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

用限定培养基分析环境因素对艰难梭菌毒素产生的调节，在葡萄糖存在下使用菌株KZ 1647，在葡萄糖不存在下使用菌株VPI 10467。在葡萄糖存在下，在限制生物素的条件下，毒素产生显著增加，亮氨酸和异亮氨酸被优先摄取，在孵育期间，毒素滴度显著增加，但细菌生长正在脱衬。添加精氨酸、谷氨酸、谷氨酰胺和赖氨酸可抑制毒素的产生。前三种氨基酸促进细菌生长，赖氨酸抑制细菌生长。亮氨酸和异亮氨酸的消耗与前三种氨基酸是渐进的，但与赖氨酸无关。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。胞质蛋白的SDS-PAGE分析显示，至少130 kDa和145 kDa的蛋白在生物素限制条件下过表达。确定了蛋白质的N-末端序列，目前正在进行分子克隆，以阐明其在毒素产生中的作用。在不存在葡萄糖的情况下，补充高水平（100 mM）的组氨酸、甲硫氨酸、缬氨酸、异亮氨酸、脯氨酸和亮氨酸，特别是异亮氨酸，显著增加毒素产生。从20到100 mM的异亮氨酸浓度增加显着增加毒素的生产，而细菌生长逐渐下降。在孵育期间，当毒素滴度显着增加，但细菌生长下降，亮氨酸和异亮氨酸优先采取的情况下，生物素有限的条件与葡萄糖。这些研究结果表明，亮氨酸和异亮氨酸可能发挥重要作用，艰难梭菌毒素生产。

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.