Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC’s ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC’s self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.

中文翻译：

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艰难梭菌富含半胱氨酸的外孢子形态发生蛋白 CdeC 表现出自组装特性，导致大肠杆菌中有组织的包涵体

艰难梭菌是一种专性厌氧、形成孢子的革兰氏阳性病原菌，被认为是全世界医院内腹泻的主要原因。最近的研究试图了解艰难梭菌孢子最外层的生物学，外孢子被认为有助于与宿主的早期相互作用。已经描述了富含半胱氨酸的蛋白质 CdeC 和 CdeM 的基本作用。然而，外孢子组装机制背后的分子细节缺失。控制艰难梭菌外孢子组装的潜在机制仍然缺乏研究，部分原因是难以获得艰难梭菌的纯可溶性重组蛋白外孢子。在这项工作中，我们观察到 CdeC 能够在大肠杆菌中形成有组织的包涵体 (IB)，其中充满了由 5 到 15 nm 的间隙隔开的层状结构；然而，CdeC 在具有更强氧化环境的大肠杆菌菌株中的表达导致 CdeC IBs 的层状组织丧失。此外，CdeC 包涵体的二硫苏糖醇 (DTT) 处理会释放 CdeC 的单体可溶形式。CdeC 不同部分的缺失不会影响 CdeC 在变性条件下聚集和形成稳定的低聚物的能力，但会影响 CdeC 的自组装性能。总体而言，这些观察结果对阐明 CdeC 在艰难梭菌外孢子组装中的作用的进一步研究具有重要意义 孢子。