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

分别用KZ1647菌株和VPI 10467菌株在有葡萄糖和无葡萄糖条件下对艰难梭菌产毒的环境因素进行了研究。在葡萄糖存在下，在毒素产量显著增加的生物素限制条件下，在毒素效价显著增加但细菌生长脱脂的潜伏期，亮氨酸和异亮氨酸被优先摄取。添加天冬氨酸、谷氨酸、谷氨酰胺和赖氨酸可抑制毒素的增加。前三种氨基酸促进细菌褐变，赖氨酸抑制细菌褐变。亮氨酸和异亮氨酸的消耗量随前三种氨基酸的增加而递增，但与赖氨酸的消耗量不同。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分析表明，在生物素限制的条件下，至少有130 kDa和145 kDa的蛋白过表达。蛋白质的N末端序列已经确定，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 mM增加到100 mM时，毒素产量显著增加，细菌生长逐渐减弱。在毒素滴度显著增加但细菌生长下降的潜伏期内，亮氨酸和异亮氨酸被优先摄取，就像在生物素限用葡萄糖的情况下一样。这些发现表明，亮氨酸和异亮氨酸可能在艰难梭菌产生毒素的过程中发挥重要作用。

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.