S-layers as functional armours: structure, function and variability in Clostridioides difficile

Surface layers (S-layers) are paracrystalline protein lattices that serve as molecular exoskeletons and environmental interfaces across diverse bacteria and archaea. Despite their widespread occurrence, S-layers remain comparatively understudied. The available models do not fully represent the structural and physiological diversity of S-layers, and fragmented methodologies limit comprehensive functional comparisons and detailed characterisation. Recently determined high-resolution structural details for multiple variants of Clostridioides difficile S-layer provide a uniquely rich model system with functional links to antimicrobial resistance, phage recognition and immune evasion. In this review, we present C. difficile S-layers as extremely diverse, adaptable and multifunctional structures. We (i) summarise known S-layer roles in C. difficile infection; (ii) present a comprehensive comparison of their extremely diverse structures, including unique features and topological differences in lattice assemblies; (iii) propose quantitative metrics for comparative, structural analysis of these S-layers; (iv) outline S-layer pore size thresholds for the diffusion of metabolites essential for C. difficile growth; and (v) explore possible mechanisms underlying S-layer function and highlight the key outstanding questions.