Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity

Abstract

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.

FIG. 1.

Purification and characterization of the hemolytic activity of the Hly-OppA protein from V. furnissii. (A) The extracellular medium from a V. furnissii culture (lane 1) was passed through Phenyl Sepharose 6 Fast Flow, Mono Q, and antibody-conjugated Sepharose 4B columns to obtain a homogeneous protein (lane 2) with a molecular mass of ∼58 kDa, as shown by sodium dodecyl sulfate-PAGE. (B) Native PAGE of purified Hly-OppA, showing a molecular mass of ∼120 kDa. (C) Hemolytic activity detected when the ammonium sulfate-precipitated protein fraction (lane 1) and antibody-conjugated purified Hly-OppA (lane 2) from native PAGE were embedded in a blood agar plate. (D) Immunoblot analysis with antiserum against Hly-OppA, revealing that both the crude (lane 1) and purified (lane 2) proteins yielded a single band. Lane M contained markers.

FIG. 2.

Dot blots demonstrating binding of the 9-mer oligopeptide library to purified Hly-OppA protein in a concentration-dependent manner (lanes 3 to 7). Lanes 1 and 2 contained Hly-OppA protein and BSA, which were positive (P) and negative (N) controls, respectively.

FIG. 3.

Vector construction for expression and knockout of the hly-oppA gene. (A) The full-length hly-oppA gene was treated with the NdeI and EcoRI restriction enzymes and inserted into a kanamycin-resistant pET28a(+) vector predigested with the same enzymes to generate pYKW1. (B) The same hly-oppA open reading frame was inserted into the NdeI/EcoRI sites of ampicillin-resistant pET3a(+) to generate pYKW3. (C) V. furnissii chromosomal DNA was used as a template for amplification of 162 bp of the 5′ end and 305 bp of the 3′ end of the hly-oppA gene. The 467-bp PCR fragment was cloned into an allelic exchange suicide vector, pCVD442, to generate the knockout plasmid pVF-hly-oppA-K. The pVF-hly-oppA-K plasmid was transferred from E. coli to V. furnissii to generate the V. furnissii hly-oppA knockout strain VFYKW1. The full-length hly-oppA gene was cloned into pCVD442 to generate pVF-hly-oppA-I and transferred to VFYKW1 to generate the V. furnissii VFYKW2 strain with hly-oppA restored.

FIG. 4.

Solute-binding activity and competitive inhibition tests using the fluorescent substrates EtBr (10 μM) and SYBR green (100 U) and various concentrations of ampicillin. The excitation (Ex) and emission (Em) wavelengths were 544 and 590 nm for EtBr and 485 and 538 nm for SYBR green, respectively. (A) Transport of EtBr in wild-type V. furnissii (•) or VFYKW1 (○). (B) Transport of EtBr in YKWEC1 or YKWEC2. □, EtBr alone; ▵, pET28a(+) alone; ⋄, pYKW1 alone; ○, pET28a(+) plus EtBr; •, pYKW1 plus EtBr. (C) Transport of SYBR green in wild-type V. furnissii (•) or VFYKW1 (○). (D) Transport of SYBR green in YKWEC1 or YKWEC2. ○, pET28a(+) plus SYBR green; •, pYKW1 plus SYBR green. (E) Ampicillin competitively inhibited transport of EtBr in YKWEC1 in a concentration-dependent manner. ▴, pYKW1 plus EtBr; •, pYKW1 plus EtBr plus 0.5 mg/ml ampicillin; ○, pYKW1 plus EtBr plus 2 mg/ml ampicillin. (F) SYBR green competitively inhibited transport of EtBr in YKWEC1 in a concentration-dependent manner. ▴, pYKW1 plus EtBr; •, pYKW1 plus EtBr plus 5 U SYBR green; ○, pYKW1 plus EtBr plus 50 U SYBR green.

FIG. 5.

Effect of the Hly-OppA protein on the V. furnissii hemolytic phenotype, erythrocyte lysis, morphology, and cytotoxicity in CHO-K1 cells. (A) Hemolytic phenotype of wild-type V. furnissii, VFYKW1, and VFYKW2 on TSA containing 5% sheep blood. (B) Erythrocyte lysis and hemoglobin release caused by purified Hly-OppA protein in the presence or absence of anti-Hly-OppA monoclonal antibody, as measured by the change in absorbance at 540 nm. Blank, PBS buffer; Negative control, BSA (0.5 μg/μl); Hly-OppA, 0.1 μg/μl Hly-OppA; Hly-OppA + anti Hly-OppA mAb, 0.1 μg/μl Hly-OppA plus 0.1 μg/μl anti-Hly-OppA monoclonal antibody; Hly-OppA + mouse serum, 0.1 μg/μl Hly-OppA plus 0.1 g/μl mouse serum; Positive control, 0.1% Triton X-100. (C and D) CHO-K1 cells were not exposed (C) or exposed (D) to the Hly-OppA protein (1 μg/ml) for 30 min at 37°C. (E) Dose-dependent cytotoxicity of the Hly-OppA protein in CHO-K1 cells. CHO-K1 cells were exposed to various concentrations of the Hly-OppA protein for 30 min, and the viability of the cells was determined using a commercial cytotoxicity assay kit. The data are the means and standard deviations from at least three independent experiments.

FIG. 6.

Scanning electron micrographs, biofilm productivity, and growth curves of wild-type V. furnissii and VFYKW1. (A) Micrograph of wild-type V. furnissii exhibiting a rod-shaped morphology. Magnification, ×10,000. (B) Diplococcus-shaped VFYKW1 with a “dehydrated string” morphology on the cellular surface. Magnification, ×10,000. (C) VFYKW2 exhibiting a rod-shaped morphology similar to that of the wild type. (D) Comparison of biofilm production by wild-type V. furnissii (V. furnissii WT) and VFYKW1 (hly-oppA mutant). OD₅₇₀, optical density at 570 nm. (E) Comparison of growth ratios of wild-type V. furnissii and VFYKW1. OD₆₀₀, optical density at 600 nm.

FIG. 7.

Susceptibility to various antibiotics of (A) the wild-type V. furnissii and VFYKW1 knockout strains and (B) recombinant strains YKWEC1 and YKWEC2. The concentrations of various antibiotics utilized in the experiment are described in Materials and Methods.

FIG. 8.

Characterization of in vitro hemolytic phenotypes of various Vibrio homologous ABC transporter proteins cloned and overexpressed in E. coli. Section 1, pRSET (negative control); section 2, pRSET-Hly-oppA from V. furnissii; section 3, pRSET-Hly-oppA from V. fluvialis; section 4, pRSET-Hly-oppA from V. vulnificus; section 5, pRSET-Hly-oppA from V. parahaemolyticus.