Phylogenomic analysis of the collagen-like BclA proteins in Clostridioides difficile

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium and a major nosocomial pathogen, notable for its high genetic diversity. C. difficile has been classified into five classical phylogenetic clades (C1-C5) and five cryptic clades (C-I to C-V), reflecting its extensive genome plasticity. In addition, C. difficile spores are considered essential for the onset, persistence, and transmission of the disease, and their exosporium layer features hair-like projections formed by the BclA family of proteins (BclA1, BclA2, and BclA3). Previous work in C1 and C2 strains has demonstrated that the absence of these proteins affects spore germination, pathogenesis, persistence, and recurrence of CDI. Nevertheless, the conservation of BclA proteins across different C. difficile clades remains unclear. In this work, genomic analysis of more than 25,000 C. difficile genomes revealed that the prevalence and variability of BclA are not consistent across classical and cryptic clades. The most represented clade in the data set was C1, where roughly 50% of the genomes possessed all three bclA genes. Pseudogenization of bclA1 and all bclA was observed in C2 and C3, respectively. Additionally, the absence of bclA1 in C4 and both bclA1 and bclA3 in C5 further demonstrated the divergence of BclA among C. difficile clades. Subsequent analysis revealed high variability in the central collagen-like region (CLR) of all three BclA and a highly conserved bclA1 pseudogenization event in most members of C2. Restoration of a full-length bclA1 from strain 630 (C1) into C. difficile R20291 (C2) slightly decreased the length of the hair-like projections. Overall, the extensive variability of bclA, primarily focused on their CLR, prevalence of a bclA1 pseudogenization, and absence of bclA in specific clades likely impact spore morphogenesis and pathogenesis across the species.IMPORTANCEClostridioides difficile is a major cause of healthcare-associated infections, with spores playing a central role in disease transmission, persistence, and recurrence. The outermost spore layer, the exosporium, is critical for interactions with the host and environment, and its structure is shaped in part by the BclA family of proteins. While previous studies have linked BclA proteins to virulence traits, their distribution and conservation across the species' genetically diverse clades have remained unexplored. This study provides the first comprehensive analysis of bclA gene prevalence and variability across more than 25,000 genomes, revealing substantial divergence among classical and cryptic clades. The frequent pseudogenization or absence of bclA genes, particularly bclA1, and high variability in the collagen-like regions suggest that BclA-mediated exosporium architecture is not universally conserved and may have clade-specific implications for spore morphology and pathogenicity. These findings highlight the need to account for phylogenetic context when studying C. difficile spore biology and open new avenues for understanding how structural variation in spores contributes to strain-specific virulence and transmission dynamics.