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

以菌株KZ 1647和菌株VPI 10467为培养基，分析了环境因素对艰难梭菌产毒的调控作用。在有葡萄糖存在的条件下，在生物素限制条件下，毒素产量显著增加，亮氨酸和异亮氨酸被优先吸收，在毒素滴度显著增加但细菌生长脱脂的孵育期间。添加天冬氨酸、谷氨酸、谷氨酰胺和赖氨酸可以抑制毒素的产生。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随着前三种氨基酸的增加而增加，而赖氨酸则没有。这些结果表明，赖氨酸的抑制机制不同于天冬酰胺、谷氨酸和谷氨酰胺。细胞质蛋白的SDS-PAGE分析显示，在生物素限制条件下，至少有130 kDa和145 kDa蛋白过表达。确定了蛋白质的n端序列，并进行了分子克隆，以阐明其在毒素产生中的作用。在没有葡萄糖的情况下，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，显著增加了毒素的产生。异亮氨酸浓度从20 ~ 100 mM增加，毒素产量显著增加，而细菌生长逐渐减少。在毒素滴度显著增加而细菌生长下降的潜伏期，与生物素限制条件下的葡萄糖一样，亮氨酸和异亮氨酸被优先吸收。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起重要作用。

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.