Isolation and Molecular Characterization of the LTA Precursor Molecule Glc₂-DAG, a Potential Target for Antibiotics

Bacterial infections present a major global health threat, often displaying resistance to various antibiotics. Lipoteichoic acid (LTA) is a vital component of bacterial cell envelopes of Gram-positive bacteria, crucial for cell integrity, cell division, and host inflammation. Due to its essential role for bacteria, LTA and its biosynthesis are also attractive drug targets, however, there is only scant molecular knowledge on LTA and its precursor molecules in membranes. Here, we report the isolation and molecular characterization of diglucosyldiacylglycerol (Glc₂-DAG), the glycolipid precursor molecule that anchors LTA in the bacterial plasma-membrane. Using a tailored growth medium and purification protocols, we isolated ¹³C-isotope labelled Glc₂-DAG from bacteria, which can then be used for high-resolution NMR studies. Using solution-state and solid-state NMR, we show an in-depth molecular characterization of Glc₂-DAG, including in native-like membranes. Our approach may help to identify antibiotics that directly target LTA precursor molecules, and it offers a tool for future investigations into the role of Glc₂-DAG in bacterial physiology.