Structural Insights into the Staphylococcus aureus DltC-Mediated D-Alanine Transfer

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a major Gram-positive pathogen, and treatment of <i>S. aureus</i> infections is often challenging due to widespread antibiotic resistance. In Gram-positive bacteria such as <i>S. aureus</i>, D-alanylation of teichoic acids (TA) reduces the net negative charge of the cell envelope and contributes to resistance to diverse antibiotics, particularly cationic antimicrobial peptides. D-alanylation is mediated by the <i>dlt</i>ABCD operon, which encodes four proteins (DltA, DltB, DltC, and DltD), all of which is essential for the multistep transfer of D-alanine to teichoic acids. Here, we present the first crystal structure of the <i>S. aureus</i> D-alanyl carrier protein DltC and analyze its interaction with DltA using AlphaFold3 and all-atom molecular dynamics simulations. We further show that single substitutions of <i>Sa</i>DltA-<i>Sa</i>DltC interface residues abolish <i>Sa</i>DltC mediated enhancement of <i>Sa</i>DltA catalysis. Together, these findings define a catalytically critical <i>S. aureus</i> DltA-DltC interface and provide a structural insight for targeting the D-alanylation pathway as a potential anti-<i>Staphylococcus</i> strategy.