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

The S-layer of C. difficile is a paracrystalline array that covers the bacterial cell but its contribution to overall disease remains unclear. A previously described spontaneous slpA-null mutant, FM2.5, with a point mutation in slpA offers the opportunity to study the role of the S-layer in vivo. Here, we confirm our previous observation that this strain is less virulent in vivo despite effectively colonising the host and producing toxin. We also show a strong in vivo selection pressure for strains that express the intact S-layer array through sequence modifications that restore slpA expression. While such modifications do not affect the overall S-layer structure, RNA-Seq analysis in vitro showed large differences in gene expression between FM2.5, the revertant strains and R20291. Despite these differences, an intact S-layer clearly provides a selective advantage of C. difficile in the host, and, for one of the revertants tested, helped restore disease severity. Bulk RNA-seq and DNA-Seq of Clostridioides difficile strains FM2-5, R20291, Rv9, Rv189 and RW2-5.