Constructing a crystal shell : biogenesis and function of the Clostridium difficile surface layer

Abstract

Clostridioides difficile is a Gram-positive, spore-forming bacteria, and the most frequent cause of antibiotic-associated diarrhoea around the world. The infection can evolve from mild diarrhoea into toxic megacolon and lead to death. In 2019, the C. difficile infection (CDI) has been declared as an urgent threat by the CDC due to its increasing rate of drug resistance and change in epidemiological profile. The C. difficile cell, like all Archea and most bacteria, is completely covered by a proteinaceous surface layer (S-layer), that primarily acts as a protective layer and a molecular sieve which still enables cell growth and division. In C. difficile, the S-layer has been implicated in interactions with the host, including cell adhesion and host immune response, making it an important target to combat infections. In C. difficile, the S-layer lattice is formed by the main protein, S-layer protein A (SlpA) which is expressed as a pre-peptide and processed into two S-layer proteins (SLP): the SLPH (HMW SLP) and SLPL (LMW SLP). These two proteins interact forming the H/L complex, that polymerises into a paracrystalline arrangement. Using protein crystallography, we determined the structure of the full length SlpA for three different homologues. These structures revealed key structural features necessary for the H/L complex as well as the main interactions involved in S-layer assembly. The interacting domains (LID and HID) from the two SLPs form an intricate folding, essential for H/L complex formation. Electron microscopy reconstructions from the intact S-layer nicely fit the 2D layer formed in crystallo, indicating that the crystal lattice reflects the in situ assembly. The polymerisation of H/L complex into an S-layer is broadly aided by electrostatic interactions formed between charged residues on the surface of SLPH. The anchorage of the S-layer onto the cell was previously shown to be performed by the interaction of the anionic polyssacharide PS-II with SlpA. In this project, we applied microscale thermophoresis and circular dichroism to determine the effect of this interaction on the protein structure and oligomerisation. Our results indicate that association of PS-II does not induce a secondary structure modification and raises the possibility of the need of a structured region for this association to happen. Additionally, computational analysis with molecular docking and simulations were conducted to understand the minimal units within the PS-II chemical composition which would potentially interact with the SlpA. Interestingly, our results indicate that although SLPH consists of three tandem cell wall binding motifs (CWB2), they are not expected to form interactions with PS-II equally. Taken together, our findings shed light on the structure, attachment and fluidity of the C. difficile S-layer and hint to explanations on the features needed for the cell to grow and divide while still protected by the S-layer.