Romero-Rodriguez, A. | Search bioRxiv

bioRxiv2025

Assessment of chemical methods in the extraction of spore surface layers in Clostridioides difficile spores

Clostridioides difficile spores are essential for initiation, recurrence, and transmission of C. difficile 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 C. difficile 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 C. difficile spore biology, standardizing their impact in spore coat and exosporium extraction for use in future studies. ImportanceThe outermost layers of C. difficile spores, the exosporium and spore coat, are essential for the spores resistance properties and initial interactions with the host. However, there is variability in extraction protocols making it difficult to compare across studies. This work evaluates the commonly used extraction methods EBB, USD, and Laemmli at removing the exosporium and spore coat and provides a foundation for improved reproducibility. Here, we identified the effectiveness of these different extraction methods allowing us to better understand these techniques to accurately analyze spore surface in C. difficile spore research.

microbiology↗

bioRxiv2020

Characterization of exosporium layer variability of Clostridioides difficile spores in the epidemically relevant strain R20291.

Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea and spores are the transmission vehicle of the disease. 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 the laminations of the spore-coat 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.

microbiology↗