Biochemical characterization of a catalase from Vibrio vulnificus , a pathogen that causes gastroenteritis

Vibrio vulnificus is a virulent human pathogen causing gastroenteritis and possibly life threatening septicemia in patients. Most V. vulnificus are catalase positive and can deactivate peroxides, thus allowing them to survive within the host. In the study presented here, a catalase from V. vulnificus (CAT-Vv) was purified to homogeneity after expression in Escherichia coli. The kinetics and function of CAT-Vv were examined. CAT-Vv catalyzed the reduction of H2O2 at an optimal pH of 7.5 and temperature of 35°C. The Vmax and Km values were 65.8±1.2 U/mg and 10.5±0.7 mM for H2O2, respectively. Mutational analysis suggests that amino acids involved in heme binding play a key role in the catalysis. Quantitative reverse transcription-PCR revealed that in V. vulnificus, transcription of CAT-Vv was upregulated by low salinity, heat, and oxidative stresses. This research gives new clues to help inhibit the growth of, and infection by V. vulnificus.


INTRODUCTION
Vibrio vulnificus is a Gram-negative marine bacterium that grows in warm water and low salinity in coastal regions (Kitamura et al., 2016;Motes et al., 1998); it survives in a planktonic form associated with the mucosal surfaces of aquatic animals (Oliver, 2015;Pajuelo et al., 2016).Wound infection or oral infection result from exposure to seawater or from the handling of shellfish contaminated with V. vulnificus, generally causing mild gastroenteritis with diarrhea, vomiting, and abdominal pain (Chung et al., 2016;Strom & Paranjpye, 2000).However, V. vulnificus in the human gut can cause life-threatening septicemia when it crosses the intestinal mucosal barrier and enters the bloodstream (Lee et al., 2008).Over 90% of infections resulting in septicemia from V. vulnificus are associated with consumption of raw and/or undercooked oysters (Oliver, 2015).Therefore, efforts have been made to eliminate V. vulnificus in postharvest processing of oysters, including cool pasteurization (Melody et al., 2008), inactivation by high hydrostatic pressure (Ye et al., 2013), irradiation (Mahmoud, 2009), and treatment with sodium hypochlorite (Ramos et al., 2012).
Catalase activity protects bacteria from disinfection by hydrogen peroxide, which is used in hospitals to clean surfaces.Nakamura et al. had shown that microbial catalase suppresses generation of hydroxyl radicals in a disinfection system in which hydroxyl radicals are artificially generated by photolysis of H 2 O 2 (Nakamura et al., 2012).Catalase also plays a protective role against UVA radiation in both, planktonic cells and biofilms of Pseudomonas aeruginosa, a versatile bacterium present in terrestrial and aquatic environments and an important opportunistic pathogen of humans (Pezzoni et al., 2014).Therefore, it is possible that inactivation of catalase can potentiate the bactericidal effects of different disinfection systems.
Despite the importance of catalase in protecting against oxidative stress and in infection, little is known about the function of catalase in V. vulnificus.Draft or complete genome sequences of several V. vulnificus strains have been reported (Chung et al., 2016;Efimov et al., 2015).These genomes generally contain one catalase.In the study presented here, recombinant, His-tagged catalase from V. vulnificus (CAT-Vv) was efficiently produced in a bacterial expression system and purified by immobilized metal affinity chromatography.The enzyme was biochemically characterized and mutants were used to analyze catalysis.The expression level of CAT-Vv in V. vulnificus was also analyzed under different conditions.

Bioinformatic analysis.
Protein function was predicted using the NCBI Conserved Domain Search (Marchler-Bauer et al., 2015).Protein molecular weight and pI were calculated with the ExPASy Server (Gasteiger et al., 2005).Multiple protein sequence alignments were performed using the Clustal Omega program (Sievers et al., 2011).Unrooted phylogenetic trees were constructed with MEGA 6.0 using the Maximum Likelihood (NJ) method and bootstrap tests carried out with 1000 iterations (Tamura et al., 2013).Signal peptides were predicted using SignalP 4.0 (Petersen et al., 2011).
Protein expression and purification.V. vulnificus ATCC 43382 was cultured at 37°C in Luria-Bertani (LB) broth supplemented with 0.85% NaCl.DNA was recovered from harvested cell pellets using the Qiagen Genomic DNA Isolation kit (Valencia, CA, USA).
V. vulnificus genomic DNA was used as a template in a polymerase chain reaction (PCR), which amplified CAT-Vv (KTL38510) using the following oligonucleotide primers: forward, 5′-CGG GGATCC CAA ACG CTT A CTC GTG A-3′ and reverse, 5′-CCG CTCGAG TTA CAT CGA CGC CA-3′.The primers introduced BamHI and XhoI restriction sites (underlined), respectively.The PCR product and vector pET28(a) were digested with BamHI and XhoI, and ligated.The construct was sequenced by the Beijing Genomics Institute (BGI; Shenzhen, China) before transformation into Escherichia coli BL21 (DE3).E. coli BL21 (DE3) cells containing the pET28-CAT-Vv plasmid were cultured in LB broth at 37°C.When OD 600 reached 0.7, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce protein expression.The cells were cultured in the presence of IPTG for 4 h with shaking, at 37°C, and then harvested and resuspended in a lysis buffer containing 50 mM Tris (pH 7.5), 300 mM NaCl, and 20 mM imidazole.The cell suspension was sonicated at 30% power for 3 min and centrifuged at 25 000 × g for 20 min, and the supernatant was loaded onto a Ni-NTA column.After washing the column with lysis buffer, CAT-Vv was eluted using an imidazole gradient (50-250 mM) in lysis buffer.Fractions containing catalase activity were pooled and dialyzed overnight against 50 mM Tris buffer containing 100 mM NaCl, pH 8.0.Purified CAT-Vv was separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie Brilliant Blue staining.Protein concentrations were estimated using the Bradford method and bovine serum albumin as a standard (Bradford, 1976).
Assays of CAT-Vv activity.Catalase activity was routinely determined spectrophotometrically at 35°C by monitoring the decrease in absorbance at 240 nm of 10 mM H 2 O 2 in 50 mM MES buffer (pH 7.5).One unit is defined as the amount of enzyme that catalyzed the oxidation of 1 µmol of H 2 O 2 min -1 under the assay conditions used here.
To determine the influence of temperature on the enzymatic activity, reactions were performed at temperatures ranging from 10 to 50°C.The influence of pH on CAT-Vv activity was determined using the protocol described above except that the Tris-HCl buffer was replaced with 50 mM MES (pH 5.0-7.5),50 mM HEPES (pH 8.0-8.5), or 50 mM glycine (pH 9.0-10.0);all of these assays were performed at the optimal temperature.
For kinetic studies, the initial velocities of the enzymatic reaction were examined by varying the concentration of H 2 O 2 (from 1 to 100 mM) under the optimal conditions (pH 7.5, 35°C, in 50 mM MES buffer).Values of the Michaelis constant (K m ) and maximal velocity (V max ) were obtained by calculations using Sigma Plot software with a linear model.Every point was determined by three separate experiments.
Quantitative reverse transcription-PCR (RT-qPCR).Culture of V. vulnificus was carried out as described above.Bacterial cells in the exponential phase were harvested by centrifugation and washed in an equal volume of phosphate-buffered saline (PBS, pH 7.0) with 0.85% NaCl.For the preparation of the low-salinity-adapted culture, the washed bacterial cells were incubated for 30 min in an equal volume of PBS containing 0.12% NaCl plus 20 amino acids to prevent interference from nutrient starvation, as previously described (Wong & Liu, 2008).To provide heat and oxidative stress, cells in the exponential phase were shocked by exposure to 42°C or 5 mM H 2 O 2 for 30 min.The cells were harvested and RNA was prepared with the TRIzol reagent (Invitrogen, CA).To ensure complete removal of any contaminating DNA, all RNA preparations were treated with DNase (Thermo Scientific Fermentas, Germany).RNA was quantified with a spectrophotometer and cDNA was synthesized with the Universal RiboClone cDNA Synthesis System (Promega, USA) per the manufacturer's protocols.Real-time PCR was performed with the CFX96 Real-Time PCR System (Bio-Rad, USA), using SYBR Green PCR Master Mix (Toyobo, Japan).The reaction products were serially diluted to find the appropriate concentration for real-time PCR analysis using the following primers: 5′-GGT ACA CGC TCA AAC GCT TA-3′ and 5′-GCG GTT ATC GAA TTC TGG TT-3′ (with amplification efficiency of 95.6%).Reactions for detection of 16S rRNA (LOSI01000001) levels were used for normalization between experimental samples and primers used were: 5′-GTC GTA GTC CGG ATT GGA GT-3′ and 5′-GTA TTC ACC GTG GCA TTC TG-3′ (with amplification efficiency 102.8%).The relative fold changes were determined from the cycle threshold (C T ) values using the ∆∆C T method.The experiments were analyzed in three independent assays, with at least three technical replicates included in each PCR to ensure reproducibility.

In silico analysis of CAT-Vv
CAT-Vv, which has not been previously characterized, was identified by homology and domain searches of the V. vulnificus genome using the NCBI Conserved Domain Search (Marchler-Bauer et al., 2015).The CAT-Vv ORF contains 1545 nucleotides and encodes a protein of 514 aa, with a theoretical pI of 8.69 and theoretical MW of 57.21 kDa (http://web.expasy.org/compute_pi/).A phy-logenetic tree of proteins derived by the neighbor-joining method for CAT-Vv and catalases from archaea, bacteria, and eukaryotes is presented in Fig. 1.The tree places catalases from archaea in one cluster, while CAT-Vv and other catalases from bacteria were more closely related to the proteins from eukaryotes.Multiple sequence alignment had shown that the amino acid sequence of CAT-Vv shows 66% and 46% identity to the catalases from Lipotes vexillifer and Lasius niger respectively, 62% to that from Pseudomonas syringe, and 52% to the Bacillus pumilus sequence.Amino acid sequence analysis indicated that CAT-Vv contains a hydrophobic stretch of about 20 amino acids at the amino-terminus, which is most likely a signal sequence for secretion, while other homologs in bacteria lack this sequence.Multiple sequence alignment (Fig. 1B) revealed the presence of conserved residues responsible for heme binding, i.e., His72, Asn139, and Tyr353, as well as amino acids that form a channel connecting the active site of the enzyme to the external surface (Loewen et al., 2015;Rahi et al., 2011).

Cloning, expression, and purification of CAT-Vv
The CAT-Vv gene without the putative secretion signal at the N-terminus was amplified from V. vulnificus genomic DNA using PCR and inserted into the expression vector pET28a which adds an N-terminal 6×Histag to the protein.Recombinant CAT-Vv was expressed in E. coli after 1 mM IPTG induction (Fig. 2).The cells were collected by centrifugation and disrupted by sonication.The supernatants were applied to a nickel-affinity column and eluted using an imidazole gradient.Active catalase-containing fractions were pooled, and purified protein was visualized as a 58 kDa protein by SDS-PAGE (Fig. 2).Based on gel filtration chromatography analysis, CAT-Vv is a tetramer (~240 kDa) composed of 58 kDa subunits.The UV/visible spectrum of the purified catalase with a Soret peak at 407 nm indicated the presence of a heme prosthetic group (Fig. 2).

Optimal temperature and pH for activity
The optimal temperature and pH of purified CAT-Vv were determined (Fig. 3).The recombinant enzyme is active above 10°C, and then its activity increases with temperature.The highest catalytic activity toward H 2 O 2 was observed at 35°C.The relative activity of CAT-Vv increased with increasing pH and reached its highest val-ue at pH 7.5, then decreased to 60% of the maximum at pH 8.5.

Thermostability of CAT-Vv
To examine the thermostability of CAT-Vv, the enzyme was incubated at 37°C, 42°C, and 45°C at pH 7.5 in MES buffer.As Fig. 3 shows, the relative activity gradually reduced with increasing temperature or with extension of incubation time.At 37°C, the relative activity of CAT-Vv remained 80% after 2 h and 70% after 4 h.The relative activity reduced to 40% after 40 min at 45°C.

Effects of point mutations on enzymatic activity
The X-ray crystal structure of catalase revealed that the enzyme forms a hydrophobic channel that provides access from the protein surface to the active site; the channels of catalase are 25-55 Å from the protein surface to the heme (Hara et al., 2007).Multiple sequence  alignment had shown that in CAT-Vv, His72 and Tyr353 may bind heme, while Val71 and Phe158 may be involved in the channel formation (Fig. 1).To confirm the function of these amino acids, they were individually mutated to Ala.The mutant proteins were expressed and purified and the activities were measured (Fig. 3).The mutants in heme-binding residues (H72A and Y353A) had barely detectable catalase activity.The activity toward H 2 O 2 of the channel point mutants (V71A and F158A) was approximately 20% of that of wild-type CAT-Vv.These experimental findings confirmed that heme binding is very important for catalysis.

Enzyme kinetics
The kinetics of recombinant wild-type CAT-Vv were analyzed using H 2 O 2 as a substrate (Fig. 4).The reaction was performed in MES buffer (pH 7.5) at 35°C with H 2 O 2 concentrations ranging from 1 to 100 mM.The Michaelis-Menten equation was used to calculate the kinetic parameters.The K m and V max values for CAT-Vv were found to be 10.5 ± 0.7 mM and 65.8 ± 1.2 U/mg, respectively.

Expression profile of the CAT-Vv gene under stress conditions
Considering previous reports that the expression of catalase genes can be induced by various stresses, we used qRT-PCR to examine expression of the CAT-Vv gene in V. vulnificus in response to three different abiotic stresses.RNA was isolated from V. vulnificus cells grown under stress.Figure 5 shows the relative expression patterns of CAT-Vv.Under low-salinity or heat treatment, the transcript level of CAT-Vv had shown an obvious increase relative to unstressed cells and peaked at 15 min, after which the expression level decreased.Under oxidative stress treatment, the transcript level of CAT-Vv reached a peak at 5 min.These results suggest that CAT-Vv plays a role in defense against various stresses.

DISCUSSION
Catalase is found in plants, animals and microbes (Sooch et al., 2014).V. vulnificus, an opportunistic human pathogen, is responsible for the overwhelming majority of reported seafood-related deaths (Jones & Oliver, 2009).In this study, we cloned, expressed, and biochemically characterized a catalase from V. vulnificus (CAT-Vv) for the first time.The recombinant enzyme, with a subunit molecular mass of 58 kDa, exists as a tetramer with a native molecular mass of 240 kDa.Activity assays of the wild-type and mutant proteins had shown that hemebinding residues are critical for activity.The native transcription level of CAT-Vv was upregulated by salinity, heat, and oxidative stress.Considering the importance of catalase in protecting V. vulnificus, we propose that CAT-Vv could be a potential target to control the growth of V. vulnificus.
Previous studies had shown that monofunctional catalases could be divided into three distinct clades based on phylogenetic analysis of the conserved core region (Klotz et al., 1997;Nicholls et al., 2000).Catalases in clade 1 are mainly from plants but also include catalases of algal and bacterial origin.Clade 2 catalases are proteins with a large-subunit, from bacteria, archaea, and fungi.This clade of catalases shows a strong tolerance to denaturation by proteolysis and heat (Chelikani et al., 2004).Clade 3 catalases are from bacteria, archaea, and eukaryotes.There is no clear functional difference between clade 1 and clade 3 catalases (Hara et al., 2007).The calculated molecular mass of CAT-Vv (56,487.7 Da) is similar to those of clade 1 and 3 catalases (55-69 kDa).Sequence alignments and phylogenetic analysis indicated that CAT-Vv exhibits a high degree of similarity with clade 1 catalases (e.g., catalases from P. syringe and B. pumilus, Fig. 1) (Carpena et al., 2003;Loewen et al., 2015).Inter-  estingly, CAT-Vv contains a predicted N-terminal signal peptide directing the secretion of the enzyme which is absent from the catalases from P. syringe and B. pumilus (Fig. 1).
The distribution of V. vulnificus in the environment is positively correlated with salinity (20 to 25 ppm) (Randa et al., 2004).Fresh water is often used to wash seafood that harbors V. vulnificus.Thus, low salinity is a common stressor for this pathogenic bacterium in both natural and food-processing environments (Wong & Liu, 2008).RpoS, the sigma factor of V. vulnificus, plays an active role in low salinity adaptation, as well as resistance to diverse environmental stresses including exposure to hydrogen peroxide and acidic conditions (Tan et al., 2010).VvhA, a virulence factor of V. vulnificus, was also reported to be involved in osmoprotection and/or cold shock response (Smith & Oliver, 2006).Catalase is a ubiquitous enzyme that destroys hydrogen peroxide formed during oxidative stress.In the study presented here, our results demonstrate that CAT-Vv is upregulated by low salinity, heat, and oxidative stress.Pezzoni and coworkers (2014) reported that an extracellular catalase is an essential factor in the resistance of P. aeruginosa biofilms to UVA exposure.In addition, degradation of the host-produced H 2 O 2 by a secreted catalase is considered essential to the success of infection (Tanabe et al., 2009).Based on this evidence, we propose that CAT-Vv plays crucial roles in the survival, antioxidant defense, and infectivity of V. vulnificus.

Figure 1 .
Figure 1.Phylogenetic and sequence analysis of CAT-Vv and other catalases.(A) Molecular phylogenetic analysis of CATs from archaea (square), bacteria (triangle), and eukaryote (diamond) by Maximum Likelihood method generated using MEGA6.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.The accession numbers of the proteins are indicated after the names of the species.(B) Sequence alignment of CAT-Vv and other homologues.The residues involved in heme binding are indicated by triangles.The amino acid residues involved in the narrow channel are highlighted by stars.The predicted secondary structure is shown at the top of the alignment.α-Helices are represented as helices, β-strands are represented as arrows, β-turns are represented as "TT," and 310-helices are represented as η.Signal peptide is labeled by the broken line.

Figure 2 .
Figure 2. Purification and spectra of CAT-Vv.(A) Purification and gel filtration chromatography of CAT-Vv.The insert shows the proteins that were electrophoresed on a 12.5% SDS-polyacrylamide gel and stained with Coomassie brilliant blue G-250.Lane M, protein marker; the molecular mass standards are indicated at the left.(B) UV/visible spectrum of CAT-Vv in 50 mM MES buffer, pH 7.5.

Figure 3 .
Figure 3. Enzyme activity assays of CAT-Vv.(A) Optimal temperature of CAT-Vv activity.(B) Optimal pH of CAT-Vv activity.Different buffers were used to obtain the different pH of solutions used in this assay.MES buffer was used for pH 5.0 and 7.5; HEPES buffer was used for pH 8.0 and 8.5; glycine buffer was used for pH 9.0 and 10.0.(C) Effect of temperature on stability of recombinant CAT-Vv.The purified enzyme was pre-incubated at 37°C (triangle), 42°C (cycle) and 45°C (diamond) for different times and the residual activities of the enzymes were measured.(D) Relative activity of wild-type CAT-Vv, CAT-Vv-V71A, CAT-Vv-H72A, CAT-Vv-F158A, and CAT-Vv-Y353A.All experiments were performed in triplicate.The error bars mean the standard deviation of three measurements.

Figure 4 .
Figure 4. Kinetics assay of CAT-Vv.The velocity data obtained with the increase of substrate concentrations were fitted to the Michaelis-Menten equation by non-linear regression calculations.

Figure 5 .
Figure 5. Expression level of CAT-Vv gene under low salinity (A), heat (B), and oxidative (C) stresses.The culture and treatment of V. vulnificus were described as in "Materials and methods."The mRNA relative quantity of CAT-Vv from V. vulnificus cells treated by different stresses was measured by RT-qPCR and indicated as fold difference from the value of the untreated cells, which was taken as 1.Error bars indicate the standard deviations from three independent experiments.