ABSTRACT
Introduction: The dlt operon encodes proteins responsible for the esterification of positively charged D-alanine on the wall teichoic acids and lipoteichoic acids of Gram-positive bacteria. This structural modification of the bacterial anionic surface in several species has been described to alter the physicochemical properties of the cell-wall. In addition, it has been linked to reduced sensibilities to cationic antimicrobial peptides and antibiotics. Methods: We studied the D-alanylation of Clostridioides difficile polysaccharides with a complete deletion of the dltDABCoperon in the 630 strain. To look for D-alanylation location, surface polysaccharides were purified and analyzed by NMR. Properties of the dltDABCmutant and the parental strains, were determined for bacterial surface's hydrophobicity, motility, adhesion, antibiotic resistance. Results: We first confirmed the role of the dltDABCoperon in D-alanylation. Then, we established the exclusive esterification of D-alanine on C. difficile lipoteichoic acid. Our data also suggest that D-alanylation modifies the cell-wall's properties, affecting the bacterial surface's hydrophobicity, motility, adhesion to biotic and abiotic surfaces,and biofilm formation. In addition, our mutant exhibitedincreased sensibilities to antibiotics linked to the membrane, especially bacitracin. A specific inhibitor DLT-1 of DltA reduces the D-alanylation rate in C. difficile but the inhibition was not sufficient to decrease the antibiotic resistance against bacitracin and vancomycin. Conclusion: Our results suggest the D-alanylation of C. difficile as an interesting target to tackle C. difficile infections.
