Sanchez Javier, Romero-Rodriguez Alba, Troncoso-Cotal Scarlett, Osborne Morgan S, Ariri Theresa, Sorg Joseph A, Paredes-Sabja Daniel
Department of Biology, Texas A&M University, College Station, Texas, U.S.A.
ANID - Millennium Science Initiative Program - Millennium Nucleus in the Biology of the Intestinal Microbiota, Santiago, Chile.
bioRxiv. 2025 Aug 19:2025.08.19.671018. doi: 10.1101/2025.08.19.671018.
spores are essential for initiation, recurrence, and transmission of infections (CDI). These outermost layers of the spore, the exosporium and spore coat, are responsible for initial interactions with the host and spore resistance properties respectively. Several spore coat /exosporium extraction methods have been utilized to study the spore surface with differing procedures making comparison across studies difficult. Here, we tested how commonly used exosporium and spore coat extraction methods, termed EBB, USD, and Laemmli, remove the spore coat and exosporium layers of spores. We assessed the impact of these extraction methods on the spore through transmission electron microscopy, phase contrast microscopy, western blotting, and lysozyme triggered cortex degradation. Transmission electron microscopy shows that treatment with EBB and USD, completely remove the spore coat and exosporium layer while leaving decoated spores intact. Western blots revealed differences in the ability to extract spore surface protein markers (CdeC, CdeM, CotA). In addition, lysozyme was able to degrade the cortex in decoated spores regardless of the treatment employed. Western blot analysis of lysozyme treated-decoated spores, reveals that EBB and USD treatment allows for detection and release of the spore core germination protease, GPR. Our results provide a comparison of commonly used extraction methods in spore biology, standardizing their impact in spore coat and exosporium extraction for use in future studies.
芽孢对于艰难梭菌感染（CDI）的起始、复发及传播至关重要。芽孢的最外层，即芽孢外壁和芽孢衣，分别负责与宿主的初始相互作用以及芽孢的抗性特性。已采用多种芽孢衣/芽孢外壁提取方法来研究芽孢表面，但不同的操作程序使得各研究间难以进行比较。在此，我们测试了常用的称为EBB、USD和Laemmli的芽孢外壁和芽孢衣提取方法如何去除芽孢的芽孢衣和芽孢外壁层。我们通过透射电子显微镜、相差显微镜、蛋白质免疫印迹以及溶菌酶引发的芽孢皮层降解来评估这些提取方法对芽孢的影响。透射电子显微镜显示，用EBB和USD处理可完全去除芽孢衣和芽孢外壁层，同时使脱衣芽孢保持完整。蛋白质免疫印迹揭示了提取芽孢表面蛋白标志物（CdeC、CdeM、CotA）能力的差异。此外，无论采用何种处理，溶菌酶都能够降解脱衣芽孢中的皮层。对经溶菌酶处理的脱衣芽孢进行蛋白质免疫印迹分析表明，EBB和USD处理能够检测并释放芽孢核心萌发蛋白酶GPR。我们的结果对芽孢生物学中常用的提取方法进行了比较，规范了它们在芽孢衣和芽孢外壁提取中的影响，以供未来研究使用。
