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首页> 外文期刊>Infection and immunity >Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae.
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Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae.

机译：霍乱毒素和霍乱弧菌识别的其他蛋白质在细胞外转运的结构信号的初步研究。

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The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis.
机译：霍乱弧菌用于霍乱毒素（CT）和其他蛋白质的细胞外转运的途径的特异性以几种不同的方式进行了检查。首先，测试霍乱弧菌分泌大肠杆菌II型不耐热肠毒素的B多肽的能力。通过电穿孔将pBluescriptKS-噬菌粒中编码LT-IIb的B多肽的基因引入霍乱弧菌。从霍乱弧菌转化子的培养物中收集培养物上清液和周质提取物，并通过酶联免疫吸附测定法测量肠毒素B亚基。结果证实霍乱弧菌分泌LT-IIa和LT-IIb的B多肽，其效率均与CT分泌的效率相当。其次，将质粒克隆导入霍乱弧菌的epsE突变体M14菌株。 M14未能将LT-IIa和LT-IIb的B多肽转运至细胞外介质，表明霍乱弧菌分泌II型肠毒素的过程与用于CT的细胞外转运的途径相同。这些数据表明在LT-IIa和LT-IIb中存在由霍乱弧菌的分泌机制识别的细胞外转运信号。此外，由于CT的B多肽与LT-IIa或LT-IIb的B多肽几乎没有（如果有的话）一级氨基酸序列同源性，因此转运信号可能是构象依赖性基序。第三，显示了CT的B亚基突变体（CT-B）在11位氨基酸上用赖氨酸替代了谷氨酸，尽管它的免疫反应性和神经节苷脂GM1结合活性与霍乱弧菌相当，但霍乱弧菌的分泌却很差。野生型CT-B。这些发现表明，Glu-11可能在CT-B的细胞外转运基序内或附近。最后，通过核苷酸序列分析确定了霍乱弧菌M14的epsE等位基因中的遗传损伤。

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