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Daptomycin pore formation is restricted by membrane lipid acyl chain composition Robert M Taylor, David Beriashvili, Scott D. Taylor, and Michael Palmer ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00138 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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ACS Infectious Diseases

Daptomycin pore formation is restricted by lipid acyl chain composition Robert Taylor, David Beriashvili, Scott Taylor1, and Michael Palmer1 Department of Chemistry, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada

1

corresponding authors. Email: [email protected] and [email protected]

1 ACS Paragon Plus Environment


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Daptomycin is a calcium-dependent lipopeptide antibiotic that is used clinically against various Grampositive pathogens. It acts on bacterial cell membranes, whose susceptibility varies with the content of phosphatidylglycerol (PG). Some studies have reported that daptomycin permeabilizes and depolarizes bacterial cell membranes, while others have found no evidence of membrane permeabilization and thus proposed different mechanisms of antibacterial action. Divergent observations have also been reported regarding the effect of daptomycin on model membranes, which were found to be permeabilized nonselectively, selectively for small cations, or not at all. While these diverging model studies did consider the functional roles of different lipid head groups, they assumed the acyl chains to be interchangeable. We here show this assumption to be erroneous. In equimolar mixtures of PG and phosphatidylcholine (PC), dimyristoyl lipids support membrane permeabilization, whereas dioleyl and palmitoleyl lipids do not, even though daptomycin does bind to and form oligomers on all of these membranes. These observations help reconcile some of the discrepant findings in the literature. – Keywords: lipopeptide antibiotics, membrane permeabilization, model membranes, phosphatidylglycerol – The lipopeptide antibiotic daptomycin requires calcium to bind to the cell membranes of Gram-positive bacteria, whereupon it forms oligomers1,2 that bring about the antibacterial effect3. Binding and oligomerization are mediated by phosphatidylglycerol (PG)4,5. This negatively charged phospholipid is much more abundant in bacterial cell membranes than in mammalian ones, which most likely is important for the comparatively low toxicity of daptomycin in humans. Diminished abundance of PG – due to either decreased biosynthesis6 or increased conversion to either lysyl-PG6 or cardiolipin7 – reduces bacterial susceptibility to the antibiotic. Binding to other membrane components, in particular to intermediates of murein biosynthesis, has been examined but not demonstrated8. Early studies found that daptomycin depolarizes bacterial cells9,10, suggesting that it renders the cell membranes permeable for the major cations. In contrast, calcein entrapped in bacterial cells does not leak out, and another organic dye (ToPro3) does not enter11, indicating a selective nature of the membrane lesion. In Staphylococcus aureus, membrane permeabilization was found to correlate with bactericidal action12,13. However, a recent study on Bacillus subtilis did not confirm this correlation, finding no potassium leakage at bactericidal daptomycin concentrations; the bactericidal effect was instead ascribed to the dislodgement of biosynthetic enzymes from the cell membrane14. Therefore, the action mechanism of daptomycin, and the role of membrane permeabilization in it, remain contentious. The diverging findings and conclusions suggest the possibility that the action mode may indeed vary depending on the membrane composition of the bacterial species in question.


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The interaction of daptomycin with specific membrane lipids has been studied using liposomes and other membrane models. All of these studies agree on the requirement of PG for activity, but they differ on the nature of the membrane-damaging effect on PG-containing membranes. Two studies that employed daptomycin concentrations well above bactericidal ones reported rapid lipid flip-flop and permeabilization for the anionic fluorescent probe calcein15 as well as membrane fusion16, suggesting the formation of fairly non-selective membrane defects and general membrane disruption. A more recent study observed that daptomycin shrank the surface of giant unilamellar vesicles17, concomitantly with the formation of clusters containing both daptomycin and lipid that sometimes appeared to detach from the vesicle surface. An organic dye (Texas Red) entrapped in the vesicles did not leak out, suggesting that the membranes were not permeabilized. In contrast, selective permeabilization of large unilamellar vesicles (LUV) toward Na+, K+ and other small cations was observed using a coupled fluorescence assay18. We reasoned that the divergence of the reported findings might be due to neglected experimental parameters. While most model studies employed mixtures of phosphatidylcholine (PC) and PG, the phospholipids used differed in acyl chain composition. In particular, cation-selective permeabilization was observed with dimyristoyl lipids18, whereas non-selective permeabilization and fusion15,16 were reported with palmitoleyl lipids, and membrane surface shrinkage without permeabilization was observed with dioleyl lipids17. We here compared the permeabilization of liposomes with each of the three acyl chain species at daptomycin concentrations similar to those required for antibacterial activity, and found that under such conditions only DMPC/DMPG liposomes are permeabilized. Lack of permeabilization of DOPC/DOPG and POPC/POPG liposomes corresponds with reduced membrane translocation and a decrease in oligomer subunit stoichiometry. The results demonstrate that lipid acyl chains significantly affect the activity of daptomycin. This finding may also be relevant to the antibacterial mode of action against different bacterial species. Daptomycin binding to membranes composed of dimyristoyl, dioleyl, and palmitoleyl lipids. The calcium-dependent membrane interaction of daptomycin can be monitored using the intrinsic fluorescence of its kynurenine-13 residue19. Figure 1 shows the results obtained with liposomes composed of equimolar mixtures of DMPC with DMPG, DOPC with DOPG, and POPC with POPG, respectively. (The chemical structures of all lipids used in this study are shown in Figure S7.) With all membranes, kynurenine fluorescence rises steeply as calcium is increased, which indicates membrane insertion of this residue. The calcium threshold is a little higher with dimyristoyl lipids than with the two other mixtures. In each case, the signal approaches saturation at calcium levels of ≥1mM. Using the polarity-sensitive dye acrylodan attached to ornithine-6 of daptomycin, we have previously shown that this residue inserts into the membrane at lower calcium concentrations than does kynurenine-1320. On DOPC/DOPG membranes, the binding of ornithine-6 appears to be shifted to slightly lower calcium concentrations also (not shown). Overall, therefore, we conclude that



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daptomycin binds somewhat more avidly to the membranes composed of dioleyl or palmitoleyl than myristoyl lipids. We reported earlier that cardiolipin also increases the membrane affinity of daptomycin21. In that study, we propose how lipids with relatively smaller head groups, such as cardiolipin, may promote daptomycin binding. Since dioleyl and palmitoleyl phospholipids have bulkier acyl chains than dimyristoyl lipids and thus relatively smaller head groups, the same explanation may apply. Biologically relevant concentrations of daptomycin permeabilize DMPC/DMPG but not DOPC/DOPG or POPC/POPG membranes. Permeabilization of liposomes towards cations was monitored with a fluorescence assay that couples the exchange of cations across the membrane to the dissipation of a pH gradient, which is detected using the pH-sensitive probe pyranine18,22. Figure 2 shows that, among the three membranes, only DMPC/DMPG membranes were appreciably permeabilized, whereas DOPC/DOPG and POPC/POPG membranes were not. Since all membranes shared the same composition of lipid head groups, the difference in susceptibility must be ascribed to the different acyl chains. A recent study by Zhang et al. that also employed the pyranine-based assay to examine membrane permeabilization by daptomycin23 was inconclusive with respect to DMPC/DMPG membranes, while detecting no permeabilization of POPC/POPG membranes. However, using another assay that employed the fluorescent probes PBFI and SBFI, which bind potassium and sodium, respectively, the same authors concluded that POPC/POPG membranes were in fact permeabilized for both ions. Since these researchers used the same concentrations of daptomycin and calcium in both assays, these findings seem contradictory. To resolve this question, we here carried out the PBFI-based assay as described23, but with some added controls that were absent from the original publication. One caveat that applies to the PBFI assay is the spectral overlap between the absorption and fluorescence of this probe with those of the kynurenine residue in daptomycin (see Figure 3A). Indeed, the sudden jump in fluorescence that occurs upon addition of daptomycin, and which had also been observed Zhang et al.23 and taken as evidence of potassium permeation, is entirely due to kynurenine rather than PBFI. This is evident from its absence when daptomycin is omitted, replaced with a daptomycin derivative (daptomycin-EW) that lacks kynurenine but retains activity on DMPC/DMPG liposomes (see Figure S3) and on on bacterial cells24, or with the (non-fluorescent) potassium ionophore valinomycin. That the signal observed with daptomycin arises from kynurenine rather than PBFI is further corroborated by observing it at two different wavelengths (see Figure S4); furthermore, incubation of daptomycin with liposomes devoid of PBFI under otherwise identical conditions elicits a similar signal (Figure S5). While we did not carry out the analogous experiments with the sodium indicator SBFI, we note that it contains the same fluorophore as PBFI, and would therefore be subject to interference by daptomycin’s intrinsic kynurenine fluorescence also. In sum, the data obtained here or before23 with PBFI and SBFI 4 ACS Paragon Plus Environment

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do not support the conclusion that POPC/POPG membranes are permeabilized toward potassium or sodium by daptomycin. Translocation and oligomer formation of daptomycin on DOPC/DOPG membranes We have previously proposed that a functional daptomycin pore is formed by an oligomer of 8 subunits, with 4 each in the inner and the outer membrane leaflet. In keeping with this hypothesis, daptomycin distributes evenly between both leaflets of pure DMPC/DMPG membranes, which are susceptible to permeabilization. In contrast, it remains confined to the outer leaflet of DMPC/DMPG membranes that also contain 10 or 20% of cardiolipin, and which are not permeabilized. On such membranes, daptomycin still forms oligomers, but the number of subunits per oligomer is reduced to 421. Since POPC/POPG and DOPC/DOPG membranes also failed to be permeabilized, we decided to examine the distribution of daptomycin between membrane leaflets and the oligomer subunit stoichiometry with these membranes also. In the experiment shown in Figure 4, NBD-daptomycin was allowed to bind to DMPC/DMPG, POPC/POPG, and DOPC/DOPG liposomes, respectively, and then exposed to the reducing agent dithionite, which selectively reacts with NBD that is present on the outer membrane leaflet and quenches its fluorescence25. Similar to previous findings21, 52% of the NBDdaptomycin bound to DMPC/DMPG membranes are amenable to immediate reduction by dithionite, and thus presumably are located on the outer leaflet. With DOPC/DOPG or POPC/POPG liposomes, 69% and 65%, respectively, are immediately reduced. Thus, daptomycin distributes evenly between the two membrane leaflets on DMPC/DMPG membranes but not on the two others, which retain a greater share on the outer leaflet.On all types of membranes, the initial steep drop is followed by a time period of slower reduction, which we ascribe to daptomycin flipping back and forth between the two leaflets21. The number of daptomycin molecules per oligomer was determined using the method described in26. As in that former study, an average value of >6 subunits was obtained on DMPC/DMPG membranes, whereas values of 4.7±0.7 and 3.6±0.9 were observed on DOPC/DOPG and POPC/POPG membranes, respectively. Both values are compatible with a true number of 4 subunits per oligomer. Conclusion. While the role of head groups in daptomycin activity has been studied before, the effect of acyl chains so far has not been considered. Conflicting reports on daptomycin behavior on model membranes containing lipids with different acyl chains prompted us to compare these models side by side. The key finding of the present study is the striking difference in susceptibility to daptomycin of DMPC/DMPG membranes, which are readily permeabilized, and DOPC/DOPG or POPC/POPG membranes, which are not, even though the antibiotic does bind to each of these membranes. Therefore, when studying daptomycin activity, the acyl chain composition of model membranes cannot be chosen solely according to convenience but must be considered in the experimental design.



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One might also consider that differences in the fatty acyl composition of bacterial membranes could account for different susceptibilities toward daptomycin permeabilization. In this context, we must note that none of our liposome models closely resemble the composition of bacterial membranes: both the acyl chains (cf. Figure S6) and the head groups27-29 of bacterial membranes are more varied, and bacterial membranes generally do not contain PC. One notable study that compared membrane properties of daptomycin-sensitive and -resistant Enterococcus faecalis and faecium strains found significant differences in head group profiles but little change in fatty acyl composition28. Moreover, as with eukaryotic cells, bacterial membrane lipids may form segregated domains30, and daptomycin appears to preferentially bind to some of these6,31,32; therefore, average lipid profiles likely do not tell the whole story. In sum, the currently available evidence does not implicate specific acyl chains in daptomycin activity on bacterial cell membranes. We have previously suggested that the oligomeric daptomycin pore is an octamer that comprises two aligned daptomycin tetramers within the inner and outer membrane leaflet, respectively21. (The number of subunits determined experimentally by FRET here and earlier26,21 is 6-7; possible reasons why the experimental subunit stoichiometry may be lower than the true one have been discussed before26.) When daptomycin is restricted to the outer leaflet of model membranes through the inclusion of cardiolipin, only tetramers are observed21. While POPC/POPG and DOPC/DOPG membranes permit the partial translocation of daptomycin, the number of subunits per oligomer is again reduced to approximately 4. A conceivable explanation is that the membrane lipid acyl chain composition prevents daptomycin tetramers located in opposite leaflets from binding to one another and thereby forming a functional pore (as illustrated in the visual abstract for this paper). Whether or not this is explanation is correct, the results do indicate that translocation of daptomycin to the inner leaflet as such is not sufficient for pore formation. It should be noted that proposed bactericidal action mechanisms that do not involve membrane permeabilization maintain that daptomycin dislodges biosynthetic enzymes14 or proteins that regulate cell-division32 from their regular sites of attachment to the cell membrane. Since these proteins are associated with the cytoplasmic membrane surface, their functional disruption by daptomycin would most likely require the antibiotic to reach the inner membrane leaflet. Thus, regardless of permeabilization, it seems important to characterize the translocation of daptomycin to the inner membrane leaflet in any membrane model that aims to replicate the effect of daptomycin on susceptible bacteria.

Methods All experiments were carried out essentially as described before, but some methods underwent minor modifications. Liposomes were prepared by polycarbonate membrane extrusion as described before1. Oligomer subunit stoichiometry was determined exactly as described earlier26. The pyranine-based permeability assay was carried out as described before18 but with 1 mM pyranine only as detailed in a


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more recent study33. The dithionite reduction assay of daptomycin membrane translocation was performed as described before21. As noted in that reference, the high calcium concentration (25 mM) was chosen because it improves the performance of this assay, possibly by overcoming electrostatic repulsion between dithionite and PG. The PBFI-based permeability assay was carried out as reported by Straus and coworkers23. Additional details are given, and some potential pitfalls discussed, in the supplementary file to this paper. References 1. Muraih, J. K.; Pearson, A.; Silverman, J. and Palmer, M.: Oligomerization of daptomycin on membranes. Biochim Biophys Acta (2011) 1808:1154-1160. DOI: 10.1016/j.bbamem.2011.01.001 2. Muraih, J. K.; Harris, J.; Taylor, S. D. and Palmer, M.: Characterization of daptomycin oligomerization with perylene excimer fluorescence: stoichiometric binding of phosphatidylglycerol triggers oligomer formation. Biochim Biophys Acta (2012) 1818:673-678. DOI: 10.1016/j.bbamem.2011.10.027 3. Zhang, T.; Muraih, J. K.; Mintzer, E.; Tishbi, N.; Desert, C.; Silverman, J.; Taylor, S. and Palmer, M.: Mutual inhibition through hybrid oligomer formation of daptomycin and the semisynthetic lipopeptide antibiotic CB-182,462. Biochim Biophys Acta (2013) 1828:302-308. DOI: 10.1016/j.bbamem.2012.10.008 4. Baltz, R. H.: Daptomycin: mechanisms of action and resistance, and biosynthetic engineering. Curr Opin Chem Biol (2009) 13:144-151. DOI: 10.1016/j.cbpa.2009.02.031 5. Taylor, S. D. and Palmer, M.: The action mechanism of daptomycin. Bioorganic and Medicinal Chemistry (2016) 24:6253-6268. DOI: 10.1016/j.bmc.2016.05.052 6. Hachmann, A.-B.; Angert, E. R. and Helmann, J. D.: Genetic analysis of factors affecting susceptibility of Bacillus subtilis to daptomycin. Antimicrob Agents Chemother (2009) 53:1598-1609. DOI: 10.1128/aac.01329-08 7. Davlieva, M.; Zhang, W.; Arias, C. A. and Shamoo, Y.: Biochemical characterization of cardiolipin synthase mutations associated with daptomycin resistance in enterococci. Antimicrob Agents Chemother (2013) 57:289-296. DOI: 10.1007/s40278-013-1779-9 8. Rubinchik, E.; Schneider, T.; Elliott, M.; Scott, W. R. P.; Pan, J.; Anklin, C.; Yang, H.; Dugourd, D.; Müller, A.; Gries, K.; Straus, S. K.; Sahl, H. G. and Hancock, R. E. W.: Mechanism of action and limited cross-resistance of new lipopeptide MX-2401. Antimicrob Agents Chemother (2011) 55:2743-2754. DOI: 10.1128/aac.00170-11



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9. Alborn, W. E. J.; Allen, N. E. and Preston, D. A.: Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrob Agents Chemother (1991) 35:2282-2287. DOI: 10.1128/aac.35.11.2282 10. Allen, N. E.; Alborn, W. E. J. and Hobbs, J. N. J.: Inhibition of membrane potential-dependent amino acid transport by daptomycin. Antimicrob Agents Chemother (1991) 35:2639-2642. DOI: 10.1128/aac.35.12.2639 11. Cotroneo, N.; Harris, R.; Perlmutter, N.; Beveridge, T. and Silverman, J. A.: Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus. Antimicrob Agents Chemother (2008) 52:2223-2225. DOI: 10.3410/f.1108799.564926 12. Mascio, C. T. M.; Alder, J. D. and Silverman, J. A.: Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob Agents Chemother (2007) 51:4255-4260. DOI: 10.1128/AAC.00824-07 13. Silverman, J. A.; Perlmutter, N. G. and Shapiro, H. M.: Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob Agents Chemother (2003) 47:2538-2544. DOI: 10.1128/AAC.47.8.2538-2544.2003 14. Müller, A.; Wenzel, M.; Strahl, H.; Grein, F.; Saaki, T. N. V.; Kohl, B.; Siersma, T.; Bandow, J. E.; Sahl, H.-G.; Schneider, T. and Hamoen, L. W.: Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc Natl Acad Sci U S A (2016) 113:E7077E7086. DOI: 10.3410/f.726900979.793526790 15. Jung, D.; Rozek, A.; Okon, M. and Hancock, R. E. W.: Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin. Chem Biol (2004) 11:949-957. DOI: 10.2210/pdb1t5m/pdb 16. Jung, D.; Powers, J. P.; Straus, S. K. and Hancock, R. E. W.: Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes. Chem Phys Lipids (2008) 154:120-128. DOI: 10.1016/j.chemphyslip.2008.04.004 17. Chen, Y.-F.; Sun, T.-L.; Sun, Y. and Huang, H. W.: Interaction of daptomycin with lipid bilayers: a lipid extracting effect. Biochemistry (2014) 53:5384-5392. DOI: 10.1021/bi500779g 18. Zhang, T.; Muraih, J.; MacCormick, B.; Silverman, J. and Palmer, M.: Daptomycin forms cation- and size-selective pores in model membranes. Biochim Biophys Acta (2014) 1838:2425-2430. DOI: 10.1016/j.bbamem.2014.05.014 19. Lakey, J. H. and Ptak, M.: Fluorescence indicates a calcium-dependent interaction between the lipopeptide antibiotic LY146032 and phospholipid membranes. Biochemistry (1988) 27:4639-4645. DOI: 10.1021/bi00413a009 8 ACS Paragon Plus Environment

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20. Taylor, R. M.; Butt, K.; Scott, B.; Muraih, J. K.; Zhang, T.; Taylor, S. D.; Palmer, M. and Mintzer, E. M.: Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145. BBA Biomembranes (2016) 1858:1999-2005. DOI: 10.1016/j.bbamem.2016.05.020 21. Zhang, T.; Muraih, J. K.; Tishbi, N.; Herskowitz, J.; Victor, R. L.; Silverman, J.; Uwumarenogie, S.; Taylor, S. D.; Palmer, M. and Mintzer, E.: Cardiolipin prevents membrane translocation and permeabilization by daptomycin. J Biol Chem (2014) 289:11584-11591. DOI: 10.1074/jbc.m114.554444 22. Clement, N. R. and Gould, J. M.: Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles. Biochemistry (1981) 20:15341538. DOI: 10.1021/bi00509a019 23. Zhang, J.; Scoten, K. and Straus, S. K.: Daptomycin Leakage Is Selective. ACS infectious diseases (2016) 2:682-687. DOI: 10.1021/acsinfecdis.6b00152 24. Lohani, C.; Taylor, R.; Palmer, M. and Taylor, S.: Solid-Phase Total Synthesis of Daptomycin and Analogs. Organic Letters (2015) 17:748-751. DOI: 10.1021/acs.orglett.5b00043 25. McIntyre, J. C. and Sleight, R. G.: Fluorescence assay for phospholipid membrane asymmetry. Biochemistry (1991) 30:11819-11827. DOI: 10.1021/bi00115a012 26. Muraih, J. K. and Palmer, M.: Estimation of the subunit stoichiometry of the membraneassociated daptomycin oligomer by FRET. Biochim Biophys Acta (2012) 1818:1642-1647. DOI: 10.1016/j.bbamem.2012.02.019 27. Houtsmuller, U. M. T. and van Deenen, L. L. M.: On the amino acid esters of phosphatidylglycerol from bacteria. Biochim Biophys Acta (1965) 106:564-576. DOI: 10.1016/00052760(65)90072-X 28. Mishra, N. N.; Bayer, A. S.; Tran, T. T.; Shamoo, Y.; Mileykovskaya, E.; Dowhan, W.; Guan, Z. and Arias, C. A.: Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content. PLoS One (2012) 7:e43958. DOI: 10.1371/journal.pone.0043958 29. op den Kamp, J. A.; Redai, I. and van Deenen, L. L.: Phospholipid composition of Bacillus subtilis. J Bacteriol (1969) 99:298-303. DOI: 10.3168/jds.s0022-0302(69)86757-3 30. López, D. and Kolter, R.: Functional microdomains in bacterial membranes. Genes & development (2010) 24:1893-1902. DOI: 10.1101/gad.1945010



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31. Hachmann, A.-B.; Sevim, E.; Gaballa, A.; Popham, D. L.; Antelmann, H. and Helmann, J. D.: Reduction in membrane phosphatidylglycerol content leads to daptomycin resistance in Bacillus subtilis. Antimicrob Agents Chemother (2011) 55:4326-4337. DOI: 10.1128/aac.01819-10 32. Pogliano, J.; Pogliano, N. and Silverman, J. A.: Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins. J Bacteriol (2012) 194:4494-4504. DOI: 10.1128/jb.00011-12 33. Taylor, R. M.; Scott, B.; Taylor, S. and Palmer, M.: An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall. ACS Infect Dis (2017) 3:462-466. DOI: 10.1021/acsinfecdis.7b00019


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Figure legends Figure 1: Daptomycin binding to LUV membranes differing in lipid composition. Large unilamellar vesicles consisting of DMPC/DMPG, DOPC/DOPG, and POPC/POPG (molar ratio of PC/PG, 1:1; total lipid concentration, 250 µM) were incubated with daptomycin at different calcium concentrations. An increase in daptomycin’s intrinsic kynurenine fluorescence (excitation, 365 nm; emission, 445 nm) indicates membrane binding. Figure 2: Permeabilization by daptomycin of LUV membranes differing in lipid composition. Large unilamellar vesicles consisting of DMPC/DMPG, DOPC/DOPG, and POPC/POPG (molar ratio of PC/PG, 1:1; total lipid concentration, 250 µM; inner pH, 6.0) were loaded with the pH-sensitive probe pyranine (1mM interior concentration) and exposed to daptomycin (2 µM) in the presence of the proton ionophore CCCP (5nM). Permeabilization towards potassium in the buffer (pH 8.0; calcium: 5 mM) dissipates the proton gradient and increases the pyranine fluorescence. Dotted lines, controls with CCCP but without daptomycin; virtually identical curves were obtained when daptomycin was used without CCCP, or both were omitted (not shown). Only DMPC/DMPG membranes are permeabilized by daptomycin. Figure 3: Experiments with PBFI-loaded POPC/POPG liposomes. A: Excitation and emission spectra of daptomycin bound to POPC/POPG membranes. The excitation and emission wavelengths used for PBFI are indicated. B: Fluorescence response of POPC/POPG liposomes loaded with the K+ probe PBFI to daptomycin and to several controls. K+ is contained in the buffer but not the liposomes. The compound in question is added at 60 seconds; Triton X-100 is added at 300 seconds. Of all tested compounds, only daptomycin produces a step change in fluorescence emission. The K+ ionophore valinomycin as well as daptomycin-EW, which contains tryptophan instead of kynurenine in position 1324, are indistinguishable from control and only exhibit increased fluorescence when the entrapped PBFI is released into the buffer by Triton X-100 (0.1% final concentration). All concentrations were exactly as given by Zhang et al.23; daptomycin-EW, like native daptomycin, was used at 4.8 µM, and valinomycin at 0.5 µM. Figure 4: Translocation of NBD-daptomycin across DMPC/DMPG, POPC/POPG, and DOPC/DOPG membranes. LUVs of the stated lipid composition were incubated with mixtures of native daptomycin and NBD-daptomycin (molar ratio, 2:1) and calcium (25 mM). NBD fluorescence was measured over time (excitation wavelength, 478 nm; emission wavelength, 520 nm; each curve is the average of three separate experiments; curves are offset by 5 seconds along the x axis to avoid overlap). The fluorescence remaining immediately after addition of the membrane-impermeant reducing agent dithionite – 48±6% on DMPC/DMPG vs. 31±3% on DOPC/DOPG and 35±1% on POPC/POPG membranes – reflects the fraction of NBD-daptomycin that is initially inaccessible and presumably translocated to the inner membrane leaflet; the subsequent slow decrease likely reflects backpartitioning from the inner to the outer leaflet.



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Supporting Information •

Detailed descriptions of experimental materials and methods

•

Additional experiments on the use of PBFI for detecting membrane permeabilization


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100

E445 (normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36


80 60 DMPC/DMPG DOPC/DOPG POPC/POPG

40 20 0 0

0.01 0.05 0.1 0.5

1

5

10

50 100

Calcium (mM)

ACS Paragon Plus Environment

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E510nm (normalized)


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100 DMPC/DMPG DOPC/DOPG POPC/POPG

80 60 40 20 0 0

60

120

180

240

300

Time (seconds)


360

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Fluorescence (normalized)

A 100 75

excitation emission PBFI ex

50

PBFI em

25 0 300

350

400

450

500

550

Wavelength (nm) B liposomes only + daptomycin + daptomycin−EW + valinomycin

100

E507 (normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36


80 60 40 20 0 0

60

120

180

240

300

Time (seconds)


360


dithionite

Fluorescence (normalized)

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DMPC/DMPG

100

POPC/POPG DOPC/DOPG

75 50

Triton X−100

25 0 0

100

200

300

400

Time (seconds)


500

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For Table of Contents Use Only


Daptomycin Pore Formation Is Restricted by Lipid ... - ACS Publications

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