Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus.

An extracellular cytolytic toxin produced by the halophilic bacterium Vibrio vulnificus was isolated free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-75, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and isoelectric focusing in an ethylene glycol density gradient. The cytolysin is a heat-labile, hydrophobic protein that is inhibited by large amounts of cholesterol, is partially inactivated by proteases and trypan blue, has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid analysis) of ca. 56,000, and has an isoelectric point of ca. 7.1. The first 10 amino-terminal amino acid residues of the cytolysin are Gln-Glu-Tyr-Val-Pro-Ile-Val-Glu-Lys-Pro. Lysis of mouse erythrocytes by the purified cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step, followed by a temperature-dependent, membrane-perturbation step(s). In addition to possessing cytolytic activity against erythrocytes from 17 animal species and against Chinese hamster ovary cells in tissue culture, the purified cytolysin preparation was lethal for mice (ca. 3 micrograms/kg, intravenous 50% lethal dose) and had vascular permeability factor activity in guinea pig skin.     

The extracellular cytolysin of Vibrio vulnificus: inactivation and relationship to virulence in mice.

The ca. 51-kDa extracellular cytolysin of Vibrio vulnificus has been proposed as a virulence factor. We inactivated the structural gene for cytolysin in fully virulent, clinical V. vulnificus strains by both transposon mutagenesis and marker exchange techniques. Inactivation of the cytolysin did not affect virulence in our mouse models. The 50% lethal dose of cytolysin-negative strains was comparable to that of the cytolysin-positive parent strains after intraperitoneal inoculation with and without iron loading. Similar results were obtained after intradermal injection: cytolysin-positive and -negative strains had the same 50% lethal dose and caused comparable tissue damage. While we cannot say that the cytolysin has no effect on the pathogenesis of V. vulnificus infections, its role appears to be of much less importance than are other factors, such as encapsulation.     

Low density lipoprotein inactivates Vibrio vulnificus cytolysin through the oligomerization of toxin monomer

Blood lipoprotein has been shown to be an important defense factor against the bacterial infection. Lipoprotein is scavenger of bacterial endotoxin (lipopolysaccharide, LPS). However, there is little evidence showing its protective action against the bacterial exotoxin. We have previously demonstrated that cholesterol inactivates Vibrio vulnificus cytolysin (VVC) through oligomerization of the toxin monomer. The aim of the present study was to investigate the relationship between VVC and low-density lipoprotein cholesterol (LDL-C). LDL induced the oligomerization of VVC in a dose-dependent manner. The oligomerization of VVC monomer with LDL was demonstrated by immunoblotting, which exhibited 200-kDa bands corresponding to a tetramer of the native VVC of relative molecular weight of 51,000. Moreover, LDL inactivated hemolytic activity of VVC in a dose-dependent manner, and this response was not changed by the modifications of LDL (heat denaturation of proteins and peroxidation of phospholipids), suggesting that LDL-C is associated with the defense action against VVC. These results suggest that LDL-C inactivates VVC through the induction of toxin oligomerization.The first two authors contributed equally to this work     

The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene.

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.     

Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin

We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.     

Siderophore production by Vibrio vulnificus

Previous studies in our laboratory, as well as clinical evidence, have suggested that increased iron levels in the host may be important in infections caused by the halophilic pathogen Vibrio vulnificus. To study iron acquisition, we induced siderophore production by growth in a low-iron medium, and biochemical testing indicated the production of both hydroxamate- and phenolate-type siderophores. The siderophores were extracted from growth filtrates with ethyl acetate (for phenolates) and phenol-chloroform-ether (for hydroxamates). These extracts enhanced the growth of V. vulnificus when the bacterium was grown in iron-limited medium. The ability of these siderophores to stimulate the growth of Salmonella typhimurium LT-2 enb-7 (a mutant deficient in the biosynthesis of enterochelin) and Arthrobacter flavescens JG-9 (a hydroxamate auxotroph) supported the conclusion that V. vulnificus produces both hydroxamate- and phenolate-type siderophores.     

Purification and characterization of Vibrio metschnikovii cytolysin.

An extracellular cytolysin produced by Vibrio metschnikovii was purified by acid precipitation, phenyl-Sepharose CL-4B chromatography, and rechromatography on a phenyl-Sepharose CL-4B column and high-performance liquid chromatography on a Mono Q (anion-exchange) column. The purified cytolysin had a molecular weight of 50,000 and an isoelectric point of 5.1. It was inactivated by heating at 60 degrees C for 5 min and was inhibited by Zn2+, Cu2+, and high concentrations of cholesterol. Lysis of calf erythrocytes by cytolysin was temperature dependent and occurred only above 18 degrees C. Moreover, no lysis was observed at high concentrations of erythrocytes, suggesting that the cytolysin lyses erythrocytes by a multihit mechanism. This cytolysin had no immunological cross-reactivities with hemolysins from other Vibrio species tested, indicating that it is a new cytolysin. V. metschnikovii cytolysin lysed erythrocytes from several animal species (calf, rabbit, guinea pig, mouse, human, sheep, chicken, and horse) and cultured cells (Vero and Chinese hamster ovary), caused fluid accumulation in the intestines of infant mice, and increased vascular permeability in rabbit skin.     

Necrotising fasciitis caused by Vibrio vulnificus

A case of necrotising fasciitis caused by Vibrio vulnificus is described. The need for early recognition and aggressive surgical treatment are highlighted, and the necrotising infections due to V vulnificus described in the published work are reviewed.     

Vibrio vulnificus septicemia

A 33-year-old Japanese male, who had a three year history of biopsy-proved liver cirrhosis, was admitted to the hospital on June, 24, 1983 with a sudden onset of fever (38.6 degrees C), chills, generalized pain, nausea, anorexia, weakness, and eruption over the entire body. The patient went into shock and died about 7 hours after admission. Blood cultures before death were positive for V. vulnificus. Postmortem microscopic examination revealed "necrotizing vasculitis" in the small and large intestines, stomach, and skin, and also showed marked toxic epidermal necrolysis. This case matches the primary septicemia caused by V. vulnificus described by Blake et al. In addition, this case suggests that the septicemia was acquired through the gastrointestinal tract, especially the small intestine, because the V. vulnificus was isolated from blood and numerous Gram-negative bacilli around the submucosal vessels were observed in the area with acute necrotizing vasculitis.     

Vibrio vulnificus endometritis

Vibrio vulnificus most frequently causes wound infections contracted after exposure to seawater or primary septicemias resulting from the consumption of raw oysters. We report a case of endometritis caused by V. vulnificus. The infection was apparently acquired during the act of sexual intercourse in seawater in an area in which V. vulnificus has been frequently isolated. The efficacy of treatment with an antimicrobial regimen which included tetracycline is discussed.     

创伤弧菌溶细胞素体外诱导人脐静脉内皮细胞凋亡及其在细胞中的定位

目的 了解创伤弧菌溶细胞素(Vibrio vulnifru,cytolysin,WC)诱导人脐静脉内皮细胞(HUVEC)凋亡作用及其机制.方法 采用PCR扩增创伤弧菌GTC333株VVC编码基因vvhA,T-A克隆后测序并构建vvhA基因原核表达系统E.coli BL21DE3pET-42a-vvhA.采用Ni-NTA亲和层析法提纯目的 重组蛋白rVVC,SDS-PAGE联合Bio-Rad凝胶图像分析系统确定rVVC表达量及提取物纯度.采用溶血试验检测rVVC溶解兔红细胞的活性.2,4-二硝基苯肼比色法和四苯硼钠比色法分别测定rVVC作用的HUVEC培养物上清中乳酸脱氢酶(LDH)和K+含量.采用流式细胞术检测rVVC诱导HUVEC凋亡的作用.将FITC标记rVVC,采用激光共聚焦技术对HUVEC中的FITC标记rVVC进行定位.结果 与GenBank中相关序列比较,所克隆的vvhA基因核苷酸和氨基酸序列相似性分别为96.09%和98.26%.1μg/ml的rVVC即可溶解兔红细胞(P<0.01).10 μg/ml的rVVC可引起HUVEC培养物上清中K+含量明显增高(P<0.01),但LDH含量无明显改变(P>0.05).1～100μg/ml的rVVC作用2 h可使HUVEC凋亡.在FITC标记rVVC作用HUVEC 5～240 min内,rVVC逐渐从细胞膜表面向膜内侧和胞浆内移行,作用30 min时多数rVVC进入细胞.结论 rVVC具有溶细胞素活性.VVC可进入HUVEC,诱导细胞凋亡是其损伤HUVEC的主要机制.     

Purification and characterization of an extracellular cytolysin produced by Vibrio damsela.

Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium Vibrio damsela were obtained free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-100, and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The cytolysin is heat labile and protease sensitive and has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of ca. 69,000 and an isoelectric point of ca. 5.6. The first 10 amino-terminal amino acid residues of the cytolysin are Phe-Thr-Gln-Trp-Gly-Gly-Ser-Gly-Leu-Thr. The cytolysin was very active against erythrocytes from 4 of the 18 animal species examined (mice, rats, rabbits, damselfish) and against Chinese hamster ovary cells and was lethal for mice (ca. 1 microgram/kg, intraperitoneal median lethal dose). Lysis of mouse erythrocytes by the cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step followed by a temperature-dependent, membrane-perturbation step(s).     

Detection of anti-Vibrio vulnificus cytolysin antibodies in sera from mice and a human surviving V. vulnificus disease.

An enzyme-linked immunosorbent assay and a cytolysin neutralization assay were used detect anti-Vibrio vulnificus cytolysin antibodies in sera from mice and a human that survived V. vulnificus disease. The detection of antibodies against the cytolysin indicated that the cytolysin is produced in vivo, and this observation is consistent with the hypothesis that the cytolysin is involved in the pathogenesis of V. vulnificus disease.     

Extracellular phospholipase A2 and lysophospholipase produced by Vibrio vulnificus.

Phospholipase A2 and lysophospholipase activities were detected in the culture supernatant fluids of a virulent strain of Vibrio vulnificus. The phospholipase A2 was inactivated by heating at 56 degrees C for 30 min, had an apparent molecular weight of greater than or equal to 80,000 (estimated by gel filtration with Sephadex G-75), and a pI of ca. 5.0. Phospholipid hydrolysis was unaffected by Ca2+ or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and was optimal at pH 5.0 to 5.5. The lysophospholipase was not affected by heating at 56 degrees C for 30 min but was inactivated at 100 degrees C and had an apparent molecular weight of greater than or equal to 80,000 and a pI of ca. 4.0. The enzymes were detected coincidentally with a previously described extracellular cytolysin of V. vulnificus; however, they were physically separable from the toxin (which did not possess phospholipase A, C, or D activity) by gel filtration with Sephadex G-75.     

Activation of the plasma kallikrein-kinin system by Vibrio vulnificus protease.

Vibrio vulnificus protease enhanced hypodermic vascular permeability when injected into the dorsal skin of a guinea pig. Enhancement of permeability was observed within 2 min, and the permeability-enhancing reaction terminated at about 10 min postinjection. The permeability-enhancing reaction was greatly augmented by simultaneous injection of a kininase II inhibitor, whereas the reaction was inhibited by soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein. Furthermore, in vitro activation of plasma prekallikrein to kallikrein by V. vulnificus protease was observed. These results indicate that V. vulnificus protease enhances vascular permeability through activation of the plasma kallikrein-kinin system which generates bradykinin, factor in edema formation.     

A fatal case of Vibrio vulnificus septicemia.

A fatal case of Vibrio vulnificus septicemia in a 60 yr old man is described. This man displayed many of the classical features seen in fulminant infections with this organism. The epidemiology of V. vulnificus infections is discussed in this report.     

Detection of extracellular toxin(s) produced by Vibrio vulnificus.

Conditions are described for the production, in high titers, a heat-labile, antigenic, extracellular toxin(s) by Vibrio vulnificus, a recently recognized human pathogen. Bacteriologically sterile culture filtrate preparations obtained from mid-logarithmic-phase cultures of the bacterium possessed cytolytic activity against mammalian erythrocytes, cytotoxic activity for Chinese hamster ovary cells, vascular permeability factor activity in guinea pig skin, and lethal activity for mice. The specific activity of toxin preparations from cultures of a virulent strain of the bacterium was ca. 25-fold more than that of toxin preparations obtained from cultures of a weakly virulent strain. The four toxic activities were inseparable by gel filtration with Sephadex G-100; however, two components, which had markedly different elution behavior but which possessed the four activities mentioned above, were obtained. The major (ca. 88% of the recovered activity) and minor components had apparent molecular weights of ca. 38,500 and greater than 150,000, respectively.