Marine‐Derived Cyclo(<scp>l</scp>‐Leucyl‐<scp>l</scp>‐Prolyl) Targets <scp>d</scp>‐Alanylation of Lipoteichoic Acid to Combat <i>Streptococcus mutans</i> UA159 Mediated Dental Cariogenesis

With the growing threat of antimicrobial resistance (AMR), antivirulence strategies present a promising alternative to traditional antibiotics, particularly in dentistry. Dental caries, a chronic biofilm-associated disease primarily driven by the AMR pathogen Streptococcus mutans, results in enamel demineralization and significant oral health challenges. This study explores the anticariogenic mechanism of marine-derived cyclo(l-leucyl-l-prolyl) (CLP), a biomolecule known to inhibit key virulence factors of S. mutans UA159. LC-MS/MS proteomic analysis revealed 30 and 71 significantly regulated proteins following 12 and 24 h of CLP treatment, respectively. Protein-protein interaction and gene ontology analyses demonstrated that CLP downregulates critical virulence proteins related to d-alanylation of lipoteichoic acid (LTA), glucan synthesis, acid production and acid tolerance, while upregulating proteins associated with translation, DNA repair and protein metabolism. KEGG pathway analysis highlighted the involvement of downregulated proteins in key metabolic pathways, including d-alanine metabolism, starch and sucrose metabolism, glycolysis and branched-chain amino acid metabolism. Given the pivotal role of d-alanine metabolism in modulating interconnected virulence pathways, a comparative analysis of in vitro virulence assays-including cell adherence, biofilm formation, acid production and cell surface charge-alongside proteomic data signify that CLP specifically targets the d-alanylation of LTA. This hypothesis was further validated by LTA and d-alanine quantification assays, which confirmed a significant reduction in d-alanine content within LTA after CLP treatment, leading to a marked attenuation of S. mutans cariogenic virulence. Additionally, qPCR and molecular docking analyses corroborated that CLP disrupts S. mutans virulence by interfering with the d-alanylation of LTA. These findings highlight CLP's potential as a novel therapeutic agent for combating dental cariogenesis by targeting S. mutans virulence, offering a promising avenue for the development of advanced anticariogenic therapies.