Investigating the Interaction Mechanisms of Clostridium difficile Toxins with Host GTPases: A Bioinformatic Approach

Background Clostridioides difficile is frequently implicated in colitis and antibiotics-induced diarrhoea in both community and hospital settings around the world, and there reports of resistance to the antibiotics of choice used in the management of its infections. Yet, there is limited information on the structural dynamics of its toxins TcdA and TcdB that could guide potential therapeutic candidates. Aim We undertook a structural insights study into the glucosyltransferase domain (GTD) of Clostridioides difficile toxin A (TcdA). Methods Structural analyses and molecular dynamics simulation (250 ns) were carried out for the various TcdA glucosyltransferase domain of 5UQL, 3SZA, 4DMW, 7U2P, and 7UBY. Results our findings highlights conserved catalytic residues (e.g., Trp101, Glu514), Mn²⁺ coordination, and substrate-binding motifs. Key residues (Lys428, Glu381) were shown to mediate RhoA engagement, while small-molecule and antibody inhibitors targeted both active and allosteric sites. Molecular dynamics revealed RMSD increases to ~ 3.6 Å across five TcdA structures over 250 ns, indicating intrinsic conformational flexibility. Complexes with RhoA or inhibitors showed altered contact profiles and dynamic behaviour, supporting functional plasticity. Principal component analysis (PCA) revealed that the ternary complex 7U2P exhibited the highest global motion, suggestive of catalytically relevant conformational changes. Contact frequency analysis confirmed stable ligand engagement in active complexes and disruption in inhibitor-bound states. Conclusion These findings underscore the structural adaptability of TcdA’s GTD and reveal potential therapeutic targets through inhibition of conserved residues or conformational states essential for substrate recognition and catalysis.