Connell T D, Metzger D J, Wang M, Jobling M G, Holmes R K
Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
Infect Immun. 1995 Oct;63(10):4091-8. doi: 10.1128/iai.63.10.4091-4098.1995.
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.
通过几种不同方式研究了霍乱弧菌用于霍乱毒素（CT）及其他蛋白质细胞外转运的途径的特异性。首先，检测了霍乱弧菌分泌大肠杆菌II型不耐热肠毒素B多肽的能力。通过电穿孔将编码LT-IIb的B多肽的基因导入pBluescriptKS-噬菌体载体中的霍乱弧菌。从霍乱弧菌转化体培养物中收集培养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，LT-IIa和LT-IIb的B多肽均由霍乱弧菌分泌，其效率与CT分泌的测量效率相当。其次，将质粒克隆导入霍乱弧菌的epsE突变体M14菌株。M14无法将LT-IIa和LT-IIb的B多肽转运到细胞外培养基中，表明霍乱弧菌分泌II型肠毒素的途径与CT细胞外转运途径相同。这些数据表明，LT-IIa和LT-IIb中存在霍乱弧菌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，该转运信号可能是一个构象依赖性基序。第三，CT的B亚基（CT-B）在氨基酸位置11处用赖氨酸取代谷氨酸的突变体，尽管其表现出与野生型CT-B相当的免疫反应性和神经节苷脂GM1结合活性，但被证明由霍乱弧菌分泌较差。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。
