Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis

Abstract The lipopeptide antibiotic daptomycin exhibits bactericidal activity against gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanism of bacterial resistance to daptomycin has gained great importance. In the present study, we found that overexpression of ugtP , which encodes a diglucosyldiacylglycerol synthase, resulted in daptomycin resistance in Bacillus subtilis . The amount of diglucosyldiacylglycerol (Glc 2 DAG) increased in the ugtP -overexpressed strain, whereas the amounts of the acidic phospholipids cardiolipin (CL) and PG and the basic phospholipid lysylphosphatidylglycerol (Lys-PG) decreased. Moreover, ugtP overexpression did not alter the cell surface charge or the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Furthermore, under daptomycin exposure, we obtained daptomycin-resistant mutants that carried ugtP mutations and showed increased amounts of Glc 2 DAG and more than 4-fold increase in the MIC of daptomycin. These results suggest that an increase in the amount of Glc 2 DAG changes the phospholipid composition and leads to daptomycin resistance without altering the bacterial cell surface charge in B. subtilis . Importance Daptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, and the emergence of daptomycin-resistant bacteria has become a problem. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP , which encodes diglucosyldiacylglycerol synthase, causes daptomycin resistance in B. subtilis , a model organism for gram-positive bacteria. Overexpression of ugtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection.