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Spore Assembly in Clostridioides difficile

艰难梭菌中的孢子组装

基本信息

批准号：
10365431
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
项目类别：
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/10365431
关键词：
Address Anaerobic Bacteria Antibiotics Bacillus subtilis Bacteria Binding Cell Wall Cell division Cephamycins Chimeric Proteins Clostridium difficile Data Dependence Disease Enzymes Foundations Frequencies Genetic Transcription Growth Health Care Costs Healthcare Systems Human Individual Infection Lead Location Medial Mediating Membrane Metabolic Molecular Morphology Mus Mutagenesis Nosocomial Infections Patients Penicillin-Binding Proteins Peptidoglycan Peptidyltransferase Play Proteins Recurrence Reporter Reproduction spores Resistance Role Testing Therapeutic Thick United States Vancomycin Work base cell growth cost disease transmission effective therapy experience glycosyltransferase gut dysbiosis healthcare-associated infections inhibitor/antagonist insight novel prevent protein function protein protein interaction recurrent infection response standard of care stressor transmission process

项目摘要

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及其造成的费用医疗保健系统估计每年花费 50 亿美元。艰难梭菌感染成本高昂且难以治疗因为它们的复发频率很高（~20%）。疾病复发取决于艰难梭菌的形成能力代谢休眠孢子，因为它们是这种专性厌氧菌的传播形式。最近的对小鼠的研究表明，防止孢子形成可以打破复发性孢子的破坏性循环艰难梭菌疾病特征的感染。虽然用头霉素阻断孢子形成可以与当前标准治疗万古霉素、头霉素联合使用可预防小鼠复发可以通过加剧肠道菌群失调而使人类对艰难梭菌感染敏感。因此，抗孢子形成需要选择性针对艰难梭菌的疗法。然而，开发此类疗法需要更深入地了解艰难梭菌如何组装孢子。头霉素通过抑制 SpoVD（孢子形成诱导的青霉素结合）来阻止孢子形成蛋白质。在枯草芽孢杆菌中，SpoVD 与 SpoVE 糖基转移酶和 SpoVB 协同作用翻转酶合成厚厚的孢子肽聚糖（PG）保护层，称为皮质。尽管我们证实艰难梭菌皮质合成需要这三个因素，我们意外地发现C. difficile 皮质合成需要这三个因素。艰难梭菌 SpoVD 和 SpoVE 调节孢子形成的最早阶段，即不对称分裂。我们也鉴定出孢子形成诱导的、调节不对称分裂的分裂体样蛋白质。这些结果强烈建议艰难梭菌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艰难梭菌使用独特的 PG 合成机制来合成营养细胞分裂期间的内侧隔膜，因为我们已经确定的分裂成分是尽管在迄今为止研究的所有其他细菌中都是必需的，但在艰难梭菌中却是可有可无的。有趣的是，孢子形成诱导的 SpoVD 可能会调节营养细胞分裂，因为 SpoVD 的丧失会使 C. 肉汤培养过程中艰难梭菌对头霉素抗生素的影响。基于这些发现，该提案旨在确定艰难梭菌在孢子形成过程中如何调节极隔膜的形成以及它如何使用一些这些成分可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传染性孢子，为开发艰难梭菌特异性抗孢子形成疗法奠定基础，以及揭示了导致艰难梭菌对细胞壁抗生素具有高水平耐药性的新机制。