The Binding Behavior of Daptomycin on the Bacterial Membranes

Abstract

Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that kills gram-positive bacteria via cell membrane distortion. It is currently approved for treatment of complicated infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant S. aureus, coagulase-negative staphylococci, penicillin-resistant streptococci and vancomycin-resistant enterococci. Recent studies showed that the bactericidal activity of DAP on the target membrane is dependent on Ca2+. DAP is also associated with membrane depolarization in the presence of phosphatidylglycerol (PG). Therefore, in this study, we aimed to investigate the binding behavior of DAP on the membrane of S. aureus cells as well as the specific interaction of DAP with target molecules. <br /> We first used highly inclined and laminated optical sheet (HILO) microscopy to visualize DAP location on the membrane of S. aureus cells. Then, we quantitatively analyzed DAP distribution on S. aureus cell membranes and its correlation with cell size and aggregate formation in a time- and concentration-dependent manner. We observed septum binding for the concentrations lower than the minimum inhibitory concentration (MIC) of DAP and for the concentration around the MIC until 10 min of incubation. However, overall membrane binding of DAP occurred at longer incubation times and higher DAP concentrations. This result was further supported by the super-resolution imaging of the localization of single DAP molecules on the membrane of S. aureus. We found that DAP accumulation correlated negatively with cell size but positively with aggregate formation. <br /> Thus, we further examined the colocalization of 5(6)-TAMRA-X, SE-labeled DAP (DAP-TMR) with the FtsW-GFP fusion protein and lipid II. FtsW is a bacterial cell division protein, which is positioned at the septum. For the short incubation interval, DAP-TMR localized to the septum and was colocalized with FtsW-GFP. For incubation times, DAP bound to the complete cell membrane but the distribution of FtsW-GFP remained unaffected. Furthermore, in cells stained with a BODIPY FL conjugate of vancomycin (Van-BDP FL), considerably less binding of DAP-TMR occurred, indicating that Van-BDP FL prevented the binding of DAP and that lipid II might be the target molecule of DAP.<br /> Finally, we used fluid supported lipid bilayers to study the binding behavior of DAP on membranes with different lipid compositions. Bilayers were prepared on coverslips by vesicle fusion. The neutral phosphatidylcholine phospholipids were used as the matrix to which PG or/and bactoprenol lipids (C55-PP, C55-P, lipid II) were added. PG as well as the three bactoprenol lipids enhanced the binding of DAP. Surprisingly, addition of PG in bactoprenol-containing membranes significantly strengthened DAP binding, indicating that the bactoprenol lipids affect the binding of DAP and that the combination of PG and bactoprenol lipids is critical for the bactericidal mechanism of DAP. This explains the preferential binding of DAP to the septum. Our findings describe a new model for the mechanism of action of DAP.