Telavancin Disrupts the Functional Integrity of the Bacterial Membrane through Targeted Interaction with the Cell Wall Precursor Lipid II

Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC₂(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous d-Ala-d-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin''s membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.The increasing prevalence of serious infections caused by gram-positive bacteria, including those caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), highlights the need for new agents with enhanced antimicrobial properties (2, 10, 21, 26, 35). One promising approach has been the development of lipoglycopeptide antibiotics, semisynthetic derivatives of glycopeptides that contain hydrophobic substituents and that possess improved antimicrobial properties (1, 4, 13, 32, 39). Telavancin, a lipoglycopeptide derivative of vancomycin, exhibits enhanced potency in vitro, concentration-dependent bactericidal activity, and activity both in vitro and in vivo against organisms that display reduced susceptibility to vancomycin (17, 18, 23, 24, 28, 31, 33, 36, 42). Telavancin has been evaluated in phase III clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia (46, 53).The bactericidal action of telavancin results from a mechanism that includes the inhibition of cell wall synthesis and the disruption of essential membrane barrier functions (25). Telavancin possesses the glycopeptide core of vancomycin, which binds with a high affinity to the acyl-d-alanyl-d-alanine (d-Ala-d-Ala) terminus of cell wall precursors through a network of hydrogen bonds and hydrophobic packing interactions (3, 45). Inhibition of cell wall synthesis by telavancin therefore involves binding to late-stage peptidoglycan precursors, including membrane-embedded lipid II. These interactions prevent both the polymerization of the precursor into peptidoglycan and subsequent cross-linking events. Telavancin also binds to bacterial membranes and causes membrane depolarization and increased membrane permeability. The mechanism by which telavancin binds to and disrupts the function of the bacterial membrane has not been determined.The present study was undertaken to further explore the interaction of telavancin with the bacterial membrane. Using a flow cytometry assay optimized for the accurate measurement of membrane potential in bacteria, we demonstrate that telavancin causes pronounced, concentration-dependent depolarization in S. aureus cells. Isolates of S. aureus expressing important and emerging resistance phenotypes, such as MRSA, heterogeneous vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA), and daptomycin-nonsusceptibile MRSA, are equally susceptible to depolarization by telavancin. We provide evidence, through multiple lines of investigation, that membrane disruption by telavancin requires binding to the bacterial specific target, lipid II. Finally, we demonstrate that telavancin does not lyse bacteria during the time course that membrane effects are assayed. Importantly, the latter observation indicates that telavancin-induced membrane depolarization is not a consequence of a weakened cell wall. The findings of the studies reported here enhance our understanding of telavancin''s mechanism of action and, in assays designed to be representative of physiological conditions, demonstrate that therapeutically relevant concentrations of telavancin inhibit essential functions of the bacterial membrane.