Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile.
Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile.
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DOI：
10.3389/fcimb.2017.00531
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发表时间：
2017
影响因子：
5.7
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Gu H;Yang Y;Wang M;Chen S;Wang H;Li S;Ma Y;Wang J
艰难梭菌是院内腹泻和伪膜性结肠炎的主要原因，仍然构成严重的卫生保健挑战。据报道，其两种主要毒力因子 TcdA 和 TcdB 的表达受到半胱氨酸的抑制，但分子机制仍不清楚。半胱氨酸脱硫酶 CdsB 影响某些细菌的毒力和感染进程。艰难梭菌菌株 630 基因组编码 CdsB 的同源物，在本研究中，我们通过构建基于 ClosTron 的同基因 cdsB 突变体来分析其在艰难梭菌 630Δerm 中的作用。当艰难梭菌在补充半胱氨酸的TY肉汤中培养时，cdsB基因在指数生长期迅速被诱导。 cdsB的失活不仅影响艰难梭菌对半胱氨酸的耐药性，而且改变了细胞内半胱氨酸降解酶的表达水平和硫化氢的产生。这表明艰难梭菌 CdsB 是主要的诱导型半胱氨酸降解酶。艰难梭菌中cdsB基因的失活也消除了毒素产生的半胱氨酸依赖性抑制，但未能消除Na2S依赖性抑制，这支持毒素产生的半胱氨酸依赖性抑制可能归因于半胱氨酸副产物的积累。我们还在 cdsB 基因上游定位了 δ54 (SigL) 依赖性启动子，并且 cdsB 表达并未响应 cdsR::ermB 或 sigL::ermB 菌株中的半胱氨酸而诱导。通过报告基因融合分析，我们鉴定了半胱氨酸依赖性 cdsB 表达所需的启动子序列。总之，这些结果表明 CdsB 是艰难梭菌中关键的诱导型半胱氨酸脱硫酶，它受 δ54 和 CdsR 调节以响应半胱氨酸，并且毒素产生的半胱氨酸依赖性调节与半胱氨酸降解密切相关。
Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.