An intact S-layer is advantageous to Clostridioides difficile within the host.

Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well known. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer revertants, following infection with the S-layer-null strain, FM2.5. Sequencing of the slpA gene revealed either correction of the original point mutation or modification of the sequence upstream of the mutation, which restored the reading frame, and translation of slpA. Selection of these strains was rapid, with up to 90% of isolates identified as revertants 24 h post infection. Two revertant isolates, RvA and RvB, showed modification of 3 and 13 amino acids respectively, compared to wild type sequence. Structural determination of SlpA from RvB revealed a different orientation of its domains, resulting in a reorganisation of the lattice assembly and changes in interacting interfaces which might result in functional differences. These revertants showed differing patterns of disease in vivo; RvA causing equivalent severity to R20291 and RvB an attenuated FM2.5-like phenotype. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates showed large changes in differentially expressed genes (DEGs) between R20291 and FM2.5 namely in TcdA/TcdB expression, in transcripts associated with sporulation and those linked to cell wall integrity, which may account for attenuation observed in vivo. In comparison, smaller differences were observed between RvA/R20291, and RvB/FM2.5 respectively, which correlated with observed disease severity in vivo. Cumulatively, these data highlight that the S-layer plays a role in C. difficile disease. ### Competing Interest Statement The authors have declared no competing interest.