Letter pubs.acs.org/journal/aidcbc
Daptomycin Leakage Is Selective Jin Zhang, Kyle Scoten, and Suzana K. Straus* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada ABSTRACT: Daptomycin is a lipopeptide antibiotic approved for use against Gram-positive organisms, including highly resistant species. A number of studies have suggested that daptomycin kills bacteria by membrane permeabilization and depolarization. Recently a model membrane system consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in a 1:1 ratio and the ionophore CCCP was proposed as a simple model to investigate the mode of action of daptomycin and resistance mechanisms at a molecular level. This study investigates how this model depends on the composition of the membrane and the role of CCCP. Results obtained from a fluorescence assay using pyranine show that daptomycin causes leakage in liposomes of limited stability and that CCCP promotes this leakage. A different model membrane system used here, which relies on ion selective dyes such as 4,4′-[1,4,10,13tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (PBFI), and 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetraammonium salt (SBFI), is a more robust alternative. Findings based on this newer model suggest that daptomycin is selective for potassium. KEYWORDS: daptomycin, lipopeptide, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (PBFI), 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetraammonium salt (SBFI), pyranine, membrane depolarization, fluorescence, leakage
D
declined on a faster time scale than membrane depolarization. This study also demonstrated that the activity of daptomycin is linked to loss of not only K+ but also Mg2+ and ATP.18 All of the studies cited here involved whole bacterial cells, making studying membrane permeabilization by daptomycin at a molecular level complex. To study daptomycin at the level of molecular detail, Zhang et al.19 recently investigated the permeability properties of the proposed membrane pores caused by daptomycin by using the liposome model system 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho(1′-rac-glycerol) (DMPG) in a 1:1 ratio. They used a pyranine-based liposome permeabilization assay, as first proposed by Clement and Gould20 and also used by other groups such as Otis et al.21 In this assay, the pH-sensitive fluorophore pyranine is loaded into vesicles at, for example, pH 6.2. The external solution consists of cations in solution at, for example, pH 7.2. Peptides that form channels in the membrane will result in cations entering the vesicles, whereas protons exit to preserve the ionic equilibrium. As a consequence, the pH near the pyranine increases, leading to an increase in fluorescence as a function of time. This very assay was recently used to confirm the permeability induced by aurein 2.3, an
aptomycin is a lipopeptide antibiotic, composed of 13 residues that include a number of D- and uncommon amino acids. Importantly, it possesses an n-decanoyl fatty acid chain at the N-terminus, which is important for the interaction of daptomycin with the membrane.1,2 Daptomycin is one of the few available antibiotics that are effective against many resistant bacterial strains, for example, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant Enterococci (VRE). Calcium, in the form of Ca2+, is essential for its activity.1,3−5 Despite its wide use, the complete mechanism of action of daptomycin is still not fully understood and debated in the literature.6−13 In recent years, a number of studies have suggested that daptomycin functions by causing membrane permeabilization and depolarization. Early studies showed that daptomycin causes the same extent of leakage of DiSC35 in S. aureus cells as the control nisin, but at a much slower rate.14 In addition, daptomycin was shown to trigger K+ efflux, in a manner analogous to valinomycin,14 a peptide carrier ionophore that selectively transports potassium ions across bacterial membranes.15 A subsequent paper demonstrated that daptomycin acts without lysing bacterial cells,16 a finding further supported by a more recent study involving Bacillus anthracis.17 The importance of the gradual membrane depolarization in daptomycin’s ability to kill bacteria was called into question by Hobbs et al.,18 as it was found that bacterial viability © XXXX American Chemical Society
Received: August 23, 2016
A
DOI: 10.1021/acsinfecdis.6b00152 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Letter
antimicrobial peptide that kills B. subtilis by pore formation and selective ion leakage.22 In the data reported by Zhang et al.,19 daptomycin was shown to permeabilize liposomes for K+, Na+, Cs+, and Mg2+ equally well. Interestingly, this finding was observed only when the ionophore carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was present, albeit in nanomolar amounts. Many of the membrane depolarization experiments in whole bacterial cells mentioned above use CCCP as a control. For instance, 10 μM CCCP on its own was found to fully depolarize S. aureus in
