Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea. C. difficile spore is a dormant state which acts as a vehicle of transmission and infection. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that spore-coat laminations are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC, and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

艰难梭菌 是革兰氏阳性厌氧肠致病菌，是抗生素相关性腹泻的病原体。 艰难梭菌孢子处于休眠状态，是传播和感染的媒介。在艰难梭菌 孢子，最外孢子囊层是与宿主相互作用的第一个障碍，应携带参与孢子与宿主相互作用的孢子配体。 艰难梭菌形成两种类型的孢子（即薄和厚的外孢子层）。在本交流中，我们有助于了解这两种外孢子形态型的生物学方面。通过透射电子显微镜，我们证明了两种孢子囊形态在孢子形成过程中同时出现，并且在无氧条件下形成了孢子被覆层。Nycodenz密度梯度允许孢子富集具有厚孢子囊层形态和极性附肢的存在。使用与外孢子蛋白BclA3，CdeA，CdeC和CdeM以及几种孢子外壳蛋白进行的翻译荧光融合，我们观察到SNAP翻译融合在R20291菌株中的表达强度和分布是高度异质的。电子显微照片表明CdeC的多拷贝表达，但CdeM却没有，SNAP翻译融合，增加了厚的外孢子形态型的丰度。总的来说，这些结果提出了关于在孢子形成过程中如何形成这些独特的孢子囊形态的进一步问题。