“…The amplicon was ligated to the pGEM-T Easy vector and transformed into the E. coli XL1-Blue strain. The complete gene sequence was cut with SalI and SphI and was ligated to the shuttle vector pSCB01, which had been digested with the same enzymes (10). The plasmid pSCB05, containing the complete sequence of oxyR, was propagated in E. coli SM10 -pir and was conjugated to the corresponding oxyR mutant to generate a complementary strain, which was selected by its chloramphenicol resistance (Table 1).…”

“…The levels of expression of genes (Table 5) were determined in the wild-type and oxyR mutant strains of V. parahaemolyticus using real-time reverse transcription-quantitative PCR (RT-qPCR) as described in our previous publications (9,10). Briefly, bacterial strains were cultivated statically in TSB-3% NaCl at 37°C, and the cultures in the exponential phase were challenged with 30 M cumene for 1.5 h. Bacterial cells were harvested by centrifugation and broken by TRIzol reagent (Invitrogen, United Kingdom), and RNA samples were extracted using an RNApure kit (Genesis Biotech Inc., Taipei, Taiwan) by following the manufacturer's instructions.…”

“…Recombinant plasmids for the ahpC1 and VPA1418 genes were used as calibration standards. The quality of the RNA samples and of the quantification protocols that were adopted for this study was evaluated by previously described methods (9,10). Statistical analysis.…”