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• W2952315077 abstract "•14-Helical β-peptides can permeabilize synthetic lipid vesicles•β-Peptide binding to synthetic vesicles is consistent with a pore formation model•Antifungal β-peptides permeabilize the plasma membrane of Candida albicans•β-Peptides disrupt the nuclear membrane and vacuole to kill fungal cells Synthetic peptidomimetics of antimicrobial peptides (AMPs) are promising antimicrobial drug candidates because they promote membrane disruption and exhibit greater structural and proteolytic stability than natural AMPs. We previously reported selective antifungal 14-helical β-peptides, but the mechanism of antifungal toxicity of β-peptides remains unknown. To provide insight into the mechanism, we studied antifungal β-peptide binding to artificial membranes and living Candida albicans cells. We investigated the ability of β-peptides to interact with and permeate small unilamellar vesicle models of fungal membranes. The partition coefficient supported a pore-mediated mechanism characterized by the existence of a critical β-peptide concentration separating low- and high-partition coefficient regimes. Live cell intracellular tracking of β-peptides showed that β-peptides translocated into the cytoplasm, and then disrupted the nucleus and vacuole sequentially, leading to cell death. This understanding of the mechanisms of antifungal activity will facilitate design and development of peptidomimetic AMPs, including 14-helical β-peptides, for antifungal applications. Synthetic peptidomimetics of antimicrobial peptides (AMPs) are promising antimicrobial drug candidates because they promote membrane disruption and exhibit greater structural and proteolytic stability than natural AMPs. We previously reported selective antifungal 14-helical β-peptides, but the mechanism of antifungal toxicity of β-peptides remains unknown. To provide insight into the mechanism, we studied antifungal β-peptide binding to artificial membranes and living Candida albicans cells. We investigated the ability of β-peptides to interact with and permeate small unilamellar vesicle models of fungal membranes. The partition coefficient supported a pore-mediated mechanism characterized by the existence of a critical β-peptide concentration separating low- and high-partition coefficient regimes. Live cell intracellular tracking of β-peptides showed that β-peptides translocated into the cytoplasm, and then disrupted the nucleus and vacuole sequentially, leading to cell death. This understanding of the mechanisms of antifungal activity will facilitate design and development of peptidomimetic AMPs, including 14-helical β-peptides, for antifungal applications. Antimicrobial peptides (AMPs) are components of innate immune systems found in a wide variety of invertebrates and vertebrates (Beutler, 2004Beutler B. Innate immunity: an overview.Mol. Immunol. 2004; 40: 845-859Crossref PubMed Scopus (865) Google Scholar, Gallo and Nizet, 2003Gallo R.L. Nizet V. Endogenous production of antimicrobial peptides in innate immunity and human disease.Curr. Allergy Asthma Rep. 2003; 3: 402-409Crossref PubMed Scopus (71) Google Scholar, Ganz, 2003Ganz T. Defensins: antimicrobial peptides of innate immunity.Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2342) Google Scholar), and have demonstrated broad spectrum activity against bacteria, fungi, viruses, and cancer cells (Brogden et al., 2003Brogden K.A. Ackermann M. McCray P.B. Tack B.F. Antimicrobial peptides in animals and their role in host defences.Int. J. Antimicrob. Agents. 2003; 22: 465-478Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, Hancock and Lehrer, 1998Hancock R.E.W. Lehrer R. 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AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense.Trends Immunol. 2009; 30: 131-141Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar, Mahlapuu et al., 2016Mahlapuu M. Hakansson J. Ringstad L. Bjorn C. Antimicrobial peptides : an emerging category of therapeutic agents.Front. Cell. Infect. Microbiol. 2016; 6: 194Crossref PubMed Scopus (986) Google Scholar, Nizet, 2006Nizet V. Antimicrobial peptide resistance mechanisms of human bacterial pathogens.Curr. Issues Mol. Biol. 2006; 8: 11-26PubMed Google Scholar, Peschel and Sahl, 2006Peschel A. Sahl H.G. The co-evolution of host cationic antimicrobial peptides and microbial resistance.Nat. Rev. Microbiol. 2006; 4: 529-536Crossref PubMed Scopus (801) Google Scholar), AMPs are, as a class, less susceptible than conventional antibiotics to the emergence of microbial resistance because of their lack of a single target (Mahlapuu et al., 2016Mahlapuu M. Hakansson J. Ringstad L. Bjorn C. Antimicrobial peptides : an emerging category of therapeutic agents.Front. Cell. Infect. Microbiol. 2016; 6: 194Crossref PubMed Scopus (986) Google Scholar). Thus, there is significant interest in developing AMPs as drugs against infectious agents since many existing antimicrobials are plagued by the emergence of resistant strains. Unfortunately, the viability of natural AMPs as therapeutics is reduced by their low structural stability and activity in physiologic environments (Chu et al., 2013Chu H.L. Yu H.Y. Yip B.S. Chih Y.H. Liang C.W. Cheng H.T. Cheng J.W. Boosting salt resistance of short antimicrobial peptides.Antimicrob. Agents Chemother. 2013; 57: 4050-4052Crossref PubMed Scopus (89) Google Scholar, Goldman et al., 1997Goldman M.J. Anderson G.M. Stolzenberg E.D. Kari U.P. Zasloff M. Wilson J.M. 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Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37.Mol. Microbiol. 2002; 46: 157-168Crossref PubMed Scopus (340) Google Scholar). A variety of synthetic or non-natural peptidomimetic compounds have been developed to exploit the antimicrobial properties of AMPs and to address their low stability (Godballe et al., 2011Godballe T. Nilsson L.L. Petersen P.D. Jenssen H. Antimicrobial beta-peptides and alpha-peptoids.Chem. Biol. Drug Des. 2011; 77: 107-116Crossref PubMed Scopus (101) Google Scholar, Lienkamp et al., 2013Lienkamp K. Madkour A.E. Tew G.N. Antibacterial peptidomimetics: polymeric synthetic mimics of antimicrobial peptides.in: Abe A. Kausch H.H. Moller M. Pasch H. Polymer Composites - Polyolefin Fractionation - Polymeric Peptidomimetics - Collagens. Springer-Verlag Berlin, 2013: 141-172Google Scholar). One such class of compounds, β-peptides, is composed of β-amino acids and can fold into protein secondary structures found in AMPs, including the α helix (Appella et al., 1997Appella D.H. Christianson L.A. Klein D.A. Powell D.R. Huang X.L. Barchi J.J. Gellman S.H. Residue-based control of helix shape in beta-peptide oligomers.Nature. 1997; 387: 381-384Crossref PubMed Scopus (588) Google Scholar, Seebach et al., 1997Seebach D. Gademann K. Schreiber J.V. Matthews J.L. Hintermann T. Jaun B. Oberer L. Hommel U. Widmer H. 'Mixed' beta-peptides: a unique helical secondary structure in solution.Helv. Chim. Acta. 1997; 80: 2033-2038Crossref Scopus (205) Google Scholar, Seebach and Matthews, 1997Seebach D. Matthews J.L. Beta-Peptides: a surprise at every turn.Chem. Commun. 1997; : 2015-2022https://doi.org/10.1039/A704933ACrossref Google Scholar), β sheet (Chung et al., 2000Chung Y.J. Huck B.R. Christianson L.A. Stanger H.E. Krauthauser S. Powell D.R. Gellman S.H. Stereochemical control of hairpin formation in beta-peptides containing dinipecotic acid reverse turn segments.J. Am. Chem. Soc. 2000; 122: 3995-4004Crossref Scopus (134) Google Scholar, Krauthauser et al., 1997Krauthauser S. Christianson L.A. Powell D.R. Gellman S.H. Antiparallel sheet formation in beta-peptide foldamers: effects of beta-amino acid substitution on conformational preference.J. Am. Chem. Soc. 1997; 119: 11719-11720Crossref Scopus (213) Google Scholar), and γ turn (Chung et al., 1998Chung Y.J. Christianson L.A. Stanger H.E. Powell D.R. Gellman S.H. A beta-peptide reverse turn that promotes hairpin formation.J. Am. Chem. Soc. 1998; 120: 10555-10556Crossref Scopus (150) Google Scholar). Structure-functional analyses of β-peptides have demonstrated that these compounds can be designed to exhibit antibacterial (Arvidsson et al., 2001Arvidsson P.I. Frackenpohl J. Ryder N.S. Liechty B. Petersen F. Zimmermann H. Camenisch G.P. Woessner R. Seebach D. On the antimicrobial and hemolytic activities of amphiphilic beta-peptides.Chembiochem. 2001; 2: 771-773Crossref PubMed Scopus (106) Google Scholar, Epand et al., 2003Epand R.F. Umezawa N. Porter E.A. Gellman S.H. Epand R.M. Interactions of the antimicrobial beta-peptide beta-17 with phospholipid vesicles differ from membrane interactions of magainins.Eur. J. Biochem. 2003; 270: 1240-1248Crossref PubMed Scopus (65) Google Scholar, Hamuro et al., 1999Hamuro Y. Schneider J.P. DeGrado W.F. De novo design of antibacterial beta-peptides.J. Am. Chem. Soc. 1999; 121: 12200-12201Crossref Scopus (396) Google Scholar, Porter et al., 2000Porter E.A. Wang X.F. Lee H.S. Weisblum B. Gellman S.H. Antibiotics - non-haemolytic beta-amino-acid oligomers.Nature. 2000; 404: 565Crossref PubMed Scopus (632) Google Scholar, Raguse et al., 2002Raguse T.L. Porter E.A. Weisblum B. Gellman S.H. Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency.J. Am. Chem. Soc. 2002; 124: 12774-12785Crossref PubMed Scopus (253) Google Scholar) and antifungal (Karlsson et al., 2009Karlsson A.J. Pomerantz W.C. Neilsen K.J. Gellman S.H. Palecek S.P. Effect of sequence and structural properties on 14-helical beta-peptide activity against Candida albicans planktonic cells and biofilms.ACS Chem. Biol. 2009; 4: 567-579Crossref PubMed Scopus (71) Google Scholar, Raman et al., 2015Raman N. Lee M.-R. Lynn D.M. Palecek S.P. Antifungal activity of 14-helical β-peptides against planktonic cells and biofilms of Candida species.Pharmaceuticals. 2015; 8: 483-503Crossref Scopus (23) Google Scholar) activities at concentrations that induce little hemolysis in mammalian red blood cells. In prior work, we demonstrated that fungicidal activity of 14-helical β-peptides against the opportunistic pathogen Candida albicans requires a globally amphiphilic structure, composed of hydrophobic and cationic faces, with approximately three helical turns (Lee et al., 2014Lee M.R. Raman N. Gellman S.H. Lynn D.M. Palecek S.P. Hydrophobicity and helicity regulate the antifungal activity of 14-helical beta-peptides.ACS Chem. Biol. 2014; 9: 1613-1621Crossref PubMed Scopus (47) Google Scholar). We observed that both antifungal and hemolytic activities increased as β-peptides became more hydrophobic, and we identified a hydrophobicity window that resulted in high antifungal activity but low hemolysis. The addition of helix-stabilizing residues, such as aminocyclohexane carboxylic acid (ACHC), increased the activity and specificity of these 14-helical β-peptides against C. albicans. While prior studies have shown that β-peptides and other synthetic AMP peptidomimetics have promise as antimicrobial therapeutics, little is known about their mechanisms of action compared with those of natural AMPs. The canonical mechanism of AMP-mediated toxicity involves permeabilization of the target cell membrane, a process that depends on both the physicochemical properties of the AMP and those of the target cell membrane. Several models have been proposed to describe the mechanisms of AMP permeabilization of cell membranes (Brogden, 2005Brogden K.A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?.Nat. Rev. Microbiol. 2005; 3: 238-250Crossref PubMed Scopus (4384) Google Scholar, Huang, 2000Huang H.W. Action of antimicrobial peptides: two-state model.Biochemistry. 2000; 39: 8347-8352Crossref PubMed Scopus (651) Google Scholar, Shai, 1999Shai Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides.Biochim. Biophys. Acta. 1999; 1462: 55-70Crossref PubMed Scopus (1577) Google Scholar, Yeaman and Yount, 2003Yeaman M.R. Yount N.Y. Mechanisms of antimicrobial peptide action and resistance.Pharmacol. Rev. 2003; 55: 27-55Crossref PubMed Scopus (2339) Google Scholar). In the “barrel-stave model,” AMP monomers bind to the cell membrane and then oligomerize and form structured pores with the hydrophobic region of the peptide oriented toward the membrane lipids and the hydrophilic region comprising the pore channel. For example, aggregation of four to six alamethicin molecules in a cell membrane has been shown to form voltage-dependent ion channels (Boheim, 1974Boheim G. Statistical analysis of alamethicin channels in black lipid membranes.J. Membr. Biol. 1974; 19: 277-303Crossref PubMed Scopus (307) Google Scholar, Gordon and Haydon, 1972Gordon L.G.M. Haydon D.A. The unit conductance channel of alamethicin.Biochim. Biophys. Acta. 1972; 255: 1014-1018Crossref PubMed Scopus (163) Google Scholar, Gordon and Haydon, 1975Gordon L.G.M. Haydon D.A. Potential-dependent conductances in lipid membranes containing alamethicin.Philos. Trans. R. Soc. Lond. Series B Biol. Sci. 1975; 270: 433-447Crossref PubMed Scopus (91) Google Scholar). In contrast, in the “toroidal model,” peptides insert into the cell membrane and associate with lipid head groups to induce lipid bending. Thus, the pore lining is composed of both the peptides and membrane lipids. Magainin 2 (Matsuzaki et al., 1995Matsuzaki K. Murase O. Miyajima K. Kinetics of pore formation by an antimicrobial peptide, Magainin-2, in phospholipid bilayers.Biochemistry. 1995; 34: 12553-12559Crossref PubMed Scopus (170) Google Scholar, Yang et al., 1998Yang L. Harroun T.A. Heller W.T. Weiss T.M. Huang H.W. Neutron off-plane scattering of aligned membranes. I. Method of measurement.Biophys. J. 1998; 75: 641-645Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), protegrin-1 (Lazaridis et al., 2013Lazaridis T. He Y. Prieto L. Membrane interactions and pore formation by the antimicrobial peptide protegrin.Biophys. J. 2013; 104: 633-642Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Yamaguchi et al., 2002Yamaguchi S. Hong T. Waring A. Lehrer R.I. Hong M. Solid-state NMR investigations of peptide-lipid interaction and orientation of a β-sheet antimicrobial peptide, protegrin.Biochemistry. 2002; 41: 9852-9862Crossref PubMed Scopus (147) Google Scholar), melittin (Yang et al., 2001Yang L. Harroun T.A. Weiss T.M. Ding L. Huang H.W. Barrel-stave model or toroidal model? A case study on melittin pores.Biophys. J. 2001; 81: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, Zhou et al., 2014Zhou L. Narsimhan G. Wu X.Y. Du F.P. Pore formation in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/cholesterol mixed bilayers by low concentrations of antimicrobial peptide melittin.Colloid Surf. B Biointerfaces. 2014; 123: 419-428Crossref PubMed Scopus (16) Google Scholar), LL-37 (Lee et al., 2011Lee C.C. Sun Y. Qian S. Huang H.W. Transmembrane pores formed by human antimicrobial peptide LL-37.Biophys. J. 2011; 100: 1688-1696Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), MSI-78 (Hallock et al., 2003Hallock K.J. Lee D.K. Ramamoorthy A. MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain.Biophys. J. 2003; 84: 3052-3060Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), and other AMPs have been shown to act via the toroidal model. Finally, in the “carpet model,” AMPs first interact with the target membrane via electrostatic interactions, and then they effectively solubilize and disrupt the membrane. Dermaceptin S (Pouny et al., 1992Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogs with phospholipid membranes.Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (569) Google Scholar), cecropin (Gazit et al., 1996Gazit E. Miller I.R. Biggin P.C. Sansom M.S.P. Shai Y. Structure and orientation of the mammalian antibacterial peptide cecropin P1 within phospholipid membranes.J. Mol. Biol. 1996; 258: 860-870Crossref PubMed Scopus (226) Google Scholar), caerin 1.1 (Pukala et al., 2004Pukala T.L. Brinkworth C.S. Carver J.A. Bowie J.H. Investigating the importance of the flexible hinge in caerin 1.1: solution structures and activity of two synthetically modified caerin peptides.Biochemistry. 2004; 43: 937-944Crossref PubMed Scopus (60) Google Scholar), ovispirin (Yamaguchi et al., 2001Yamaguchi S. Huster D. Waring A. Lehrer R.I. Kearney W. Tack B.F. Hong M. Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR spectroscopy.Biophys. J. 2001; 81: 2203-2214Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and other AMPs have been suggested to follow the carpet model. Little is known about how antifungal peptidomimetic 14-helical β-peptides may induce membrane lysis and promote cell death in C. albicans. In addition to permeabilizing the cell membrane, some AMPs act on intracellular targets to induce microbial death. Modes of intracellular action include binding nucleic acids (e.g., buforin II [Park et al., 1998Park C.B. Kim H.S. Kim S.C. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions.Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (689) Google Scholar] and tachyplesin [Yonezawa et al., 1992Yonezawa A. Kuwahara J. Fujii N. Sugiura Y. Binding of Tachyplesin-I to DNA revealed by footprinting analysis - significant contribution of secondary structure to DNA binding and implication for biological action.Biochemistry. 1992; 31: 2998-3004Crossref PubMed Scopus (125) Google Scholar]) and inhibiting cell-wall synthesis (mersacidin [Brotz et al., 1995Brotz H. Bierbaum G. Markus A. Molitor E. Sahl H.G. Mode of action of the lantibiotic mersacidin - inhibition of peptidoglycan biosynthesis via a novel mechanism.Antimicrob. Agents Chemother. 1995; 39: 714-719Crossref PubMed Scopus (112) Google Scholar]), nucleic acid and protein synthesis (pleurocidin [Patrzykat et al., 2002Patrzykat A. Friedrich C.L. Zhang L.J. Mendoza V. Hancock R.E.W. Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli.Antimicrob. Agents Chemother. 2002; 46: 605-614Crossref PubMed Scopus (275) Google Scholar], PR-39 [Boman et al., 1993Boman H.G. Agerberth B. Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine.Infect. Immun. 1993; 61: 2978-2984Crossref PubMed Google Scholar], and HNP-1 [Lehrer et al., 1989Lehrer R.I. Barton A. Daher K.A. Harwig S.S.L. Ganz T. Selsted M.E. Interaction of human defensins with Escherichia coli - mechanisms of bactericidal activity.J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (582) Google Scholar]), and enzymatic activity (histatins [Nishikata et al., 1991Nishikata M. Kanehira T. Oh H. Tani H. Tazaki M. Kuboki Y. Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis.Biochem. Biophys. Res. Commun. 1991; 174: 625-630Crossref PubMed Scopus (72) Google Scholar, Puri and Edgerton, 2014Puri S. Edgerton M. How does it kill?: Understanding the candidacidal mechanism of salivary histatin 5.Eukaryot. Cell. 2014; 13: 958-964Crossref PubMed Scopus (119) Google Scholar], pyrrhocoricin [Gagnon et al., 2016Gagnon M.G. Roy R.N. Lomakin I.B. Florin T. Mankin A.S. Steitz T.A. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition.Nucleic Acids Res. 2016; 44: 2439-2450Crossref PubMed Scopus (107) Google Scholar, Otvos et al., 2000Otvos Jr., L. O I. Rogers M.E. Consolvo P.J. Condie B.A. Lovas S. Bulet P. Blaszczyk-Thurin M. Interaction between heat shock proteins and antimicrobial peptides.Biochemistry. 2000; 39: 14150-14159Crossref PubMed Scopus (286) Google Scholar], and drosocin [Otvos et al., 2000Otvos Jr., L. O I. Rogers M.E. Consolvo P.J. Condie B.A. Lovas S. Bulet P. Blaszczyk-Thurin M. Interaction between heat shock proteins and antimicrobial peptides.Biochemistry. 2000; 39: 14150-14159Crossref PubMed Scopus (286) Google Scholar]). Prior studies have focused on interaction of AMPs with the cell membrane, but their effects on intracellular compartments, such as the nucleus, vacuole, and mitochondria have not been studied. In addition, there is little understanding of the membrane-disrupting mechanism of peptidomimetic β-peptides and the fates of these compounds inside the target cell. Here, we studied interactions between 14-helical β-peptides and small unilamellar vesicles (SUVs) to elicit the membrane-disrupting mechanism and tracked fluorescently labeled β-peptide to understand the interactions between peptide and intracellular organelles. To elucidate mechanisms of β-peptide interactions with fungal cells, we first employed SUVs to investigate how membrane composition influences β-peptide adhesion to, and the subsequent permeabilization of, the vesicles. Next, we used confocal laser scanning microscopy (CLSM) and super-resolution structure illumination microscopy (SR-SIM) to validate that cooperative binding of β-peptides observed in SUVs also occurs in C. albicans cells. Finally, time-lapse microscopy tracking of fluorescently labeled β-peptides demonstrated that, after permeabilizing the cell membranes, the β-peptides lysed the nucleus and vacuole, resulting in cell death. Our results are consistent with pore-mediated mechanisms of membrane permeabilization and suggest that disruption of intracellular organelles also plays a role in the fungicidal activity of β-peptides. The improved understanding of mechanisms of β-peptide interactions with the cell membrane and organelles will enhance efforts to further develop these compounds as active and selective antifungal agents. To study the potential mechanisms of antifungal β-peptide toxicity, we synthesized β-peptides 1 (NH2-(ACHC-β3hVal-β3hLys)3-CONH2), 2 (NH2-(ACHC-β3hVal-β3hArg)3-CONH2), and 3 (NH2-(ACHC-β3hAal-β3hLys)3-CONH2) (Figure 1). We previously demonstrated that β-peptides 1, 2, and 3 share structural properties important in antifungal activity, including charge, number of β-amino acid residues, amphiphilicity, and helical stability (Lee et al., 2014Lee M.R. Raman N. Gellman S.H. Lynn D.M. Palecek S.P. Hydrophobicity and helicity regulate the antifungal activity of 14-helical beta-peptides.ACS Chem. Biol. 2014; 9: 1613-1621Crossref PubMed Scopus (47) Google Scholar, Raman et al., 2015Raman N. Lee M.-R. Lynn D.M. Palecek S.P. Antifungal activity of 14-helical β-peptides against planktonic cells and biofilms of Candida species.Pharmaceuticals. 2015; 8: 483-503Crossref Scopus (23) Google Scholar). However, the hydrophobicity of these β-peptides (as determined by reversed-phase high-performance liquid chromatography retention time) (Browne et al., 1982Browne C.A. Bennett H.P.J. Solomon S. The isolation of peptides by high-performance liquid chromatograpy using predicted elution positions.Anal. Biochem. 1982; 124: 201-208Crossref PubMed Scopus (212) Google Scholar, Jiang et al., 2008Jiang Z.Q. Kullberg B.J. van der Lee H. Vasil A.I. Hale J.D. Mant C.T. Hancock R.E.W. Vasil M.L. Netea M.G. Hodges R.S. 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The more hydrophobic β-peptides 1 and 2 exhibit a minimum inhibitory concentration (MIC) against C. albicans of 8 μg/mL, while the MIC of less-hydrophobic β-peptide 3 is 512 μg/mL (Table S1). We also synthesized nitrobenzoxadiazole (NBD)-labeled analogs of β-peptides 1, 2, and 3 (1-NBD, 2-NBD, and 3-NBD) and Cy5-labeled β-peptide 1 (1-Cy5) (Figure 1) to facilitate quantification of β-peptide partition coefficients into synthetic lipid vesicle membranes and to visualize the interactions between the β-peptides and live C. albicans cells, respectively. NBD is a small, polar fluorophore with a low dipole moment (Gazit et al., 1994Gazit E. Lee W.J. Brey P.T. Shai Y.C. Mode of action of the antibacterial cecropin B2-a spectrofluorometic study.Biochemistry. 1994; 33: 10681-10692Crossref PubMed Scopus (173) Google Scholar, Pouny et al., 1992Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogs with phospholipid membranes.Biochemistry. 1992; 31: 12416-12423Crossref PubMed Scopus (569) Google Scholar, Pouny and Shai, 1992Pouny Y. Shai Y. Interaction of D-amino acid incorporated analogs of paradoxin with membranes.Biochemistry. 1992; 31: 9482-9490Crossref PubMed Scopus (82) Google Scholar, Rapaport and Shai, 1991Rapaport D. Shai Y. Interaction of fluorescently labeled pardaxin and its analogs with lipid bilayers.J. Biol. Chem. 1991; 266: 23769-23775PubMed Google Scholar). After NBD labeling of β-peptides 1, 2, and 3, peptide retention times increased, indicating that the NBD label increased the hydrophobicity of these β-peptides (Table S1; Figure S1). The MICs of β-peptides 1-NBD and 2-NBD were within a factor of 2 of the MICs of the corresponding unlabeled β-peptides (Table S1; Figure S2), while the MIC of 3-NBD was 8-fold lower than the MIC of β-peptide 3 (Table S1, Figure S2), likely resulting from the increase in β-peptide hydrophobicity upon adding the NBD label. β-Peptide 3-NBD still exhibited a 4- and 8-fold greater MIC than 1-NBD and 2-NBD, respectively. While NBD labeling had little effect on β-peptide hydrophobicity and activity, we are unable to rule out possible influence of the NBD label on mechanism of action. To characterize mechanisms of antifungal β-peptide toxicity, we investigated biophysical interactions of fluorescently labeled β-peptides with model phospholipid membranes and C. albicans cell membranes, as well as the subsequent localization of β-peptides to intracellular compartments. We used the leakage of calcein from synthetic SUVs to assess the specificity of β-peptide disruption of phospholipid membranes composed of phosphatidylglycerol (PG) (anionic phospholipid), phosphatidylcholine (PC) (neutral phospholipid), and phosphatidylethanolamine (PE) (neutral phospholipid). We prepared SUVs with different phospholipid compositions to characterize the influence of β-peptide on the disruption of SUVs containing zwitterionic (PC:PE) and anionic (PC:PG) phospholipids. After preparation, the SUV size and phospholipid ratio were quantified using dynamic light scattering and 31P nuclear magnetic resonance (Table S2). Magainin 2, a pore-forming antibacterial peptide, and melittin, a pore-forming non-selective AMP, were used as controls for membrane lysis. Magainin 2 induced substantial calcein leakage only from the model bacterial membrane vesicles (PC:PG 60:40) (Tamba and Yamazaki, 2005Tamba Y. Yamazaki M. Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability.Biochemistry. 2005; 44: 15823-15833Crossref PubMed Scopus (187) Google Scholar) (Figures 2 and S3A–S3C). In contrast, melittin induced calcein leakage from all SUVs tested (Figures 2 and S3A–S3C), as expected given its broad activity against bacterial, fungal, and mammalian cells. The phospholipid selectivities of magainin 2 and melitti" @default.
• W2952315077 created "2019-06-27" @default.
• W2952315077 creator A5021033521 @default.
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• W2952315077 date "2019-02-01" @default.
• W2952315077 modified "2023-10-15" @default.
• W2952315077 title "14-Helical β-Peptides Elicit Toxicity against C. albicans by Forming Pores in the Cell Membrane and Subsequently Disrupting Intracellular Organelles" @default.
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• W2952315077 doi "https://doi.org/10.1016/j.chembiol.2018.11.002" @default.
• W2952315077 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6386598" @default.
• W2952315077 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30581136" @default.
• W2952315077 hasPublicationYear "2019" @default.
• W2952315077 type Work @default.

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