The nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved coat protein, SpoIVA, and the clostridial-specific coat protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and decrease functional spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to help drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficile's SpoIVA ATPase motifs impair functional spore formation. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but retain binding to ATP enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation.

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SpoIVA ATPase母题在艰难梭菌孢子形成过程中的作用

医院病原体艰难梭菌是一种形成孢子的专性厌氧菌，取决于其耐气孢子的形式来传播感染。功能性孢子的形成取决于围绕发育中的孢子的蛋白质层的组装，即被膜。在艰难梭菌中，外壳组装取决于保守的外壳蛋白SpoIVA和梭菌特异性外壳蛋白SipL，它们直接相互作用。破坏其相互作用的突变会导致被膜定位错误并减少功能性孢子的形成。在枯草芽孢杆菌中，SpoIVA是一种ATPase，它使用ATP水解来帮助驱动其在前孢子周围的聚合。SpoIVA ATPase活性的丧失损害枯草芽孢杆菌SpoIVA包裹前孢子，并激活消除这些缺陷细胞的质量控制机制。由于艰难梭菌缺乏这种机制，因此我们测试了艰难梭菌的SpoIVA ATPase主题中的突变是否会损害功能性孢子的形成。有趣的是，预计将废除SpoIVA与ATP结合的ATPase基序残基的突变降低了SpoIVA-SipL相互作用，而预计ATPase残基的突变将破坏ATP水解但保留与ATP的结合增强了SpoIVA-SipL的相互作用。当SIPL已知降低结合SpoIVA突变与组合spoIVA突变预计会阻止SpoIVA与ATP结合，严重加剧了孢子的形成。由于此表型是等位基因特异的，因此我们的数据暗示SipL识别SpoIVA的ATP结合形式，并突出了这种相互作用对功能艰难梭菌孢子形成的重要性。