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Study on the mechanism of toxin production by Clostridium difficile

艰难梭菌产毒机制研究

基本信息

批准号：
06670286
负责人：
NAKAMURA Shinichi
金额：
$ 1.41万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670286/
关键词：
C.difficile defined medium amino acid vitamin toxin A toxin B toxin biotin

项目摘要

The mechanism of toxin production by Clostridium difficile was investigated from the stand poit of nutritional requirements. By the single-amino acid omission and addition methods with a basal defined medium consisting of 18 amino acids, it was found that valine, cysteine, tryptophan, proline, methionine, isoleucine, leucine, glycine and threonine were essential for good toxin production.Further, by qualitative analysis, a defined medium (6xMADM) consisting (g/L) of tryptophan 0.6, methionine 1.2, valine 1.8, isoleucine 1.8, proline 1.8, leucine 2.4, glycine 0.2, threonine 0.4 and cysteine 0.5 was found to be fairly effective for stimulating toxin production. In a comparison of 6xMADM and m-BHI,which is excellent comple medium for toxin production, 13 of 20 strains tested produced the same amounts of toxin in both media, suggesting that the 9 amino acids mentioned above were particularly important for toxin production. By single-vitamin omission method with 6xMADM,it was found that pyridoxine, pantothenate and biotin were essential for good growth and toxin production and that biotin deficiency might stimulate toxin production. The effect of biotin was analyzed in detail with a strain KZ 1647. When toxin production was examined in relation to biotin concentration, it was found that with decreasing concentration of biotin bacterial growth decreased, but toxin production was remarkably increased, particularly with 0.05 nM biotin.The time course of toxin production in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 18 out of 19 strains tested, suggesting that the effect is a general phenomenon amongst toxigenic C.difficile strains.These findings suggest that biotin play an important role in the development of C.difficile colitis.

从营养需求的角度探讨了艰难梭菌产毒的机理。用18种氨基酸组成的基本培养基，采用单氨基酸省略法和添加法，发现缬氨酸、半胱氨酸、色氨酸、脯氨酸、蛋氨酸、异亮氨酸、亮氨酸、甘氨酸和苏氨酸是产生良好毒素所必需的。进一步，通过定性分析发现，由（g/L）色氨酸0.6、蛋氨酸1.2、缬氨酸1.8、异亮氨酸1.8、脯氨酸1.8、亮氨酸2.4、甘氨酸0.2、苏氨酸0.4和半胱氨酸0.5组成的确定培养基（6xMADM）对刺激毒素产生相当有效。在比较6xMADM和m-BHI（一种极好的产生毒素的复合培养基）时，20株菌株中有13株在两种培养基中产生相同数量的毒素，这表明上述9种氨基酸对毒素的产生特别重要。采用6xMADM单维生素遗漏法，发现吡哆醇、泛酸和生物素是其良好生长和毒素产生所必需的，生物素缺乏可能刺激毒素的产生。以菌株kz1647为研究对象，详细分析了生物素的作用。当检测毒素产量与生物素浓度的关系时，发现随着生物素浓度的降低，细菌生长减少，但毒素产量显著增加，特别是0.05 nM生物素。生物素限制条件下毒素产生的时间过程与生物素富集条件下相似。生物素对毒素产生的影响在19个测试菌株中的18个中也被观察到，这表明这种影响是产毒艰难梭菌菌株中的普遍现象。这些发现提示生物素在艰难梭菌结肠炎的发生发展中起重要作用。