Spore Assembly in Clostridioides difficile

艰难梭菌中的孢子组装

• 批准号: 10676850
• 负责人: Aimee Shen
• 金额: $ 43.74万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-14 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676850
• 关键词: Address; Anaerobic Bacteria; Antibiotics; Bacillus subtilis; Bacteria; Binding; Cell Wall; Cell division; Cephamycins; Chimeric Proteins; Clostridium difficile; Data; Dedications; Dependence; Disease; Enzymes; Foundations; Frequencies; Genetic Transcription; Growth; Health Care Costs; Healthcare Systems; Homologous Gene; Human; Individual; Infection; Location; Medial; Mediating; Membrane; Metabolic; Molecular; Morphology; Mus; Mutagenesis; Nosocomial Infections; Patients; Penicillin-Binding Proteins; Peptidoglycan; Peptidyltransferase; Play; Proteins; Recurrence; Recurrent disease; Reporter; Reproduction spores; Resistance; Role; Testing; Therapeutic; Thick; United States; Vancomycin; Work; cell growth; cost; disease transmission; effective therapy; experience; glycosyltransferase; gut dysbiosis; healthcare-associated infections; inhibitor; insight; novel; prevent; protein function; protein protein interaction; recurrent infection; response; standard of care; stressor; transmission process

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.

艰难梭菌是美国医院感染的主要原因， 卫生保健系统估计每年50亿美元。C.难治性感染费用昂贵且难以治疗 因为它们以高频率（~20%）复发。疾病复发取决于C。difficile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。最近 对小鼠的研究表明，防止孢子形成可以打破反复出现的 以C.艰难病虽然用头霉素阻止孢子形成， 当与万古霉素、目前的标准治疗、头霉素联合使用时， 能使人类对C.通过加剧肠道生态失调来治疗艰难感染。因此，抗孢子形成 选择性靶向C.困难是需要的。然而，开发这种疗法需要 更深入地了解C.艰难梭菌聚集成孢子。 头霉素通过抑制SpoVD（一种孢子形成诱导的青霉素结合）来阻断孢子形成 蛋白在枯草芽孢杆菌中，SpoVD与SpoVE糖基转移酶和SpoVB协同工作。 翻转酶合成一层厚厚的孢子肽聚糖（PG）保护层，称为皮层。而 我们证实C. difficile皮层合成需要这三个因子，我们意外地发现C. 艰难梭菌SpoVD和SpoVE调节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C. difficile使用独特的孢子诱导PG合成机器来合成 极隔;这种机制可以选择性地针对防止孢子形成。 我们的数据进一步表明C.艰难梭菌使用独特的PG合成机制来合成 在营养细胞分裂过程中的中隔膜，因为我们已经确定的分裂体成分是 白素C difficile，尽管在迄今为止研究的所有其他细菌中是必需的。有趣的是， 孢子形成诱导的SpoVD可能调节营养细胞分裂，因为SpoVD的丧失使C. 在肉汤培养过程中，头孢霉素抗生素难以产生。根据这些调查结果，本提案力求 确定C. difficile在孢子形成过程中调节极隔膜的形成，以及它如何利用一些 这些成分增强C.艰难梭菌在营养生长期间对细胞壁抗生素的抗性。 这些目标的实现将为C.艰难汇编 侵染性孢子，为C.艰难梭菌特异性抗孢子形成疗法，和 揭示了新的机制，有助于C。艰难梭菌对细胞壁抗生素的高水平耐药性。