Daptomycin Binds but does not Translocate across PC:PG Membranes

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Mar 1, 2016 - tational order parameter in the bacteria cell wall lipid bilayer of living wild .... bound with roughly the same affinity to POPC bilayers as the ...
Tuesday, March 1, 2016 Due to their activity against different pathogens and low toxicity to host cells, antimicrobial peptides (AMPs) are a promising approach to address the bacterial antimicrobial resistance. Biophysical methods have been used to observe the permeabilizing effect of AMPs on model membranes with the same lipid composition as the bacteria cell wall (BCW). In order to fully understand the antimicrobial properties of these peptides, it also necessary to investigate how AMPs interact with other relevant bacterial cell wall components including peptidoglycan and lipopolysaccharide. One way to better understand AMP interactions with non-lipidic components is to observe their effects on the complete bacterial cell wall. 2H Nuclear Magnetic Resonance (2H NMR) can be used to measure the orientational order parameters of lipid acyl chain segments. In this work, we have used 2H NMR to study the effect of AMPs on lipids in the membranes of whole bacteria. We use two techniques to prepare 2H labeled bacteria with isotope-labeled lipids introduced into the bacterial cell wall. These preparation techniques allow us to use 2H NMR to compare the orientational order parameter in the bacteria cell wall lipid bilayer of living wild type cells with and without AMP present. The effects two AMPs, either MSI-78 or CAME (Cepropin A (1-8) and melittin (1-10)), on both gramnegative (Escherichia coli) and gram-positive (Bacillus Subtilis) bacteria have been studied. In the presence of both MSI-78 and CAME, the 2H NMR spectral shape changes in ways that correspond to a decrease in the lipid acyl chain order parameter. 2065-Pos Board B209 Selective Membrane Disruption Mechanism of an Antibacterial g-Aapeptide Defined by EPR Spectroscopy Pavanjeet Kaur1, Yaqiong Li2, Jianfeng Cai2, Likai Song1. 1 National High Magnetic Field Laboratory and Florida State University, Tallahassee, FL, USA, 2University of South Florida, Tampa, FL, USA. g-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. How g-AApeptides interact with bacterial membranes and alter lipid assembly and properties are unclear, but such information is essential in order to understand their antimicrobial activities. Using electron paramagnetic resonance (EPR) techniques, we characterized the membrane interaction and destabilizing activities of a lipo-cyclic-g-AApeptide. The analyses revealed that the g-AApeptide binding increases the membrane permeability of POPC/POPG liposomes, which mimics negatively-charged bacterial membranes. Moreover, the g-AApeptide interacts strongly with POPC/POPG liposomes, thereby inhibiting membrane fluidity and reducing solvent accessibility around the lipid head group region. Furthermore, binding of the g-AApeptide induces significant lipid-lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon the g-AApeptide binding for liposomes mimicking mammalian cell membranes, consisting of neutral lipids and cholesterol. Our findings suggest that the g-AApeptide interact and disrupt bacterial membranes through a ‘‘carpet-like’’ mechanism. The results illustrated that the intrinsic features of g-AApeptides are important for their ability to selectively disrupt bacterial membranes, the implications of which extend to developing new antibacterial biomaterials. 2066-Pos Board B210 Selectivity of Antimicrobial Peptides: Association to Bacterial and Eukaryotic Cells and Cell-Density Dependence Filippo Savini1, Vincenzo Luca2, Daniela Roversi1, Alessio Boccedi1, Renato Massoud3, Yoon-kyung Park4, Maria Luisa Mangoni2, Lorenzo Stella1. 1 Department of Chemical Science and Technologies, University of Rome Tor Vergata, Roma, Italy, 2Department of Biochemical Sciences, Sapienza University of Rome, Roma, Italy, 3Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Roma, Italy, 4Department of Biotechnology, Chosun University, Gwangju, Korea, Republic of. Several questions limit the applicability of antimicrobial peptides (AMPs) as a new class of antibiotics against the problem of multidrug resistant bacteria. AMPs are screened for their direct in vitro bactericidal activity, mediated by association to and perturbation of bacterial membranes. However, they act also as immunomodulators, and it is debated which function is predominant at peptide and bacteria concentrations relevant for in vivo conditions. In vitro, AMPs are toxic to mammalian cells only at concentrations higher than those needed for bactericidal activity. This selectivity is presumably determined by the difference in lipid composition of membranes of the two cell types, as studies on liposomes show a higher affinity for bilayers mimicking bacterial membranes. However, it might be just an experimental artifact of

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the in vitro assay conditions that employ lower cell densities for bacteria than for red blood cells (RBCs), which are also larger. Recently, by fluorescence spectroscopy we characterized the association of the cathelicidin PMAP-23 to bacterial cells, and determined for the first time the number of membrane-bound peptide molecules necessary to kill a bacterial cell (106 peptides per E. Coli cell) [ACS Chem. Biol. 2014 9:2003]. We now measured the dependence of killing activity on bacterial cell density: as a consequence of the binding equilibrium, total micromolar peptide concentrations are needed even with extremely low bacterial counts. We also extended our method to measure peptide binding to RBCs, showing that also in this case a high coverage of the cell surface is needed to cause lysis. Finally, we investigated the cell-density dependence of hemolytic activity. Overall, these data clarify the complex dependence of AMP activity and selectivity on the density of bacterial and human cells, and open new questions for their behavior in vivo. 2067-Pos Board B211 PSD1 Antimicrobial Activity Against Candida Albicans Planktonic Cells and Biofilms So´nia Gonc¸alves1, Patrı´cia M. Silva1, Ma´rio R. Felı´cio1, Luciano N. de Medeiros2, Eleonora Kurtenbach2, Nuno C. Santos1. 1 Instituto de Medicina Molecular, Lisboa, Portugal, 2Instituto de Biofı´sica Carlos Chagas Filho, Rio de Janeiro, Brazil. Psd1 is a defensin isolated from Pisum sativum seeds, with antimicrobial peptide (AMP) characteristics. Candida albicans is an important human pathogen, causing opportunistic infections. We tested the effects of Psd1 against this pathogen biofilms and planktonic cells. Three C. albicans variants were studied, including a mutant deficient in glucosylceramide synthase, conferring resistance to Psd1 antifungal action. Psd1 effects on cell morphology, membrane roughness and cell stiffness were studied. Differences in the planktonic cells and biofilm formation were observed for the fungal variants studied. Flow cytometry and confocal microscopy showed that cell death is time-dependent and accelerate by increasing Psd1 concentrations. Increased cell death and surface alterations, with membrane disruption and leakage of cellular contents, were observed by atomic force microscopy (AFM), together with an inhibition or eradication of fungal biofilms. These results showed some important key for Psd1-fungal membrane interaction against a relevant fungal human pathogen, aiming at its possible use as a natural antimycotic agent. 2068-Pos Board B212 Self-Assembling and Ion Transport Properties of Membrane Active Peptides Driven by Formation of a Fluorous Interface Normand Voyer1, Raphae¨l Godbout1, Se´bastien Le´gare´2, Maud S.V. Auger1, Franc¸ois Otis1, Claudia Carpentier1, Patrick Lagu¨e2, Miche`le Auger1. 1 Chemistry, Universite´ Laval, Quebec City, QC, Canada, 2Biochemistry, Microbiology and Bioinformatics, Universite´ Laval, Quebec City, QC, Canada. Ion channel proteins are complex architectures vital for numerous physiological processes and involved in many diseases. With the aim of developing simpler supramolecular systems mimicking ion transport properties of channel proteins, we will describe novel helical peptides designed to self assemble in membranes into well defined superstructures capable of facilitating the translocation of ions. The driving force for self assembly is the formation of a stable fluorous interface created by interdigitation of fluorinated side chains of unnatural aminoacids incorporated at specific positions of the helical peptide. We will present the synthesis, the characterization and the ion transport abilities of a prototypical fluorinated peptide. We will also describe preliminary biophysical and computational studies done to delineate the membrane active structure. 2069-Pos Board B213 Daptomycin Binds but does not Translocate across PC:PG Membranes Mark Kreutzberger, Antje Pokorny, Paulo F. Almeida. Chemistry and Biochemistry, Univ North Carolina Wilmington, Wilmington, NC, USA. The lipopeptide daptomycin is a last-resort antimicrobial in multiresistant Staphylococcus aureus infections. Despite its importance, little is understood about the interaction of daptomycin with lipid membranes and the subsequent events leading to antibacterial effects. In this work we asked three fundamental questions: (1) Does daptomycin bind to giant unilamellar vesicles (GUVs) composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG)? (2) Does it cause dye flux across the membrane? (3) Does the lipopeptide itself translocate across the membrane? Fluorescence confocal microscopy was used to visualize the binding of daptomycin to GUVs, dye flux into those

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Tuesday, March 1, 2016

vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca2þ. Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca2þ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca2þ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca2þ (2 mM), daptomycin was distributed evenly on the GUV membrane. Supported by NIH Grants GM072507 and AI088567. 2070-Pos Board B214 Are Leucine and Isoleucine Equivalent in Binding of Amphipathic Peptides to Membranes Mia A. Rosenfeld, Shatima Stokes, Jonathan H. Diaz, Paulo F. Almeida, Antje Pokorny. Chemistry and Biochemistry, Univ. North Carolina Wilmington, Wilmington, NC, USA. Lysette is a 22 amino acid peptide derived from staphylococcal d-lysin that forms an amphipathic a-helix when bound at membrane-water interfaces. We previously found that the experimentally determined DG of binding for lysette is more favorable than that predicted by the Wimley-White interfacial hydrophobicity scale by about 4 kcal/mol. Closer investigation of the amino acid composition of other peptides that are well described by the WimleyWhite interfacial scale, such as melittin, led us to hypothesize that a preponderance of isoleucine (Ile) over leucine residues (Leu), as found in lysette, may be responsible for the deviation from the Wimley-White prediction. To test our hypothesis, we designed lysette variants lysette-I and lysette-L. In lysette-I, all Leu residues were replaced by Ile, and in lysette-L, all Ile were replaced by Leu. We also designed a melittin variant, iso-melittin, in which all Leu residues were replace by Ile. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic phosphatidylcholine (POPC) vesicles were determined by stopped-flow fluorescence and circular dichroism. If the hypothesis were correct, Lysette-I and iso-melittin should bind significantly better to zwitterionic bilayers than their Leu-rich counterparts, if the helicities of the lipid-bound peptides were not influenced by the Leu-Ile substitution. We found that the lysette-I bound significantly better to POPC vesicles than the lysette-L variant, as predicted. In the case of melittin, however, iso-melittin bound with roughly the same affinity to POPC bilayers as the original Leurich melittin. The results are discussed in terms of peptide structures in solution and when bound at the water-lipid interface, and the location of the lipid-bound states in the membrane. This work was supported in part by NIH grants AI088567 and GM072507. 2071-Pos Board B215 pHLIPÒ: Uses in Measuring Cell Surface pH, Imaging Tumors, and Delivering Therapeutics Donald Engelman1, Yana K. Reshetnyak2, Oleg A. Andreev3. 1 Dept Molec Biophys/Biochem, Yale University, New Haven, CT, USA, 2 Physics, University of Rhode Island, Kingston, RI, USA, 3Physics, University of Rhode Island, Kingston, RI, USA. Acidity is a general property of tumors, and may serve as a biomarker that is not susceptible to resistance by selection. The discovery of pHLIPÒs (pH (Low) Insertion Peptides) provides a path to exploit this biomarker, and has led to the use of related peptides to study peptide insertion across bilayers, to selectively target cargoes to tumors and other acidic tissues in vivo, and to deliver molecules across tumor cell plasma membranes. A pHLIPÒ is unfolded on the surface of a membrane at normal pH, and folds to form a transmembrane helix when the pH is lowered. Tumor acidity is expected to be enhanced at cell surfaces by the electrochemical potential. Using pHLIPÒ to position pH-sensing dyes, it has been possible to document the lower surface pH, and to show that glucose further enhances acidity. We will present data on these key observations. Imaging agents, such as fluorescent labels or PET isotopes, can be positioned at the surfaces of tumor cells. Accumulation of these labels may allow uses in diagnosis and image-guided surgery. We will show examples. pHLIPÒ peptides also have the potential to target delivery of therapeutic molecules into cells. A remarkable opportunity may be afforded to expand the chemical range of such molecules, since translocation succeeds for agents that are large and polar. We will show examples of the translocation

of PNAs and of Amanitin, each of which is shown to inhibit tumor growth in vivo. 2072-Pos Board B216 The Role of Multivalency in Inhibition of Bacillus Anthracis and Clostridium Botulinum Binary Toxins by Cationic PAMAM Dendrimers Goli Yamini1, Veronica Wright1, Holger Barth2, Ekaterina M. Nestorovich1. 1 Biology, The Catholic University of America, Washington, DC, USA, 2 Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany. Multivalent interactions between oligomeric receptors and multivalent ligands not only play an important role in many biochemical processes, but are also investigated and employed for rational drug design and development. Multivalent ligands often possess a high affinity for multivalent binding sites, significantly greater than that of a single functional group interacting with a single binding site. A number of protein toxins, including binary bacterial toxins, have recently been successfully neutralized by novel synthetic multivalent blockers. This study explores the potentially universal antitoxin activity of dendrimers, which are the repeatedly branched polymers with all bonds emanating from a central core. Dendrimers are unique highly branched macromolecules, where each consecutive growth step represents a new dendrimer ‘‘generation’’ with an increased diameter and double the number of reactive surface functional groups. We use several generations of polyamido amine (PAMAM) intact dendrimers and their imperfect forms, known as dendrons, to investigate their blocking activity against the pore-forming components of two binary bacterial exotoxins, Clostridium Botulinum C2 and Bacillus anthracis anthrax (PA63 and C2IIa, respectively). While aiming for different cytosolic targets, these binary toxins rely on similar cellular uptake mechanisms that include formation of oligomeric cation selective pores under acidic endosomal conditions. These pores are essential for delivery of enzymatic components of the internalized toxins from endosomes into the cytosol of target cells. By single channel reconstitution and high-resolution recording in planar bilayer lipid membranes, we show that PAMAM dendrimers obstruct PA63 and C2IIa at nM concentrations. We also strive to understand the role of attractive interaction between the dendrimer/dendron blockers and the ion channel molecules. 2073-Pos Board B217 Ordering Effect Induced by SARS-CoV Fusion Peptides on Membranes Containing Negatively Charged Lipids Might be Important to Membrane Fusion Luis G.M. Basso1, Morteza Jafarabadi2, Alex I. Smirnov2, Antonio J. Costa-Filho1. 1 Department of Physics, University of Sao Paulo, Ribeira˜o Preto, Brazil, 2 Department of Chemistry, North Carolina State University, Raleigh, NC, USA. The S2 subunit of the spike glycoprotein from SARS coronavirus (CoV) contains internal membranotropic domains that play important roles to the viral and host cell membrane fusion. These functional domains, which include a so-called fusion peptide (FP) and an internal FP, are exposed to membrane interactions upon a specific trigger. Although membrane fusion has been broadly studied in recent years, many aspects of the molecular mechanism behind the virus-host cell membrane fusion remain unknown, including conformational changes of the lipid bilayers during peptide-membrane interactions. Here we employed spin-labeling Electron Spin Resonance (ESR), 31P oriented sample solid state (OSSS) Nuclear Magnetic Resonance (NMR), and Differential Scanning Calorimetry (DSC) to address these questions of fusion peptide - membrane interactions from the membrane perspective. DSC results showed that the peptides strongly perturb the thermotropic behavior of zwitterionic and negatively-charged lipid vesicles, with the largest effects seen for the latter. Not only the charge but also the lipid headgroup structure seems to be relevant for the energetics of the interaction. ESR showed that both peptides were capable of increasing lipid packing and head group ordering only in the presence of negatively charged lipids, which was further corroborated by NMR on aligned lipids deposited into nanoporous anodic aluminum oxide substrates. The observed effects are well correlated to those caused by well-known membrane fusion promoters and are in contrast to those promoted by membrane fusion inhibitors. The combined effect of an increased chain-packing energy, which induces bending moment in the bilayer, and membrane surface ordering, which is related to lipid dehydration, might promote negative curvature for negatively charged lipidcontaining membranes, thus, helping to overcome the high kinetic barrier of the membrane fusion.