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Colonization factor of Vibrio cholerae O1

霍乱弧菌 O1 定植因子

基本信息

批准号：
09670291
负责人：
IWANAGA Masaaki
金额：
$ 1.98万
依托单位：
University of the Ryukyus
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670291/
关键词：
Vibrio cholerae O1 colonization factor hemagglutinin OmpU LPS フコース結合蛋白

项目摘要

Sugar chain of LPS, OmpU, FSHA and NAGV14 pili of Vibrio cholerae were investigated to identify colonization factor(s) and protective antigen(s) of cholera vibrios.1) LPS sugar chain: When cholerae vibrios (V. cholerae O1 and O139) were treated with anti-LPS Fab, the adhesive ability was lowered but the level was not significant. Heat- or formaline-killed organisms maintaining the sugar chain did not adhere to the intestinal epithelium. A few strains with normal LPS which did not adhere to the intestinal epithelium were found. From these findings, LPS sugar chain was eliminated from the candidates of colonization factors of cholerae vibrios.2) OmpU: OmpU was purified and characterized. The purified OmpU adhered to the intestinal epithelium, however, the adhesion was not inhibited by treating the organisms with anti-OmpU and treating the intestine with purified OmpU. The antibody did not agglutinate the organisms, suggesting OmpU is not surface exposed. Therefore, OmpU is not supposed to function as a colonization factor.3) FSHA: We failed to purify L-fucose sensitive hemagglutinin (FSHA), therefore, genetic approach was made. Transposon (Tn5) was randomly inserted to the chromosome of Vibrio cholerae O1 strain 86B3, and hemagglutinin negative mutant was screened. Now we are on the process of identifying the position of hemagglutination gene.4) NAGV14 pili: NAGV14 pilus is a sole element which adhere to the intestinal epithelium found in V. cholerae. Gene analysis was carried out by using degenerate PCR, inverse PCR, PCR walking, and LA PCR cloning kit, based on the N-terminal amino acid sequence of the subunit protein of the pili. In conclusion, the gene encoding NAGV14 pili was not found in cholera vibrios but some restricted strains of Vibrio cholerae which are likely enteropathogenic.

通过对霍乱弧菌的LPS、OmpU、FSHA和NAGV 14皮利的糖链分析，确定霍乱弧菌的定植因子和保护性抗原。热或福尔马林杀死的微生物保持糖链不坚持肠上皮。结果显示，在大肠杆菌中，有少数菌株的LPS正常，且不粘附肠上皮，提示LPS糖链已被排除，可能是霍乱弧菌定植因子的候选者。2）OmpU：OmpU的纯化和鉴定。纯化的OmpU粘附于肠上皮，然而，用抗OmpU处理生物体和用纯化的OmpU处理肠不能抑制粘附。抗体不凝集微生物，表明OmpU未暴露于表面。因此，OmpU不应该作为定植因子发挥作用。3）FSHA：我们未能纯化L-岩藻糖敏感性血凝素（FSHA），因此，进行了遗传方法。将转座子Tn 5随机插入霍乱弧菌O 1株86 B3的染色体上，筛选出血凝素阴性的突变株。4）NAGV 14皮利：NAGV 14菌毛是霍乱弧菌肠上皮细胞的唯一粘附因子。根据皮利亚基蛋白的N端氨基酸序列，采用简并PCR、反向PCR、PCR步移和LA PCR克隆试剂盒进行基因分析。因此，NAGV 14皮利编码基因在霍乱弧菌中未发现，但在一些可能具有肠道致病性的限制性霍乱弧菌中发现。