Allele-linked divergence in SlpA and TcdB drives distinct immune and cytotoxic responses that distinguish ST01 from non-ST01 strains in Clade 2 Clostridioides difficile

Among the five MLST clades that define the global population structure of the bacterial pathogen <i>Clostridioides difficile</i>, Clade 2 has received special attention due to the global spread, clinical severity and hospital prevalence of ST01 strains. To identify features potentially contributing to the historically attributed higher virulence and epidemic potential of ST01 strains, we compared a range of phenotypic traits across the infection cycle between clinical Clade 2 ST01 and non-ST01 strains from ST41, ST47, ST67, ST154 and ST638. We found no significant differences in canonical virulence-associated characteristics such as spore adherence, motility, biofilm formation and resistance to a disinfectant. However, ST01 strains exhibited distinct profiles in surface layer protein A (SlpA)-mediated immune activation and toxin B (TcdB)-induced cytotoxicity that were consistent with allelic divergence. These findings highlight the need to reconsider current paradigms of Clade 2 hypervirulence and underscore the importance of allele-specific phenotypic variation in developing targeted public health strategies.