Activities of Alkyl Hydroperoxide Reductase Subunits C1 and C2 of Vibrio parahaemolyticus against Different Peroxides

Abstract

Alkyl hydroperoxide reductase subunit C gene (ahpC) functions were characterized in Vibrio parahaemolyticus, a commonly occurring marine food-borne enteropathogenic bacterium. Two ahpC genes, ahpC1 (VPA1683) and ahpC2 (VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of Vibrio vulnificus. The responses of deletion mutants of ahpC genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the ahpC1 mutant and ahpC1 ahpC2 double mutant in liquid medium was significantly inhibited by organic peroxides, cumene hydroperoxide and tert-butyl hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary ahpC1 gene. Inconsistent detoxification of H₂O₂ by ahpC genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an ahpC2 gene partially restored the peroxidase effect in the double ahpC1 ahpC2 mutant at 22°C. This investigation reveals that ahpC1 is the chief peroxidase gene that acts against organic peroxides in V. parahaemolyticus and that the function of the ahpC genes is influenced by incubation temperature.

INTRODUCTION

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium which frequently causes food-borne gastroenteritis in some Asian countries (1) as well as globally since the isolation of the first pandemic O3:K6 strains in 1996 (2). This bacterium inhabits seawater and is often isolated from seafood (1). Most clinical isolates of this pathogen are hemolytic on Wagatsuma agar (Kanagawa phenomenon-positive, KP⁺) and produce the major virulence factor, thermostable direct hemolysin (TDH).

Pathogenic bacteria such as V. parahaemolyticus have evolved sophisticated mechanisms to survive oxidative stresses caused by their metabolic activities, host defense systems, or environmental factors. Various reactive oxygen species (ROS) such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH) form in bacteria (3–5), and ROS are known to cause damage in all cellular components, including protein, DNA, and membrane lipids.

Several common antioxidative factors are typically used to detoxify ROS. One is alkyl hydroperoxide reductase subunit C (AhpC), which is the catalytic subunit of a family of peroxidases collectively known as peroxyredoxins or thiol peroxidases (TPx family) (6). A genome search of V. parahaemolyticus RIMD2210633 (7) revealed several putative AphC factors, including the VPA1683 and VP0580 genes, which have been designated ahpC1 and ahpC2, respectively (8).

The antioxidative activity of pathogenic bacteria is related to their survival, growth, and virulence under some environmental stresses. Our earlier studies revealed enhanced production of AhpC in V. parahaemolyticus cells subjected to concurrent cold stress and starvation (9). Our most recent study demonstrated that, at 4°C, ahpC2 has a stronger protective effect than ahpC1 and that ahpC2 is primarily responsible for inducing and maintaining the viable but nonculturable state (VBNC) of V. parahaemolyticus (8). However, the functions of ahpC1 and ahpC2 of V. parahaemolyticus have not been studied at temperatures that allow cell growth. In this investigation, the involvement of ahpC1 and ahpC2 in the growth of this pathogen under the challenge of extrinsic peroxides was investigated using mutant and complementary strains.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The V. parahaemolyticus strain KX-V231 (KP⁺, serotype O3:K6) used in this study was a clinical specimen isolated in Thailand. The ahpC mutants and complementary strains were constructed in our previous study (Table 1) (8). The bacterial cultures were frozen in the beads of Microbank cryovials and stored at −85°C (Pro-Lab Diagnostics, Austin, TX, USA). The bacteria were cultured at 37°C in either tryptic soy agar (Becton-Dickinson Diagnostic Systems, Sparks, MD, USA) supplemented with 3% sodium chloride (TSA–3% NaCl) or in tryptic soy broth–3% NaCl (TSB–3% NaCl). A 100-μl aliquot of the 16-h broth culture was inoculated into 5 ml of fresh TSB–3% NaCl and incubated at 37°C with shaking at 160 rpm for 2 h to allow cells to enter the exponential phase (approximately 10⁸ CFU/ml). This culture was then used as the inoculum in the following experiments. Chloramphenicol (final concentration of 5 μg/ml) was added to the medium as needed to cultivate the V. parahaemolyticus strains containing the complementary genes or cloning vector.

TABLE 1.

Bacterial strains and plasmids used in this study

V. parahaemolyticus strain or plasmida	Description
Strains
KX-V231	Wild type, serotype O3:K6, KP+, clinical isolate
ΔahpC1 strain	KX-V231 ΔahpC1
ΔahpC2 strain	KX-V231 ΔahpC2
ΔahpC1 ΔahpC2 strain	KX-V231 ΔahpC1 ΔahpC2
ΔahpC1 ΔahpC2/ahpC1 strain	ΔahpC1 ΔahpC2 strain complemented with ahpC1 from pSCB02
ΔahpC1 ΔahpC2/ahpC2 strain	ΔahpC1 ΔahpC2 strain complemented with ahpC2 from pSCB03
ΔahpC1 ΔahpC2/V strain	ΔahpC1 ΔahpC2 strain containing pSCB01
KX-V231/V	KX-V231 containing pSCB01
Plasmids
pSCB01	Derived from pBR328 and pDS132, mobRP4 Apr Cmr Tcr
pSCB02	pSCB01 with Tc::ahpC1 promoter-ahpC1
pSCB03	pSCB01 with Tc::ahpC2 promoter-ahpC2

^aAll strains were constructed as described previously (8). V, cloning vector.

Antioxidative activity on agar medium.

The V. parahaemolyticus cultures (100 μl) in the exponential phase were spread on a Mueller-Hinton agar (Becton-Dickinson) plate. A paper disc (6 mm; Creative Media Products, Taiwan) that had absorbed 10 μl of 260 μM H₂O₂ (Santoku Chemical Industries, Tokyo, Japan), 160 μM cumene (Alfa Aesar, Ward Hill, MA, USA), or 160 μM tert-butyl hydroperoxide (t-BOOH; Tokyo Kasei Chemicals, Tokyo, Japan) was then placed on the plate. After incubation at 37°C or 22°C for 24 h or at 12°C for 144 h, the sizes of the inhibition zones were measured.

Antioxidative activity in liquid medium.

The V. parahaemolyticus cultures in the exponential phase (200 μl) were dispensed into the wells of a microtiter plate, to which a final concentration of 125 μM H₂O₂, 50 μM cumene, or 160 μM t-BOOH was added, and incubated statically at 37°C or 22°C for 8 h or at 12°C for 72 h. To measure bacterial growth, absorbance was measured at 590 nm with an MRXII microplate reader (Dynex Technologies, Chantilly, VA, USA).

Survival of V. parahaemolyticus in oyster homogenate.

Refrigerated raw oysters were shipped to the laboratory and dehulled promptly upon arrival. Thirty grams of oyster meat was homogenized in 270 ml of phosphate buffered-saline (PBS) with a Waring blender operated at high speed for 2 min. The oyster homogenate was then stored at 4°C. Cell counting on a thiosulfate-citrate-bile salts-sucrose (TCBS) agar plate revealed no indigenous Vibrio cells in this homogenate. Ten milliliters of the oyster homogenate was inoculated with a 0.2-ml culture of V. parahaemolyticus in the exponential phase to a concentration approximating 10⁶ CFU/ml and incubated at 4°C for 30, 60, and 90 min and 24 h. The survivors on the TCBS plate were then counted.

Statistical analysis.

Experiments were performed in triplicate, and the data in each experiment were obtained from triplicate determinations. For data analysis, one-way analysis of variance (ANOVA) or a t test was performed at a significance level of an α of 0.05 using SPSS for Windows, version 11.0 (SPSS Inc., Chicago, IL, USA).

RESULTS

Analyses of ahpC genes and putative AhpC proteins.

The ahpC1 and ahpC2 genes of V. parahaemolyticus KX-V231 were cloned into pGEMT-Easy and sequenced (8). Their sequences were identical to those of the strain RIMD221066 (7).

The alignment analysis of the putative protein sequences revealed that both ahpC genes of V. parahaemolyticus encode thiol peroxidases, which contain two cysteine residues and have high similarity to homologous proteins of other Vibrio species (see Fig. S1 in the supplemental material). Two conserved cysteine residues are present at positions 45 and 164 in AhpC1 and at positions 50 and 171 in AhpC2 of V. parahaemolyticus. The putative AhpC1 proteins comprise 185 amino acid residues in V. parahaemolyticus and Vibrio vulnificus and have a similarity score of 89%. The AhpC2 of V. parahaemolyticus exhibited similarity scores of 93%, 91%, and 72% with the AhpC2 proteins of V. vulnificus, Vibrio cholerae, and Salmonella enterica serovar Typhimurium, respectively (see Fig. S1).

Antioxidative activities on agar medium.

At an incubation temperature of 37°C, the sizes of most inhibition zones formed by the wild-type and ahpC mutants against H₂O₂ and cumene did not significantly differ. The exception was the double mutant ΔahpC1 ΔahpC2 strain in which the inhibition zone was significantly larger than that of the wild-type strain against t-BOOH (Fig. 1A). At 22°C, the inhibition zone against H₂O₂ and the two organic peroxides was significantly larger in the ΔahpC1 ΔahpC2 strain than in the wild-type strain (Fig. 1B). When the assay was performed at 12°C for 144 h, bacterial growth was not significant near the disc loaded with t-BOOH. At 12°C, inhibition zones against H₂O₂ were smaller in the ΔahpC2 and ΔahpC1 ΔahpC2 strains than in the wild-type strain and ΔahpC1 strain. However, the inhibition zones against cumene were larger in the ΔahpC1 strain than in the wild-type strain (Fig. 1C).

FIG 1.

Susceptibility of wild-type and ahpC mutant strains of V. parahaemolyticus to peroxides. In this experiment, 10 μl of 260 μM H₂O₂, 160 μM cumene, or 160 μM t-BOOH was absorbed by a paper disc. The disc was then placed on a bacterial lawn on a Mueller-Hinton agar plate; the inhibition zone was observed after incubation at 37°C (A) or 22°C (B) for 24 h or after incubation at 12°C for 144 h (C). Open bar, wild-type KX-V231 strain; dotted bar, ΔahpC2 strain; striped bar, ΔahpC1 strain; solid bar, ΔahpC1 ΔahpC2 strain. The asterisk indicates a mean value that significantly differed from that for the wild-type strain in a t test at a P value of <0.05.

The effects of complementary ahpC1 and ahpC2 genes in the ΔahpC1 ΔahpC2 double mutant were assayed and compared to those of the same strain containing a cloning vector. Comparisons performed in agar medium supplemented with chloramphenicol revealed that the inhibition zone formed by the double mutant ΔahpC1 ΔahpC2 strain against organic peroxide cumene at 37°C or 12°C was significantly smaller in the presence of the complementary ahpC1 gene than in the presence of the ahpC2 gene or the cloning vector (see Fig. S2 in the supplemental material). Against H₂O₂, the strains did not significantly differ at 37°C. At 12°C, however, the inhibition zone against H₂O₂ was smaller for the ΔahpC1 ΔahpC2 strain containing the cloning vector than for strains complemented with the ahpC1 gene (see Fig. S2).

Antioxidative activities in liquid medium at 37°C.

The antioxidative activities of V. parahaemolyticus strains were assayed in TSB–3% NaCl supplemented with various peroxides at 37°C. The growth of the wild-type strain was similar to that of the ahpC mutants in the medium without additional peroxides (Fig. 2A), and the growth of these strains was unaffected by the presence of H₂O₂ (Fig. 2B).

FIG 2.

Growth of wild-type strain and ahpC mutants of V. parahaemolyticus in TSB–3% NaCl supplemented with different peroxides and incubated at 37°C, as follows: control without peroxides (A), 125 μM H₂O₂ (B), 50 μM cumene (C), and 160 μM t-BOOH (D). ahpC2 mutant, ΔahpC2 strain; ahpC1 mutant, ΔahpC1 strain; ahpC12, ΔahpC1 ΔahpC2 strain.

In the presence of cumene or t-BOOH, the growth of the wild-type strain did not significantly differ from that of the ΔahpC2 strain (Fig. 2C and D). The growth of the ΔahpC1 and ΔahpC1 ΔahpC2 strains was significantly slowed in the presence of cumene, and their absorbance levels reached as high as the absorbance of the wild-type strain after 8 h of incubation (Fig. 2C). The addition of t-BOOH did not markedly affect the growth of the strains under the experimental conditions (Fig. 2D).

Effect of incubation temperature on antioxidative activity.

Assays of the antioxidative activity of the V. parahaemolyticus wild-type and mutant strains in TSB–3% NaCl at incubation temperatures of 22°C and 12°C revealed that the growth of these strains was not significantly affected by H₂O₂ (data not shown). In contrast, the organic peroxides markedly inhibited growth of the ahpC mutant strains.

In the presence of cumene, the wild-type strain and the ΔahpC2 strain showed similar growth rates at 22°C (Fig. 3A) and at 12°C (Fig. 3C). In contrast, growth of the ΔahpC1 and ΔahpC1 ΔahpC2 strains in the presence of cumene was low at 22°C (Fig. 3A) and negligible at 12°C (Fig. 3C). At 22°C, the growth of the ΔahpC1 ΔahpC2 strain that had been inhibited by cumene was fully restored by the presence of the complementary ahpC1 gene and partially restored by the ahpC2 gene (Fig. 3B). At an incubation temperature of 12°C, the growth of the ΔahpC1 ΔahpC2 strain that had been inhibited by cumene partially recovered in the presence of the complementary ahpC1 gene but not in the presence of the ahpC2 gene (Fig. 3D).

FIG 3.

Effect of incubation temperature on growth of the wild-type strain and ahpC mutants of V. parahaemolyticus under cumene challenge. Strains were cultured in TSB–3% NaCl supplemented with 50 μM cumene and incubated at 22°C (A and B) or 12°C (C and D). For the experiments shown in panels B and D, 5 μg/ml chloramphenicol was added to the culture medium. WT/V, wild-type KX-V231 containing the cloning vector pSCB01; ahpC2 mutant, ΔahpC2 strain; ahpC1 mutant, ΔahpC1 strain; ahpC12, ΔahpC1 ΔahpC2 strain; ahpC12/C2, ΔahpC1 ΔahpC2 strain complemented with ahpC2; ahpC12/C1, ΔahpC1 ΔahpC2 strain complemented with ahpC1; ahpC12/V, ΔahpC1 ΔahpC2 strain containing the cloning vector pSCB01.

In the presence of t-BOOH, the growth rates of the wild-type strain and the ΔahpC2 strain were similar at 22°C (Fig. 4A) and were also similar at 12°C (Fig. 4C). In the ΔahpC1 and the ΔahpC1 ΔahpC2 strains (Fig. 4C), bacterial growth was weak at 22°C (Fig. 4A) and not statistically significant at 12°C (Fig. 4C). The growth of the ΔahpC1 ΔahpC2 strain inhibited by t-BOOH was significantly recovered by the presence of a complementary ahpC1 or ahpC2 gene at 22°C (Fig. 4B). At the incubation temperature of 12°C, the growth of the ΔahpC1 ΔahpC2 strain inhibited by t-BOOH was fully restored by the presence of the complementary ahpC1 gene but not by the ahpC2 gene (Fig. 4D).

FIG 4.

Effect of incubation temperature on growth of wild-type strain and ahpC mutants of V. parahaemolyticus under t-BOOH challenge. Strains were cultured in TSB–3% NaCl that was supplemented with 160 μM t-BOOH and incubated at 22°C (A and B) or 12°C (C and D). For the experiments shown in panels B and D, 5 μg/ml chloramphenicol was added to culture medium. Strains are as described in the legend of Fig. 3.

Survival of V. parahaemolyticus strains in refrigerated oyster homogenate.

Survival rates of the wild-type strain, the ΔahpC1 mutant, and the ΔahpC1 ΔahpC2 mutant were determined after refrigeration in oyster homogenate for 30, 60, and 90 min and 24 h. Levels of V. parahaemolyticus bacteria did not significantly differ (see Fig. S3 in the supplemental material).

DISCUSSION

V. parahaemolyticus is generally associated with seafood, which is typically stored at low temperature. In this study, deletion of the ahpC genes did not affect the survival of this bacterium in a refrigerated oyster homogenate (see Fig. S3 in the supplemental material) or after freeze-thaw treatment (data not shown). This suggests that, of the various antioxidative factors, these ahpC genes alone may not significantly affect the survival of this pathogen in food. Nevertheless, this study demonstrated the protective function of these ahpC genes against oxidative stress under defined laboratory conditions.

Some bacteria have two ahpC genes, which reportedly have different functions in some species. In Legionella pneumophila, ahpC1 is expressed after the exponential phase, and ahpC2 is expressed during the early exponential phase. Levels of mRNA are 7 to 10 times higher in the ahpC1 strain than in the ahpC2 strain. In the exponential phase, ahpC2 expression is significantly increased in the ahpC1 mutant, whereas that of ahpC1 is unchanged in the ahpC2 mutant, which indicates that the ahpC2 system compensates for oxidative stress (10). In V. vulnificus, AhpC1 is a typical NADH-dependent peroxiredoxin and forms a peroxide reductase system with AhpF (11), whereas AhpC2 promotes the growth of V. vulnificus at high salinity by scavenging ROS in cells (12). These investigations generally agree that ahpC1 is the chief functional peroxidase, whereas ahpC2 is an alternative peroxidase that functions under special stress conditions.

The experiments in this study suggest that AhpC1 is the chief functional peroxidase in V. parahaemolyticus cultured in a rich medium at suitable incubation temperatures. Our previous study indicated that the deletion of ahpC1 significantly harmed colony formation in V. parahaemolyticus strains cultured on an agar plate incubated at 22 or 30°C but not at 37°C (8). The inhibition zone assay in the present study could not effectively differentiate among the ahpC gene functions, whereas the growth in liquid medium clearly demonstrated that ahpC1 is the primary peroxidase (Fig. 2 to 4).

An earlier study of Escherichia coli found that H₂O₂ treatment stimulated expression of catalase, ahpC, and related genes, which are members of the OxyR regulon (13). In a catalase/peroxidase mutant of E. coli, expression of L. pneumophila ahpC1 or ahpC2 restored H₂O₂ resistance (10). In V. vulnificus, ahpC1 is the enzyme typically used to detoxify H₂O₂ and t-BOOH and is associated with the virulence of V. vulnificus (11). In fact, AhpC is known to localize in the cytoplasm and to have a wide range of potential substrates, including H₂O₂, organic peroxides, and peroxynitrite (14). The experimental results in the present study revealed that the AhpC proteins of V. parahaemolyticus protected against organic peroxides under the specified experimental conditions (Fig. 2 to 4). No detoxifying effect of these AhpC proteins against H₂O₂ was identified in the liquid culture medium (Fig. 2B), while an inconsistent effect in the inhibition zone experiment (Fig. 1) and a weak effect in colony-forming ability (8) were demonstrated in an agar medium.

The present study also demonstrated that incubation temperature is probably a critical factor in the function of ahpC genes in V. parahaemolyticus. Wang et al. (8) reported that ahpC2 has a protective effect in this pathogen, especially when V. parahaemolyticus is incubated at 4°C in a starvation medium. The present study revealed a critical protective role of ahpC1 against organic peroxides in experiments performed at 12 or 22°C (Fig. 3 and 4) but not at the common incubation temperature of 37°C (Fig. 2). An earlier comparison of AhpC production in different bacterial species at high or low incubation temperatures revealed protective effects of ahpC on Anabaena sp. under exposure to high temperature (47°C) (15). Expression levels of AhpC proteins in Acidithiobacillus ferrooxidans (16) and in Shewanella putrefaciens (17) are also increased by incubation at low temperatures of 2 to 15°C. Another study reported that the promoter of the putative ahpC gene in the Shewanella species is highly responsive to incubation at a low temperature of 4°C (18). In V. parahaemolyticus, incubation temperature may affect the expression, stability, and activities of catalases and AhpC proteins. At 37°C, compensatory effects of catalase or AhpC apparently limit the deleterious effects of ahpC mutations in V. parahaemolyticus (19, 20).

Thermal regulation of the expression and function of these antioxidative factors may involve rpoS, oxyR, toxRS, and other factors. The OxyR (VP2752) regulon is known to regulate expression levels of catalase genes and ahpC genes, which exhibit compensatory patterns in several bacteria (21), whereas the rpoS gene (VP2553) is a general stress response regulator (22, 23). ToxRS (VP0819-VP0820) is a two-component regulator that is common to Vibrio species, and its significant role in stress response has been demonstrated in V. parahaemolyticus (24). In V. parahaemolyticus, VPA1682 is a putative MarR family protein and homologous to the organic peroxide sensor and transcriptional regulator of both Bacillus subtilis (25) and some Gram-negative bacteria (26, 27). It may also function as a negative OhrR-like regulator (28). The regulation of various ahpC genes or other antioxidative factors in V. parahaemolyticus has not been studied.

In conclusion, this study demonstrates the different functions of ahpC1 and ahpC2 in V. parahaemolyticus. The ahpC1 gene is apparently the functional peroxidase gene with the largest protective effect against organic peroxides. The protective activity of ahpC genes is crucial to the growth of this pathogen under extrinsic oxidative stress, particularly at 12°C and 22°C and, to a lesser extent, at 37°C. The ahpC2 gene may provide an alternative peroxidase for partially restoring the peroxidase effect in the double ΔahpC1 ΔahpC2 mutant at 22°C.