Expression, Purification And Characterization Of The Tlh Hemolysin Of Vibrio Campbellii

As one of the most important problems in cultured marine animals, bacterial pathogens have caused severe economic loss with the rapid development of aquaculture. Vibriosis is a common and widespread epidemic disease caused by Vibrio spp., which are normal inhabitants in aquatic environments. Because of the wide epidemic, rapid spreading, high incidence and high mortality, the study of vibriosis becomes the focus of the aquaculture disease. Vibrio campbellii is widely distributed in coastal and estuarine waters. It is a Gram-negative, facultative anaerobic marine bacteria, which is recognized as a serious pathogen among the Vibiro spp. for marine aquaculture animals. V. campbellii can cause the outbreak of the red leg-disease of shrimp (Penaeus orientalis), and it can cause abalone suffering from the pyosepticemia when the temperature is higher than 16℃. In addition, it causes the disease of the snails and juvenile fish. However, we know little about the pathogenicity mechanism of V. campbellii. Hemolysin, which is an exotoxin that attacks blood cell membranes and causes cell rupture,is arguably the most widely distributed toxin among pathogenic Vibrios.Our previous study-showed that V. campbellii VIB285 contained a heat-labile hemolysin (TLH) gene similar with the VHH hemolysin gene in V. harveyi. The nucleotide sequence of tlh shared 79.81% and 79.73% identity to the vhhA and vhhB of V. harveiy, the deduced amino acids of TLH shared 85% sequence identity to the VHH hemolysin of V. harveiy and it had a lipase domain (Lipase_GDSL) at the 147-406 amino acid sites, indicating that the TLH hemolysin may be a phospholipase. In this study, the open reading frame of tlh (1254 bp) from V. campbellii VIB285 was amplified using a specific primer set (VCAF2 and VCAR2). The forward primer (VCAF2:5'-CGGAATTCATGAATAAGACCATTACGTTACTTAGT-3') begins from the initiation codon and an EcoRI site was added at the 5'end of the gene, and the reverse primer (VCAR2:5'-CGCTCGAGGAATGGATGATTCGAAAGTTGGTC-3'). ends before the stop codon, and a Xhol site was addede. The PCR product was excised and inserted into the EcoRI/Xhol-cut expression vector pET-26b (+). The ligated plasmid was transformed into E. coli BL21 (DE3) for expression of the full-length tlh gene. The recombinant TLH hemolysin was successfully expressed in E. coli strain BL21 (DE3) as His-tag fused protein, with the induction of IPTG at a final concentration of 1 mM and the incubation temperature of 37℃for 3 h. After induction, the recombinant E. coli and supernatant showed strong hemolytic activity against flounder erythrocyte on fish blood agar plate and phospholipase activity on 1% egg yolk emulsion plate. The poly His tagged TLH hemolysin was purified by Ni-NTA His-Bind Resin according to the manufacturer's instructions. The molecular weight of the recombinant TLH hemolysin was about 42 kDa as assessed by SDS-PAGE.The purified TLH (0.41 mg/mL) showed strong hemolytic activity against flounder erythrocyte (the diameter of hemolysis circle on fish blood agar plate was 16 mm) and phospholipase activity (the diameter of the inner turbid zong on 1% egg yolk emulsion plate was 11 mm, and the outer clear ring was 15mm). Temperature stability tests showed that the optimum temperature for the hemolytic activity of the TLH hemolysin was 37℃, and the TLH hemolysin was relatively stable at temperature lower than 37℃. When incubated at 45℃and 55℃for 30 min, the activity of the TLH hemolysin loss 50% and 75%, respectively. Furthermore, the hemolytic activity was destroyed entirely by treatment for 30 min at 75℃, indicating it is a heat-labile hemolysin. The Optimum pH for the hemolytic activity of the TLH hemolysin was pH6. When the pH was 5 or 7, the activity of the TLH hemolysin loss 50%; the TLH hemolysin was relatively stable in pH5-10, and it resulted in rapid decline in its stability when the pH was higher than 10 or lower than 5. The effect of cations on hemolysis was determined by addition of various concentrations of metallic cations (Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Ba2+) as the chloride forms in 20 mM TBS (pH 7.5). It was found that only a few divalent cations can cause the decrease of the hemolytic activity, such as Ca2+ and Co2+, but monavalent cations and other divalent cations did not affect the hemolytic activity. Moreover, pathogenicity study showed that, TLH had lethal effect on zebrafish when injected intraperitoneally, with an LD50 dose of 2.158μg/g fish. The main symptom of the injected zebrafish was abdominal swelling and systemic bleeding. To our knowledge, this is the first report on purification and characterization of TLH hemolysin from V. campbellii. Purified TLH could be useful for vaccine development and a diagnostic tool for vibriosis.