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首页> 外文期刊>Infection and immunity >Purification and characterization of an extracellular secretogenic non-membrane-damaging cytotoxin produced by clinical strains of Vibrio cholerae non-O1.
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Purification and characterization of an extracellular secretogenic non-membrane-damaging cytotoxin produced by clinical strains of Vibrio cholerae non-O1.

机译：非霍乱弧菌临床株产生的细胞外分泌性非膜破坏性细胞毒素的纯化和鉴定。

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Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.
机译：一些临床的非O1霍乱弧菌菌株会产生一种细胞外因子，引起快速而戏剧性的细胞毒性反应，表现为中国仓鼠卵巢（CHO）和HeLa细胞的细胞变圆而没有膜损伤。进行这项研究是为了确定非膜破坏性细胞毒素（NMDCY）的身份，该毒素不受霍乱毒素，肠毒素性大肠杆菌的热不稳定毒素，El Tor溶血素，志贺样毒素I和志贺样毒素II，表明NMDCY与上述毒素和溶血素没有明显的免疫关系。脑心浸液和AKI培养基支持NMDCY的最大产量;发现AKI培养基的培养上清液没有溶血素活性，而在脑心脏输液中，溶血素与NMDCY共同产生。在37°C摇动条件下，将培养基的pH调节至8.5，观察到AKI培养基中NMDCY的最大产量。通过三步纯化程序将NMDCY纯化至均质，该步骤可将细胞毒素的比活性提高1.7 X 10（5）倍。纯化毒素的变性分子量为35,000，并且细胞毒素不耐热并且对胰蛋白酶敏感。细胞毒素的纯化显示出肠毒性活性，这由其在兔回肠环中积聚液体的能力所反映。 NMDCY的细胞毒性和肠毒性活性均可被针对纯化细胞毒素的抗血清抑制或中和，而未被免疫前血清所抑制。纯化的NMDCY和抗血清之间的免疫扩散测试给出了一个清晰的沉淀蛋白条带，该条带显示出完全相同的反应，而在免疫印迹分析中，在35 kDa区域中观察到了清晰的单个条带。我们的结果表明，NMDCY所表达的细胞毒性和肠毒性活性似乎与霍乱弧菌非O1菌株产生该细胞毒素的疾病有关。

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