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Analysis of regulation by environmental factors of toxin production by Clostridium difficile

环境因素对艰难梭菌产毒的调控分析

基本信息

批准号：
08457085
负责人：
NAKAMURA Shinichi
金额：
$ 5.25万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457085/
关键词：
C.difficile pseudomembranous colitis toxin toxin production biotin amino acid stress protein gene cloning 細菌毒素 Clostriclium difficile 合成培地

项目摘要

The regulation by environmental factors of toxin production by Clostridium difficile was analyzed with the defined media, using strain KZ 1647 in the presence of glucose and strain VPI 10467 in the absence of glucose. In the presence of glucose, with biotin-limited conditions under which the toxin production was remarkably increased, leucine and isoleucine were taken up preferentially, during the incubation period when toxin titers increased markedly but pacterial growth was deolining. The enhanced toxin production was inhibited by addition of aspargine, glutamic acid, glutamine and lysine. Bacterial browth was accerelated with the former three amino acids but suppressed with lysine. Consumption of leucine and isoleucine was progressive with the former three amino acids but not with lysine. These findings suggest that an inhibition mechanism of lysine is different from that of asparagine, glutamic acid and glutamine. SDS-PAGE analysis of the cytosolic proteins revealed that at least 130 kDa and 145 kDa proteins were overexpressed under biotin-limited conditions. The N-terminal sequences of the proteins were determined and the molecular cloning is under way to elucidate the role in toxin production. In the absence of glucose, supplementation with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. During the incubation period when toxin titers increased markedly but bacterial growth was declining, leucine and isoleucine were taken up preferentially as in the case of biotin-limited conditions with glucose. These findings suggest that leucine and isoleucine may play an important role in toxin production by C.difficile.

使用确定的培养基，在存在葡萄糖的情况下使用菌株KZ 1647，在不存在葡萄糖的情况下使用菌株VPI 10467，分析环境因素对艰难梭菌毒素产生的调节。在葡萄糖存在下，在生物素限制的条件下，毒素产量显着增加，在孵化期间，毒素滴度显着增加，但细菌生长脱酸，亮氨酸和异亮氨酸被优先吸收。添加天冬酰胺、谷氨酸、谷氨酰胺和赖氨酸可抑制毒素产生的增加。细菌浓度与前三种氨基酸有关，而与赖氨酸有关。前三种氨基酸的亮氨酸和异亮氨酸的消耗是渐进的，但赖氨酸的消耗却不是渐进的。这些发现表明赖氨酸的抑制机制不同于天冬酰胺、谷氨酸和谷氨酰胺。胞浆蛋白的 SDS-PAGE 分析表明，在生物素限制条件下，至少 130 kDa 和 145 kDa 蛋白过度表达。蛋白质的 N 末端序列已确定，分子克隆正在进行中，以阐明其在毒素产生中的作用。在缺乏葡萄糖的情况下，补充高水平（100 mM）的组氨酸、蛋氨酸、缬氨酸、异亮氨酸、脯氨酸和亮氨酸，特别是异亮氨酸，会显着增加毒素的产生。将异亮氨酸浓度从 20 mM 增加到 100 mM，毒素产生显着增加，而细菌生长逐渐减少。在孵化期间，当毒素滴度显着增加但细菌生长下降时，亮氨酸和异亮氨酸被优先吸收，就像在生物素限制条件下使用葡萄糖一样。这些发现表明亮氨酸和异亮氨酸可能在艰难梭菌的毒素产生中发挥重要作用。