The ability of antimicrobial peptides (AMPs) to target and lyse the harmful microbial membrane over that of a host's is a unique characteristic making these innate immune effectors promising candidates to fill the growing therapeutic void resulting from antibiotic drug resistance. This discriminatory behavior is believed to strongly depend on the chemical and structural properties of the lipids that comprise the cell membrane. For instance, the selectivity of AMPs can be based on the electrostatic attraction of these predominately cationic peptides for the bacterial membrane surface heavily populated with negatively charged lipid components. We have previously shown that zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayers display concentration-dependent structural transformations induced by protegrin-1 (PG-1) that progress from fingerlike instabilities at bilayer edges, to the formation of pores, and finally to a network of wormlike micelles. The increasing degree of membrane structural disruption in charge-neutral membranes demonstrates that a more complex interaction than that suggested by a simple electrostatic argument is needed to explain AMP selectivity. We propose that in addition to an electrostatic consideration, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes rather than those of the host. We have tailored our investigations to utilize membrane components which eukaryotes and prokaryotes contain in drastically different proportions, specifically the presence and absence of cholesterol respectively. In these results we have employed a variety of biophysical techniques to elucidate how increasing cholesterol content in solid-supported DMPC bilayers retards the ability of PG-1 to induce membrane disruption. Atomic force microscopy was used to assess the propensity for pore formation, while neutron reflectivity and oriented circular dichroism studies were advantageous in providing molecular level detail on the location and orientation of PG-1 with respect to the membrane.

453-Pos Board B208 The Interaction Between the Antimicrobial Peptide K-Hya1 and Model Membranes: Distinct Action in Neutral or Negatively Charged Bilayers Thais A. Enoki1, Karin A. Riske2, Katia R. Perez2, Esteban N. Lorenzon3, Eduardo M. Cilli3, M. Teresa Lamy1. Institute of Physics, Sao Paulo University, Sao Paulo, Brazil, Department of Biophysics, Federal University of Sao Paulo, Sao Paulo, Brazil, Institute of Physics, Sao Paulo State University, Sao Paulo, Brazil. Antimicrobial peptides (AMPs) are important molecules for the innate immune defense system of plants and animals. The antimicrobial peptide Hylina1 (Hya1) was isolated from arboreal South African frog Hypsiboas albopunctatus. In this work, we investigate the interaction of an analogue peptide, KHya1 (KIFGAIWPLALGALKNLIK-NH2) with model membranes composed of DPPC (dipalmitoyl phosphatidylcholine), DPPG dipalmitoyl phosphatidylglycerol) and DPPC:DPPG 1:1 or of POPC (palmitoyl oleoyl phosphatidylcholine) and POPC:POPG (palmitoyl oleoyl phosphatidylglycerol) with different techniques. Differential Scanning Calorimetry (DSC) profiles show distinct environments in the bilayer with anionic lipids, suggesting a disturbed region due to peptide adsorption, and an unaltered region. On the other hand, in neutral membranes an average perturbation is observed, which suggests a superficial interaction. Steady-state fluorescence spectroscopy of the intrinsic Trp residue shows a deeper insertion of this residue into charged bilayers as compared with neutral membranes. Dye-leakage experiments show that membrane charge also modulates the kinetics of membrane permeabilization, which is much faster for charged bilayers. Optical microscopy of giant unilamellar vesicles (GUVs) in the fluid phase revealed a different mechanism of action of the peptide in the presence or absence of negatively charged lipids. K-Hya1 induces small perturbations in POPC vesicles, causing membrane permeabilization without morphological changes. On the other hand, the peptide induces permeabilization accompanied by a large increase in surface area of POPC:POPG vesicles, suggesting the opening of several pores in the bilayer. Taken together, the results clearly show a peptide-bilayer interaction modulated by the presence of negatively charged lipids. Data can be interpreted as a different orientation of the peptide in the bilayer: parallel to the surface in neutral membranes and stable and crossed in anionic membranes.

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Defensins are small, multifunctional cationic peptides. They typically contain six conserved cysteines whose three intramolecular disulfides stabilize a largely beta-sheet structure. This review of human alpha-defensins begins by describing their evolution, including their likely relationship to the Big Defensins of invertebrates, and their kinship to the beta-defensin peptides of many if not all vertebrates, and the beta-defensins found in certain non-human primates. We provide a short history of the search for leukocyte-derived microbicidal molecules, emphasizing the roles played by luck (good), preconceived notions (mostly bad), and proper timing (essential). The antimicrobial, antiviral, antitoxic, and binding properties of human alpha-defensins are summarized. The structural features of alpha-defensins are described extensively and their functional contributions are assessed. The properties of HD6, an enigmatic Paneth cell alpha-defensin, are contrasted with those of the four myeloid alpha-defensins (HNP14) and of HD5, the other alpha-defensin of human Paneth cells. The review ends with a decalogue that may assist researchers or students interested in alpha-defensins and related aspects of neutrophil function.

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.

Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.

Antimicrobial peptides (AMPs) are a class of small (less than 100 amino acid residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. To understand the mechanism of membrane disruption by AMPs, we investigated, via atomic force microscopy, topological changes induced by protegrin-1 (PG-1), an 18-residue, cationic, β-sheet AMP isolated from pig leukocytes, in supported phospholipid bilayers (SPBs). Lipid mixtures of dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylcholine (DOPC), and cholesterol were used to mimic eukaryotic cell membranes while bacterial cell membranes were emulated by substitution of cholesterol for dioleoylphosphatidylethanolamine (DOPE). We have previously shown that AMP disruption of zwitterionic dimyristoylphosphatidylcholine (DMPC) SPBs induce concentration dependent structural transformations that progress from fingerlike instabilities at bilayer edges, to the formation of sievelike nanoporous structures, and finally to a network of wormlike micellar structures. The observed transformations suggest that the peptides act to lower the interfacial energy of the bilayer in a manner similar to detergents. Detergent solubilization of membranes encompass processes such as pore formation, blebbing, budding, and vesiculation that share common saddle-splay ("negative Gaussian") curved topologies. We pose that membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups enhance the efficacy of AMP disruption while those membranes containing cholesterol retard disruption. Results have shown that cholesterol incorporation shifts the disruption susceptibility of a bilayer to a higher peptide dosage regime while an opposite effect is observed in the presence of PE. The observed trend sheds light on a lingering debate as to how nature has evolved AMPs to discriminate between host and pathogen.

θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel β-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents.

ABSTRACT Retrocyclins are humanized versions of the θ-defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by “piggyback phagocytosis,” model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

ABSTRACT Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.

Pore formation affects the structural integrity of the lipid membrane, compromising its ability to act as a semi-permeable barrier and eventually leading to cell death. We present results obtained via atomic force microscopy (AFM) when lipid bilayer patches made dimyristoylphosphatidylcholine (DMPC) with increasing cholesterol content were subjected to the action of an cationic antimicrobial peptide (AMP), Protegrin-1 (PG-1). The cholesterol content was varied from 0% to 30%, where the lower and the upper bounds represent the cholesterol content in bacterial and mammalian cells, respectively.