Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus

Abstract

The objective of this study was to further elucidate the role of membrane potential in the mechanism of action of daptomycin, a novel lipopeptide antibiotic. Membrane depolarization was measured by both fluorimetric and flow cytometric assays. Adding daptomycin (5 g/ml) to Staphylococcus aureus gradually dissipated membrane potential. In both assays, cell viability was reduced by >99% and membrane potential was reduced by >90% within 30 min of adding daptomycin. Cell viability decreased in parallel with changes in membrane potential, demonstrating a temporal correlation between bactericidal activity and membrane depolarization. Decreases in viability and potential also showed a dose-dependent correlation. Depolarization is indicative of ion movement across the cytoplasmic membrane. Fluorescent probes were used to demonstrate Ca 2؉ -dependent, daptomycin-triggered potassium release from S. aureus. Potassium release was also correlated with bactericidal activity. This study demonstrates a clear correlation between dissipation of membrane potential and the bactericidal activity of daptomycin. A multistep model for daptomycin's mechanism of action is proposed.Daptomycin is a novel lipopeptide antibiotic in late-stage clinical development for the treatment of serious gram-positive infections. Daptomycin exhibits rapid in vitro bactericidal activity against clinically significant strains of gram-positive pathogens including hemolytic streptococci, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci (4,10,12,14,19,22,23). Daptomycin acts at the cytoplasmic membrane of susceptible bacteria (8), as demonstrated by binding and fractionation studies. Additionally, the activity of daptomycin is dependent on the presence of physiologic levels of free calcium ions (50 mg/liter).Debate over daptomycin's mechanism of action has continued for more than a decade. One hypothesis suggests that daptomycin bactericidal activity is mediated by inhibition of lipoteichoic acid (LTA) biosynthesis (7,8). However, a recent investigation has failed to find evidence of a role for LTA in the mechanism of action of daptomycin in S. aureus or Enterococcus faecalis, suggesting that the in vitro bactericidal activity of daptomycin is independent of LTA biosynthesis (V. Laganas, J. Alder, and J. A. Silverman, submitted for publication). A second proposed mechanism of action for daptomycin is that the antibiotic causes dissipation of bacterial membrane potential, resulting in disruption of multiple aspects of cellular function (1, 2). Bactericidal activity via disruption of membrane potential is the proposed mechanism of action for a variety of antimicrobial peptides, including the pore-forming antibiotic nisin (18,20). We wished to further investigate the role of bacterial membrane potential in the mechanism of action of daptomycin. In this study, we demonstrate a significant correlation between membrane depolarization and bactericidal activity. Furthermore, we demonstrate that one possible mechanism of membrane depol...

The action mechanism of daptomycin

“…58 Similarly, Silverman et al employed a K + -sensitive fluorescent probe to demonstrate that 5 µg/ml daptomycin can trigger K + release from S. aureus suspended in Hepes buffer (pH 7.2). 73 K + release is accompanied by a collapse of membrane potential, as evidenced by the release of the membrane-permeant cation tetraphenylphosphonium; in contrast, the transmembrane pH gradient remains unaffected, as shown by the unaltered cellular accumulation of acetylsalicylic acid (ASA), a weak acid organic acid whose protonated form equilibrates across the membrane. 74 Daptomycin inhibits the cellular uptake of amino acids by active transport, which depends on the membrane potential.…”

“…79 From the above observations, the question naturally arises whether membrane permeabilization also controls the time course of bactericidal action. While this question was answered in the affirmative by Silverman et al, 73 Hobbs et al maintain that the loss of cell viability precedes that of biosynthetic capacity. However, their reported kinetics of cell killing and biosynthetic collapse differ only by some minutes.…”

“…Accordingly, the proposal by Silverman et al 73 that daptomycin also forms oligomeric membrane pores or channels was quite readily accepted, although at the time no experimental evidence of oligomer formation had been obtained. More recently, Muraih et al 45 demonstrated oligomer formation on liposomes and on bacterial membrane vesicles by FRET, using the Kyn (13) residue of unlabelled daptomycin as the donor and an extrinsic NBD moiety attached to Orn (6) as the acceptor (NBDdaptomycin, Figure 3).…”

Structure and antibacterial activities of new cyclic peptide antibiotics, pargamicins B, C and D, from Amycolatopsis sp. ML1-hF4

“…8 Our previous studies revealed that PRG-A exerted rapid bactericidal activity against staphylococci and enterococci via a mechanism that involved disruption of the membrane integrity of the target cells, 9 a mode of action distinct from that of daptomycin. 9,10 During the process of optimization of PRG-A production, we discovered new active components PRG-B, -C and -D (Figure 1) in the culture broth of the PRG-A-producing strain Amycolatopsis sp. ML1-hF4.…”

Membrane-Targeting DCAP Analogues with Broad-Spectrum Antibiotic Activity against Pathogenic Bacteria

“…5 Daptomycin is a clinical antibiotic that disrupts the membrane potential by inserting into the membrane, creating Ca 2+ -dependent pores, and releasing intracellular ions. 8 Disrupting the transmembrane potential using daptomycin may also mislocalize membrane-associated proteins that are involved in essential cellular processes, ultimately leading to membrane permeability and cell death. 9 We recently discovered 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl) propane-1,3-diol (DCAP, 1, Figure 1) in a high-throughput screen designed to identify small molecule inhibitors of MipZ, an ATPase that regulates the location of the division site in Caulobacter crescentus.…”