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標題: 探討半胱胺酸蛋白酶功能區對於困難梭狀桿菌毒素 B 之毒力影響
Investigation of the effect of the cysteine protease domain on the virulence of Clostridium difficile toxin B
作者: 許文金
Bunkim Kouy
關鍵字: 困難梭狀桿菌
毒素 B 半胱胺酸蛋白酶功能區
Clostridium difficile
hypervirulent strain
toxin B cysteine protease domain
autoprocessing activity
in vitro cleavage
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摘要: 困難梭狀桿菌之毒素 A (TcdA) 及毒素B (TcdB),是困難梭狀桿菌疾病致病的主要因素,由tcdA 與 tcdB 負責表達,該疾病通常與抗生素所造成的院內腹瀉相關。先前的研究發現TcdB 是決定困難梭狀桿菌感染病的重要因子。TcdA 及 TcdB 分別由 2710 及2366 胺基酸所組成,均有四個功能區域,分別是 glucosyltransfera- ses domain (GTD)、cysteine protease domain (CPD)、translocation domain (TD)、combined repetitive oligopeptides (CROPs)。CPD 在 TcdB 自體蛋白水解過程中具有決定性的作用,通過自體蛋白水解的方式將GTD 水解並釋放到胞質溶膠細胞質中,GTD抑制Rho GTPase 使其失去活性,而引起細胞骨架受到破壞,最終導致細胞死亡。先前的研究提出,TcdB蛋白質序列之變異可能是高毒力株困難梭狀桿菌致病嚴重程度之重要決定因素。本實驗針對十一個來自高毒力株及一般菌株的TcdB CPD之自體蛋白水解活性進行研究。根據本實驗的蛋白質序列比對結果顯示,ribotype (RT) 012 (630)、RT 001 (ATCC9689)、RT 002 (TNHP20)、 RT 014 (TNHP68) 和 RT087 (ATCC43255) 並無差異。然而,屬於高毒力株的 RT 017 (TNHP82),RT 027 (R20291) 及 RT 078 lineage胺基酸序列均有突變之現象,與一般菌株的胺基酸序列相似性百分比為 90.56 %、97.24 %、96.85 %。不僅如此,在體外水解活性試驗顯示,RT 017 (TNHP82) 和 RT 078 lineage在相同條件下,其自體蛋白水解活性更是比一般菌株來得高。 其中 RT 017 (TNHP82) 的自體蛋白水解活性rate為 0.94 μg/min, RT 078 lineage的自體蛋白水解活性rate 為0.16 μg/min,而一般菌株的自體蛋白水解活性rate 僅 0.07 μg/min。值得注意的是,RT 027 (20291) 具有最高的自體蛋白水解活性,該結果顯示於重組蛋白質純化結果。綜上所述,TcdB CPD 胺基酸之突變可影響並導致高毒力株和一般菌株之間自體蛋白水解速率差異。
Clostridium difficile (C. difficile) is the leading definable cause of antibiotic-associated diarrheal disease in hospitals due to its toxin A (2710 amino acids) and toxin B (2366 amino acids), encoded by tcdA and tcdB, respectively. Both consist of multiple function domains including glucosyltransferases domain (GTD), cysteine protease domain (CPD), translocation domain (TD), combined repetitive oligopeptides (CROPs). Previous study showed that TcdB is the key component of C. difficile virulent. CPD plays a decisive role in mediating autoprocessing and results in the release of GTD into the cytosol. GTD inactivates Rho family GTPase and leads to cytoskeleton disruption and eventual cell death. It has been reported that the variation in TcdB sequence could be in the hypervirulence strains that results an important determining factor in C. difficile associated disease severity. In this study, the autoprocessing activity of the TcdB from hypervirulent strains including ribotype (RT) 017 (TNHP82), RT 078 (M120), RT 027 (R20291) and historical stains including RT 012 (630), RT 001 (ATCC9689), RT 002 (TNHP20), RT 014 (TNHP68), and RT 087 (ATCC43255) were evaluated. TcdB CPD sequence analysis found that historical stains RT 012 (630), RT 001 (ATCC9689), RT 002 (TNHP20), RT 014 (TNHP68), and RT 087 (ATCC43255) share identical residues. However, the sequence homology of hypervirulent strains RT 017 (TNHP82), RT 078 lineage, and RT 027 (R20291), showed significant differences compared to historical stain which were 90.56 %, 96.85 %, 97.24 %, respectively. Moreover, in vitro cleavage assay has demonstrated that RT 017 (TNHP82) and RT 078 lineage possessed higher autoprocessing efficiency than others. The autoprocessing activity rate of RT 017 (TNHP82) was 0.94 µg/min. Simultaneously, the autoprocessing activity rate of other strains were 0.16 µg/min for RT 078 lineage strains and 0.07 µg/min for historical stains. It was notable that RT 027 (20291) had the highest autoprocessing activity, the result consistent with that observed during protein purification. Our data indicated that sequence variations in TcdB CPD of historical and hypervirulent strains could affect autoprocessing activity.
文章公開時間: 2021-11-09
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