Spore Assembly in Clostridioides difficile

艰难梭状芽胞杆菌是美国医院感染的主要原因和成本 医疗保健系统估计为每年50亿美元。艰难梭菌感染费用昂贵，治疗困难。 因为它们的复发频率很高(~20%)。疾病的复发取决于艰难梭菌的形成能力 代谢休眠的孢子，因为它们是这种专性厌氧菌的传播形式。近期 对小鼠的研究表明，防止孢子形成可以打破反复发作的破坏循环 艰难梭菌病的特征感染。而用头孢霉素类药物阻断孢子形成可以 预防小鼠复发与万古霉素，目前的护理标准，头孢霉素类 可通过加剧肠道生物失调而使人类对艰难梭菌感染敏感。因此，抗孢子化 需要有选择性地针对艰难梭菌的疗法。然而，开发这样的疗法将需要 对艰难梭菌如何组装孢子有了更深入的了解。 头孢菌素通过抑制孢子形成诱导的青霉素结合的SpoVD来阻止孢子的形成 蛋白。在枯草芽孢杆菌中，SpoVD与SpoVE糖基转移酶和SpoVB协同工作 Flippase合成一层厚厚的孢子肽聚糖(PG)保护层，称为皮质。而当 我们证实艰难梭菌的皮质合成需要这三个因素，我们意外地发现C. 艰难芽孢杆菌SpoVD和SpoVE调控孢子形成的最早阶段，即不对称分裂。我们也 鉴定了孢子形成诱导的、调节不对称分裂的分裂体样蛋白。这些结果 强烈建议艰难梭菌使用一种独特的芽胞形成诱导PG合成机来合成 极隔；这种机械可以选择性地阻止孢子的形成。 我们的数据进一步表明艰难梭菌使用一种独特的PG合成机制来合成 营养细胞分裂时的内侧隔膜，因为我们已经鉴定的分裂组成分是 在艰难梭菌中是必不可少的，尽管在迄今为止研究的所有其他细菌中都是必不可少的。有趣的是， 孢子形成诱导的SpoVD可能调节营养细胞的分裂，因为SpoVD的丢失使C. 肉汤培养对头孢霉素类抗生素的耐药性。基于这些发现，这项提案寻求 确定艰难梭菌在产孢子过程中如何调节极隔膜的形成，以及它如何利用 这些成分可以增强艰难梭菌在营养生长过程中对细胞壁抗生素的抵抗力。 完成这些目标将定义艰难梭菌组装的重要新机制 侵染性孢子，为开发艰难梭菌特异性抗孢子疗法奠定了基础，以及 揭示艰难梭菌对细胞壁抗生素产生高水平抗药性的新机制。

Clostridioides difficile is the leading cause of nosocomial infections in the United States and costs the healthcare system an estimated $5 billion/yr. C. difficile infections are costly and difficult to treat because they recur at high frequency (~20%). Disease recurrence depends on C. difficile’s ability to form metabolically dormant spores because they are the transmissive form of this obligate anaerobe. Recent studies in mice have shown that preventing spore formation can break the damaging cycle of recurrent infection that characterizes C. difficile disease. While blocking spore formation with cephamycins can prevent recurrence in mice when combined with vancomycin, the current standard-of-care, cephamycins can sensitize humans to C. difficile infections by exacerbating gut dysbiosis. Thus, anti-sporulation therapies that selectively target C. difficile are needed. Developing such therapies, however, will require a deeper understanding of how C. difficile assembles a spore. Cephamycins block spore formation by inhibiting SpoVD, a sporulation-induced penicillin-binding protein. In Bacillus subtilis, SpoVD works in concert with the SpoVE glycosyltransferase and SpoVB flippase to synthesize a thick, protective layer of spore peptidoglycan (PG) known as the cortex. While we confirmed that C. difficile cortex synthesis requires these three factors, we unexpectedly found that C. difficile SpoVD and SpoVE regulate the earliest stage of spore formation, asymmetric division. We also identified sporulation-induced, divisome-like proteins that regulate asymmetric division. These results strongly suggest that C. difficile uses a unique sporulation-induced PG synthesis machine to synthesize polar septa; this machinery could be selectively targeted to prevent spore formation. Our data further suggest that C. difficile uses a distinct PG synthesis machinery to synthesize medial septa during vegetative cell division because the divisome components we have identified are dispensable in C. difficile, despite being essential in all other bacteria studied to date. Interestingly, sporulation-induced SpoVD may modulate vegetative cell division because loss of SpoVD sensitizes C. difficile to cephamycin antibiotics during broth culture. Based on these findings, this proposal seeks to determine how C. difficile regulates polar septum formation during sporulation and how it uses some of these components to enhance C. difficile’s resistance to cell wall antibiotics during vegetative growth. Completing these aims will define an important new mechanism by which C. difficile assembles infectious spores, lay the foundation for developing C. difficile-specific anti-sporulation therapies, and reveal novel mechanisms that contribute to C. difficile’s high level of resistance to cell wall antibiotics.