VirB4- and VirD4-Like ATPases, Components of a Putative Type 4C Secretion System in Clostridioides difficile

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and has attracted intense interest due to its roles in the pathogenesis of infectious diseases. In the current investigation, we uncovered three distinct gene clusters in Clostridioides difficile strain 630 encoding proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis revealed that VirB4- and VirD4-like proteins of the CTn4 locus, on the one hand, and those of the CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in the physiology of C. difficile. Our further study on VirB4- and VirD4-like products encoded by CTn4 revealed that both proteins possess Mg²⁺-dependent ATPase activity, form oligomers (most likely hexamers) in aqueous solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. The DNA-binding activity of VirD4 strongly decreased with the W241A variant. Mutations in the nucleotide sequences encoding presumable Walker A and Walker B motifs decreased the stability of the oligomers and significantly but not completely attenuated the enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs neither attenuated the enzymatic activity of the protein nor influenced the oligomerization state of the ATPase. IMPORTANCE C. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include the toxins TcdA, TcdB, and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered possible virulence factors of C. difficile. In the current paper, we describe the structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to its significance for basic science, can potentially aid the development of antivirulence drugs suitable for the treatment of C. difficile infection.