Expression Of Vibrio Vulnificus VvhA Cytolysin Genes And The Cytotoxicity Of The Fusion Protein

Background and Objective Vibrio vulnificus, first described in 1979,is a halophilic gram-negative marine bacterium, causes infectious diseases in humans, especially in people who are immunocompromised or have underlying conditions such as liver cirrhosis, hemochromatosis or alcoholism. It mainly produces two types of disease: Primary sepsis via gastrointestinal infection, and severe wound infection. The primary sepsis caused by Vibrio vulnificus are observed with a mortality rate of more than 50%. And the wound infection caused by Vibrio vulnificus usually causes severe necrosis of the tissue within 24 hours. The pathogenic mechanism of Vibrio vulnificus infection remains unclear. Vibrio vulnificus cytolysin, produced by most pathogenic strains, is a water-soluble polypeptide with a Mr of 50,851. The purified cytolycin preparations were cytotoxic to Chinese hamster ovary (CHO) cells, hemolytic for mammalian erythrocytes. As a only extracellular cytotoxin, the cytolycinn plays an important part to contribute tothe virulence of Vibrio vulnificus. And because of its pore-forming character, the cytolycin has caused great interest in study of its cytotoxicity mechanism which remains unknow.For the objective of making a foundation for the further study of the pathogenic mechanism of Vibrio vulnificus and the future study of preparing Vibrio vulnificus genetic engineering vaccine and detection kit, in this study, the recombinant expression vector pET-32a(+)-whA-BL21DE3 was used to express whA fusion protein in E.coli BL21DE3 induced by IPTG. After purification and recovery, the expressed whA fusion protein was examined by SDS-PAGE. The cytotoxic effect of whA fusion protein was identified by trypan blue exclusion test. The effect of whA fusion protein to the contents of LDH and K+ inside cells were observed via 2, 4-dinitro-phenylhydrazine(2, 4-DPNH)chromatometry and tetraphenylboron sodium (TPhBNa) chromatometry respectively. The apoptosis induced by whA fusion protein was detected and quantitatively analyzed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) binding propidium iodide (PI) staining, and the morphologically observation of the apoptosis induced by whA fusion protein was undertaken by transmission electron microscope. At last whA fusion protein was labeled with FITC, and a positioning study of whA fusion protein in the cells was followed by laser confocal technique.Methods The recombinant expression vector pET-32a(+)-whA-BL21DE3was used to express whA fusion protein in E.coli BL21DE3 induced by IPTG. NTA-Ni His-Binding Resin was used to purify the products, which were verified by SDS-PAGE. The purified whA fusion protein was recovered by mean of dialysis, then was condensed, and at last was degermed by filtration. Then HUVEC cell line was cultured in 1640 medium with whA fusion protein or with both of whA fusion protein and tetraethyl ammonium (TEA). Then in order to identify the cytotoxic effect of whAfusion protein the cell line was examined by trypan blue exclusion test, and the means of 2, 4-dinitro-phenylhydrazine(2 , 4-DPNH)chromatometry and tetraphenylboron sodium (TPhBNa) chromatometry were used respectively to study the effect of whA fusion protein to the contents of LDH and K+ inside cells. Furthermore, the apoptosis induced by whA fusion protein was detected and quantitatively analyzed by flow cytometry using Annexin V -fluorescein isothiocyanate (FITC) binding propidium iodide (PI) staining, and the morphologically observation of the apoptosis induced by whA fusion protein was undertaken by transmission electron microscope. To make a further study, the purified whA fusion protein was labeled with FITC. Then HUVEC cell line was cultured in 1640 medium with the FITC labeled whA fusion protein, and at last a positioning study of whA fusion protein in the cells was followed by laser confocal technique.Results IPTG with the dosage of 1.0,0.5 and 0.1 mmol/L was able to efficientlyinduce the expression of whA fusion protein in pET-32a(+)-whA-BL21DE3 system, respectively. The product of whA fusion protein was mainly presented in ultrasonic precipitate and the output was approximate 30%-40% of the total bacterial proteins. When the cytotoxic effect of whA fusion protein to HUVEC cells was detected by trypan blue exclusion test The fusion protein killed HUVEC cells in a dose-dependent manner, more than 60% of the cells being dead by treatment with whA fusion protein at the concentration of O.lmg/ml or above, which were higher significantly than those in the normal cells(P<0.01). Compared with the normal cells, the changes of the enzymatic activity of LDH in the culture medium of the HUVEC cells treated with whA fusion protein was insignificantly (P>0.05). Compared with the normal cells, the concentrations of K+ in the culture medium of the HUVEC cells treated with whA fusion protein alone or both of whA fusion protein and TEA together were highersignificantly(P<0.01), but the concentrations of K+ between each group of the whA fusion protein treated cell had no significant difference(P>0.05). The apoptosis rates in the HUVEC cells induced by whA fusion protein were higher significantly than those in the normal cells(P<0.01). And whA fusion protein induced apoptosis of the HUVEC cells in a dose-dependent and also time-dependent manner. Typical morphological characters appeared in the HUVEC cells treated with whA fusion protein, but no character of cell necrosis had been found. The normal HUVEC cells and the HUVEC cells cultured in the FITC dialysed solution looked spreading out on the coverslip, and had no fluorescence under laser confocal scanning. The HUVEC cells which cultured in the FITC dialysed solution and treated with O.lmg/ml whA fusion protein for 4 h looked smaller and less spreading out, and had no fluorescence under laser confocal scanning. Under laser confocal scanning, the HUVEC cells which treated with O.lmg/ml whA fusion protein for 2min to 4 h respectively looked smaller and less spreading out, and had flavo-green both on the cellular membranes and inside the cells. Moreover, under laser confocal scanning, to compare with the HUVEC cells treated with O.lmg/ml whA fusion protein for 2min —20 min which had just small amounts of fluorophores on the cellular membranes and inside the cells, the fluorescence enhanced and the fluorophores became bigger along with the increase of time while the cells were treated for 30 min—4 h.Conclusion SDS-PAGE demonstrated that the constructed expressionsystem pET-32a(+)-whA-BL21DE3 of our laboratory was able to efficiently produce the target fusion protein presented with the form of inclusion body even if induced by IPTG at lower concentration of O.lmmol/L, and the output was approximate 30%-40% of the total bacterial proteins. As an expression product of pET-32a(+>whA-BL21DE3, whA fusion protein was cytotoxicity. And the cytotoxicity of whA fusion protein and natural Vibrio vulnificus cytolysin was alike.The intracellular K+ flow extracellularly when HUVEC cells were treated with whA fusion protein, while no intracellular LDH had leaked. The extracellular K+ flow of HUVEC cells caused by whA fusion protein appears quick and brief, and was not in a time-dependent manner, and did not be affected by Ca2+ dependent K+ channel blocker TEA. The cell damage of HUVEC cells caused by whA fusion protein is mainly via apoptosis induced by whA fusion protein. VvhA fusion protein could not only bind onto the cellular membrane but also went into HUVEC cells. Furthermore, whA fusion protein would gather together on the cellular membrane or in the HUVEC cells.