Jack Blazyk

Antimicrobial Activities and Structures of Two Linear Cationic Peptide Families with Various Amphipathic β-Sheet and α-Helical Potentials

ABSTRACT Many naturally occurring antimicrobial peptides comprise cationic linear sequences with the potential to adopt an amphipathic α-helical conformation. We designed a linear 18-residue peptide that adopted an amphipathic β-sheet structure when it was bound to lipids. In comparison to a 21-residue amphipathic α-helical peptide of equal charge and hydrophobicity, this peptide possessed more similar antimicrobial activity and greater selectivity in binding to and inducing leakage in vesicles composed of bacterial membrane lipids than vesicles composed of mammalian membrane lipids (J. Blazyk, R. Weigand, J. Klein, J. Hammer, R. M. Epand, R. F. Epand, W. L. Maloy, and U. P. Kari, J. Biol. Chem. 276:27899-27906, 2001). Here, we compare two systematically designed families of linear cationic peptides to evaluate the importance of amphipathicity for determination of antimicrobial activity. Each peptide contains six lysine residues and is amidated at the carboxyl terminus. The first family consists of five peptides with various capacities to form amphipathic β-sheet structures. The second family consists of six peptides with various potentials to form amphipathic α helices. Only those peptides that can form a highly amphipathic structure (either a β sheet or an α helix) possessed significant antimicrobial activities. Striking differences in the abilities to bind to and induce leakage in membranes and lipid vesicles were observed for the two families. Overall, the amphipathic β-sheet peptides are less lytic than their amphipathic α-helical counterparts, particularly toward membranes containing phosphatidylcholine, a lipid commonly found in mammalian plasma membranes. Thus, it appears that antimicrobial peptides that can form an amphipathic β-sheet conformation may offer a selective advantage in targeting bacterial cells.

A new monofluorinated phosphatidylcholine forms interdigitated bilayers.

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.

Interactions between the antimicrobial peptide, magainin 2, and Salmonella typhimurium lipopolysaccharides.

Using FT‐IR spectroscopy, the effects of magainin 2 on the thermotropic behavior of LPS isolated from wild‐type (SL3770) and LPS‐mutant strains of Salmonella typhimurium are characterized and compared. The mutant strains include Ra (SL3749), polymyxin‐sensitive Rb2(s) (SH5014), polymyxin‐resistant Rb2(r) (SH5357) and Rc (HN202) LPS chemotypes, whose polysaccharide chains differ in length but possess an identical number of phosphorylation sites. In all cases, magainin 2 causes a concentration‐dependent disordering of the LPS fatty acyl chains. Differences in disordering of LPS correlate more closely with the charge on the LPS molecule (determined by high‐resolution 31P NMR) rather than with the length of the LPS sugar side chain, contradicting the currently accepted model for the interaction of cationic antibiotics with the Gram‐negative cell envelope.

Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT‐IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature‐dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is −7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram‐negative cell envelope and may act as a molecular sponge to protect the plasma membrane.

An Automated Temperature Control and Data Acquisition System for Fourier Transform Infrared Studies: Application to Phase Changes in Hydrocarbons and Lipid Bilayers

As a means of facilitating temperature-dependent FT-IR studies of liquid samples, a simple, accurate, efficient, and versatile system for automated thermoelectric temperature control and UNIX-based data acquisition is described. With the use of this system, phase changes in an n-alkane, pentadecane, and an aqueous dispersion of a phospholipid, dipalmitoylphosphatidylcholine, were characterized. The combination of precise temperature regulation, ability to quantitate very small changes in the infrared bands, and careful control of the thermal history of the sample reveals subtle changes in the physical structure of these molecules in a reproducible manner.

Effect of magainin, class L, and class A amphipathic peptides on fatty acid spin labels in lipid bilayers

Magainins and other antimicrobial peptides increase ion flux across the membrane. They may do this by forming some type of pore or by perturbing lipid organization due to peptide lying on the bilayer surface. In order to determine if magainins perturb the lipid sufficiently to permeabilize the bilayer, their effect on the motion of fatty acid and lipid spin labels in phosphatidylcholine/phosphatidylglycerol (PC/PG) lipid vesicles was determined. Their effect was compared to two synthetic peptides, 18L and Ac-18A-NH(2), designed to mimic the naturally occurring classes of lytic (class L) and apolipoprotein (class A) amphipathic helices, respectively. We show that although magainins and 18L both had significant effects on lipid chain order, much greater than Ac-18A-NH(2), there was no correlation between these effects and the relative ability of these three peptide classes to permeabilize PC/PG vesicles in the order magainins=Ac-18A-NH(2) >> 18L. This suggests that the perturbing effects of magainins on lipid chain order at permeabilizing concentrations are not directly responsible for the increased leakage of vesicle contents. The greater ability of the magainins to permeabilize PC/PG vesicles relative to 18L is thus more likely due to formation of some type of pore by magainins. The greater ability of Ac-18A-NH(2) relative to 18L to permeabilize PC/PG vesicles despite its lack of disordering effect must be due to its ability to cause membrane fragmentation. Effects of these peptides on other lipids indicated that the mechanism by which they permeabilize lipid bilayers depends both on the peptide and on the lipid composition of the vesicles.

Methods for Assessing the Structure and Function of Cationic Antimicrobial Peptides

Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic alpha-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of antimicrobial peptides and the production of more potent analogues. To date, however, few of these peptides have been shown to have clinical efficacy, especially for systemic use, in large part due to insufficient selectivity between target and host cells. Recently, we developed a new strategy in the design of antimicrobial peptides. These linear cationic peptides, which form amphipathic beta-sheets rather than alpha-helices, demonstrated superior selectivity in binding to the lipids contained in bacterial vs. mammalian plasma membranes. Here we describe methods to evaluate the structure and function of cationic antimicrobial peptides.

Determination of the lipid composition of Salmonella typhimurium outer membranes by 31P NMR

Abstract High-resolution 31 P NMR spectroscopy is used to determine the phospholipid composition and the relative amounts of lipopolysaccharide (LPS) and phospholipid in the outer membrane (OM) of wild-type Salmonella typhimurium and rfb, rfa and pmrA mutants, which produce LPS molecules with a deficient sugar side chain. LPS mutations do not alter the phospholipid composition of the outer membrane and have only a limited effect on the relative levels LPS and phospholipids. PE comprises 85–95 mol % of the OM phospholipids. The mole fraction of LPS and phospholipids in the OM is 0.3–0.4 and 0.6–0.7, respectively. LPS mutations do not influence the fatty acid composition of either LPS or OM phospholipids. These data show that 31 P NMR is a powerful tool for identifying and quantitating bacterial membrane lipids and provide a foundation for understanding the molecular interactions of these lipids with cationic antimicrobial peptides, such as defensins, magainins and polymyxin B.

Complementary Effects of Host Defense Peptides Piscidin 1 and Piscidin 3 on DNA and Lipid Membranes: Biophysical Insights into Contrasting Biological Activities

Piscidins were the first antimicrobial peptides discovered in the mast cells of vertebrates. While two family members, piscidin 1 (p1) and piscidin 3 (p3), have highly similar sequences and α-helical structures when bound to model membranes, p1 generally exhibits stronger antimicrobial and hemolytic activity than p3 for reasons that remain elusive. In this study, we combine activity assays and biophysical methods to investigate the mechanisms underlying the cellular function and differing biological potencies of these peptides, and report findings spanning three major facets. First, added to Gram-positive (Bacillus megaterium) and Gram-negative (Escherichia coli) bacteria at sublethal concentrations and imaged by confocal microscopy, both p1 and p3 translocate across cell membranes and colocalize with nucleoids. In E. coli, translocation is accompanied by nonlethal permeabilization that features more pronounced leakage for p1. Second, p1 is also more disruptive than p3 to bacterial model membranes, as quantified by a dye-leakage assay and (2)H solid-state NMR-monitored lipid acyl chain order parameters. Oriented CD studies in the same bilayers show that, beyond a critical peptide concentration, both peptides transition from a surface-bound state to a tilted orientation. Third, gel retardation experiments and CD-monitored titrations on isolated DNA demonstrate that both peptides bind DNA but p3 has stronger condensing effects. Notably, solid-state NMR reveals that the peptides are α-helical when bound to DNA. Overall, these studies identify two polyreactive piscidin isoforms that bind phosphate-containing targets in a poised amphipathic α-helical conformation, disrupt bacterial membranes, and access the intracellular constituents of target cells. Remarkably, the two isoforms have complementary effects; p1 is more membrane active, while p3 has stronger DNA-condensing effects. Subtle differences in their physicochemical properties are highlighted to help explain their contrasting activities.

Structure-Function Relationship Investigations of Neuropeptide Y Bound to Hydrated Lipid Bilayers

Neuropeptide Y (NPY) is a neuroendocrinic peptide which belongs to the neuropeptide Y family. It activates Y-receptors, which are G-protein coupled receptors (GPCRs), and regulates various physiological processes. Furthermore, NPY and some of its fragments have antimicrobial and immunochemical properties. Several biophysical studies of NPY in the presence of model membranes indicate that it interacts strongly with various lipids. Notably, the structure of micelle-bound NPY has provided insights about how the so-called membrane catalysis model of Schwyzer applies to NPY. This model proposes that the membrane induces a “bio-active conformation” of the peptide that has the required orientation to bind the receptor selectively.Characterizing the structural features of NPY under physiologically relevant conditions is important to better understand its multiple functions. Since variations in the lipid composition of biological membranes can have drastic effects on the activity of NPY and its fragments, we have used a combination of solid-state NMR techniques to probe these peptides under changing conditions. In order to investigate the structure, dynamics, topology and depth of insertion of the peptides in the membrane-bound state, we have incorporated the peptides in various aligned lipid mixtures and collected 15N and 31P solid-state NMR spectra from the peptides and lipids, respectively. Circular dichroism studies have also been performed to assess the global structures of the peptides under sample conditions mirroring the ones used for the NMR studies. Finally, we have investigated the antimicrobial activity of the peptides on Gram-positive and Gram-negative bacteria. The results of these studies could be significant in understanding the modes of action of NPY as a multifunctional peptide active at various bacterial and mammalian lipid membranes.