AUTHOR=Lacotte Pierre-Alexandre , Denis-Quanquin Sandrine , Chatonnat Eva , Le Bris Julie , Leparfait David , Lequeux Thierry , Martin-Verstraete Isabelle , Candela Thomas 

TITLE=The absence of surface D-alanylation, localized on lipoteichoic acid, impacts the Clostridioides difficile way of life and antibiotic resistance

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1267662

DOI=10.3389/fmicb.2023.1267662

ISSN=1664-302X

ABSTRACT=<sec><title>Introduction</title><p>The <italic>dlt</italic> 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.</p></sec><sec><title>Methods</title><p>We studied the D-alanylation of <italic>Clostridioides difficile</italic> polysaccharides with a complete deletion of the <italic>dltDABC</italic>operon in the 630 strain. To look for D-alanylation location, surface polysaccharides were purified and analyzed by NMR. Properties of the <italic>dltDABC</italic>mutant and the parental strains, were determined for bacterial surface’s hydrophobicity, motility, adhesion, antibiotic resistance.</p></sec><sec><title>Results</title><p>We first confirmed the role of the <italic>dltDABC</italic>operon in D-alanylation. Then, we established the exclusive esterification of D-alanine on <italic>C. difficile</italic> 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 <italic>C. difficile</italic> but the inhibition was not sufficient to decrease the antibiotic resistance against bacitracin and vancomycin.</p></sec><sec><title>Conclusion</title><p>Our results suggest the D-alanylation of <italic>C. difficile</italic> as an interesting target to tackle <italic>C. difficile</italic> infections.</p></sec>