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Proc Natl Acad Sci U S A. Sep 1, 2009; 106(35): 14972–14977.
Published online Aug 13, 2009. doi:  10.1073/pnas.0904465106
PMCID: PMC2736466
Immunology

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine

Abstract

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure–activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Nav channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structure-function homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of anti-infective or therapeutic peptides that selectively target microbial or abnormal host cells.

Keywords: channel, defensin, host defense, toxin

Cysteine-stabilized antimicrobial polypeptides are thought to function as a first line of host defense. One of the most well-characterized groups of such molecules is the β-defensins from myeloid and epithelial tissues of mammalian and avian species. The β-defensins contain a highly conserved cysteine-array that affords structural rigidity and promotes hypervariability for accelerated evolutionary adaptation.

Several parallels suggest structural, functional, and evolutionary commonalities between defensins and other host defense and/or offense molecules, such as toxins. Like antimicrobial peptides, many toxins are small, cysteine-stabilized, and cationic (1, 2), and share a striking degree of conservation with host defense peptides that contain a γ-core motif (3). One group of toxins with particularly close homology to β-defensins is the crotamine-myotoxin family from South American rattlesnakes (4, 5). As in β-defensins, toxin expression is often targeted for mucosal or extracorporeal secretion (6). Such toxins typically induce rapid paralysis resulting in local myonecrosis in skeletal muscle. Crotamine-like toxins are thought to induce such effects through targeting of ion channels and altering membrane transport or conductivity (5).

Understanding structural and mechanistic reciprocity is of direct relevance to immunologic roles and potential therapeutic development of antimicrobial and cytotoxic peptide-based therapies. Prior studies have noted overall diversity in primary structures of β-defensin and crotamine-like toxin families, but conserved cysteine-array and γ-core motifs (4). In the present study, evolutionary, structural, and mechanistic investigations were performed to address the hypothesis that reciprocal relationships exist between antimicrobial and cytotoxic host defense peptides.

Results

Phylogenetic, structural, mechanistic, and target interaction comparisons between hBD-2 and crotamine were investigated through complementary methods.

Phylogenetic Analysis.

Crotamine and related serpentine toxins formed a monophyletic group with robust statistical confidence (Fig. 1). A single subclade in this group was represented by crotasin, a β-defensin from the nonvenomous somatic tissues of Crotalus durissus terrificus (7). A sister group was comprised of 2 crotamine-like proteins (CLP) from the venom of the bearded dragon Pogona barbata (6). The next most closely related sequences are β-defensins from various avian species. Notably, the serpentine and avian sequences form a unified clade within the Sauropsida. These findings suggest a divergence of peptides optimized for antimicrobial versus cytotoxic functions appeared concomitant with the divergence of synapsids (mammals) from sauropsids (aves/reptiles) (SI Text and Figs. S1 and S2).

Fig. 1.
Phylogenetic parallels between crotamine and hBD-2. Neighbor-joining tree visualization (36) of the hBD-2/crotamine polypeptide family. Branch significance was validated by bootstrap analysis.

Structural and Biophysical Comparison.

Three-dimensional (3D) alignment between hBD-2 and crotamine revealed a striking degree of identity (Fig. 2A). The greatest degree of 3D alignment (RMSD ≤2) occurs between respective α-helical and γ-core regions. Thus, evolutionary selective pressures have favored conservation of 3D structure in the face of limited sequence identities (28%) of the 2 peptides (2). Despite striking conformational homology, biophysical analyses revealed significant physicochemical differences between hBD-2 and crotamine that likely relate to differences in target preference. For example, the solvent accessible surface area of hBD-2 (≈70% lacking charged residues; Fig. 2B) is markedly more hydrophobic than that of crotamine.

Fig. 2.
Structure and biophysical comparison of crotamine and hBD-2. (A) 3D alignment of crotamine/hBD-2 was performed by combinatorial extension (34). Coloration is per secondary structure schema: β-Sheet (blue); turn (gray); α-helix (red), molecular ...

Antimicrobial Activity.

Crotamine antimicrobial activities paralleled those of hBD-2 at both experimental pH values against most organisms (P > 0.05; Fig. 3). The only exception to this observation was for the prokaryote Staphylococcus aureus at pH 7.5, where hBD-2 had significantly greater efficacy (P < 0.05). Notably, both crotamine and hBD-2 had marked activity against the eukaryotic pathogen Candida albicans at pH 7.5 or 5.5. Generally, hBD-2 had greater efficacy at pH 7.5, except against C. albicans, where pH had no discernable impact.

Fig. 3.
Comparative antimicrobial efficacies and pH optima of crotamine and hBD-2. Antimicrobial activity of crotamine and hBD-2 were determined using radial diffusion against a panel of Gram-positive and Gram-negative bacteria and fungi. Antimicrobial activity ...

Cytotoxic Properties.

To assess relative cytotoxicities of crotamine versus hBD-2, flow cytometry was used to compare membrane electrophysiology (ΔΨ), permeabilization, and phosphatidylserine accessibility in bacteria and 2 eukaryotic cell systems.

Membrane Energetics.

Membrane potential (ΔΨ) was evaluated using 3,3-dipentyloxacarbocyanine (DiOC5), a charged lipophilic dye that emits a fluorescent signal proportional to ΔΨ. The validity of this method to assess channel activity approaches the reliability of classical patch clamp methods, as borne out by a compelling body of evidence (810). Overall, hBD-2 and crotamine altered ΔΨ in several target organisms versus untreated controls (Fig. 4 A–D, Figs. S3 and S4, and SI Text). In Escherichia coli, hBD-2 caused cell membrane hyperpolarization, as evidenced by increases in the overall percentage of cells polarized, as well as their mean channel fluorescence (P < 0.05 versus control). Neither peptide had a detectable affect on membrane ΔΨ of S. aureus (Fig. 4B). In C. albicans, both hBD-2 and crotamine caused marked increases in membrane potential (respective 2.2- and 2.8-fold increases in mean channel fluorescence; P < 0.01; Fig. 4C). Importantly, addition of the Nav channel inhibitor tetrodotoxin (TTX) mitigated hBD-2-induced hyperpolarization of E. coli, but had little impact on activities of hBD-2 on membrane potential in other cells.

Fig. 4.
Reciprocal activities of crotamine and hBD-2 on distinct prokaryotic and eukaryotic cells. Comparative flow cytometric analysis of membrane energetics (DiOC5), membrane permeabilization (PI), and phosphatidylserine accessibility (annexin V) in A E. coli ...

Membrane Permeabilization.

Peptide permeabilization of cells was measured using the intercalating dye propidium iodide (PI). This fluorescent probe enters permeabilized cells and binds to double-stranded nucleic acids, but is excluded from cells with normal membrane integrity. Defensin hBD-2 caused marked permeabilization of E. coli and S. aureus (P < 0.01; Fig. 4 A and B). Interestingly, crotamine did not increase E. coli permeability, but significantly increased the ratio of permeated S. aureus cells (P < 0.05; Fig. 4 A and B). Moreover, in the eukaryote C. albicans, hBD-2 or crotamine caused significant permeabilization (respective 99- or 95-fold increases in percent of cells permeabilized; P < 0.01; Fig. 4C). In human umbilical vein endothelial cells (HUVECs), neither hBD-2 nor crotamine caused significant increases in cell permeability versus controls (Fig. 4D). As with membrane energetics, the Nav channel blocker TTX revealed peptide-specific consequences on target cell permeability. In bacteria, TTX caused a complete abrogation of cell permeabilization by hBD-2 (no significant difference from control). Conversely, TTX enhanced permeabilization of S. aureus by crotamine (P < 0.05), but did not do so for E. coli. In C. albicans, TTX only modestly increased permeabilization by hBD-2 or crotamine.

Phosphatidylserine (PS) Accessibility.

One of the earliest markers of cellular transition to an apoptotic state is the translocation of PS from the inner to the outer leaflet of the plasma membrane (11). There, PS is accessible to staining by fluorescent-labeled annexin V, a phospholipid-binding protein with specificity for PS. In bacteria, neither hBD-2 nor crotamine caused significant PS accessibility (Fig. 4 A and B). Likewise, hBD-2 did not lead to increased PS accessibility in C. albicans in the presence or absence of TTX. However, crotamine did induce a substantial increase in PS accessibility in C. albicans, which was not affected by TTX (Fig. 4C). Interestingly, both study peptides influenced PS accessibility in HUVECs. In these cells, hBD-2 or crotamine alone caused significant increases in PS accessibility (30-fold and 24-fold, respectively; P < 0.01; Fig. 4D). However, consistent with other mechanistic results, TTX inhibited this effect of hBD-2, but not of crotamine.

Predicted Interactions with Kv Channels.

Docking studies were performed to compare crotamine versus hBD-2 interactions with prokaryotic and eukaryotic Kv channels, including this model of the C. albicans Kv channel.

Crotamine.

Crotamine preferentially targeted eukaryotic over prokaryotic Kv channels [interaction energies (E; kcal mol−1): Kv1.2, −1119; CaKv, −618; KcsA, −355]. Moreover, crotamine interacted with the eukaryotic Kv1.2 and CAKv channels so as to completely occlude their apertures (Fig. 5). For Kv1.2, crotamine residues Arg31 and Tyr1 participated in electrostatic and hydrogen-bonding interactions with Asp375 and Tyr373 of the channel pore. A similar interaction was observed for crotamine and the fungal CAKv channel, although the opposite facet of the peptide was involved in this interaction. In this association, peptide residues Lys27 and Trp32 occupied the channel via electrostatic and hydrogen bonding interactions with aspartic acid residue Asp452 of the channel pore. In contrast, the predicted interaction between crotamine and the prokaryotic KcsA channel did not occlude the channel pore (Fig. 5).

Fig. 5.
Computational modeling of predicted interactions between crotamine or hBD-2 with Kv1.2, CaKv, and KcsA Kv channels. Residue-specific interactions between crotamine (green; A–D) or hBD-2 (orange; I–L) and respective tetrameric Kv channels ...

hBD-2.

While its binding energies were similar, the orientation and localization of hBD-2 were distinguishable from those of crotamine and eukaryotic Kv channels at the molecular level. Notably, the hBD-2 backbone did not occlude either eukaryotic channel pore. Rather, hBD-2 targeted the outer region of these channels through residues of its β1-β2 loop (Arg22 and Phe19; Figs. 2 and and5).5). In the prokaryotic KcsA model, hBD-2 did not interpose the channel aperture, but localized to partially occlude this pore. Furthermore, the interaction between hBD-2 and KcsA was mediated by Arg22 and Arg23 from the β1-β2 loop of hBD-2 (Figs. 2 and and55).

Discussion

Prior reports have noted structural homology among certain antimicrobial peptide and toxin classes (4, 12). However, structural, mechanistic, and evolutionary relatedness among these molecules is less clear. If such reciprocal relationships exist, they could provide vital insights into molecular origins of innate immunity, overcome previous barriers to development of nontoxic peptide therapeutics, and reveal agents for targeting abnormal host cells.

Structure-function reciprocity of hBD-2 and crotamine was predicted based upon congruent conformational homology, propensity for membrane disruption, proclivity for cationic charge at neutral pH, and affinities for electronegative microbial surface targets. Supporting these predictions, the current data demonstrate hBD-2 and crotamine to have parallel yet distinct structure-activity relationships: (i) Charge and hydrophobic topologies consistent with conserved antimicrobial and cytotoxic functions; (ii) similar antimicrobial spectra and pH optima; (iii) prokaryotic (hBD-2) or eukaryotic (crotamine) preference with respect to membrane disruptive activities; (iv) an ability to evoke asymmetric membrane phosphatidylserine expression consistent with cytotoxic injuries leading to programmed cell death; (v) a preferential trophism of crotamine for eukaryotic (CAKv or Kv1.2) and hBD-2 for prokaryotic (KcsA) channel targets, respectively; and (vi) antagonism of hBD-2 for TTX-sensitive ion channels. Together, these findings substantiate the hypothesis that overall 3D structural homology provides a framework for reciprocal antimicrobial and cytotoxic propensities of hBD-2 and crotamine, respectively. Moreover, these results point to residue-specific interactions as potentially critical to differentiating antimicrobial versus cytotoxic proclivities of these peptides.

Kv, NaKv, and Nav channels represent an evolutionary continuum of ion-selective channels bridging prokaryotes to eukaryotes (1317). TTX, a specific inhibitor of Nav channel function (17), was used to probe potential differential effects of crotamine and hBD-2 on target cell Nav-related functions. Generally, TTX mitigated effects of hBD-2, but failed to inhibit crotamine functions, and in some cases enhanced crotamine action. This pattern of results suggests hBD-2 and crotamine preferentially target Nav, NaKv, or other TTX-sensitive targets in eukaryotes, and orthologous targets in prokaryotes (13, 14). Whether such mechanisms may be direct or indirect or exclusively involve Nav or NaKv channels is not yet clear. It is conceivable that hBD-2 antagonizes cell functions distinguishable from or in addition to TTX-sensitive Nav or NaKv channels. For example, hBD-2 and crotamine may perturb lipid membrane integrity in relation to ion-channel function in certain target cells. Investigations to determine how hBD-2 or other host defense peptides may antagonize voltage-sensitive ion channels or related targets are ongoing. The observation that defensin-like peptides may interact with Nav channels is not entirely unprecedented. Rogachevskii et al. suggested an interaction between human defensin NP-1 and the slow Nav channels of rat ganglial neurons (18). Likewise, plant γ-thionin defensins inhibit the sodium current in cultured GH3 cells (19). If so, such interactions may begin to explain toxicity in animal models wherein bolus systemic administration of defensin-like peptides leads to neurotoxic- and cytotoxic-like effects (2022).

Mechanistic findings above suggested biologically distinct interactions of hBD-2 or crotamine with Nav versus Kv channels. Therefore, as a complement to these studies, quantitative modeling was used to compare hBD-2 versus crotamine interactions with prototypic Kv channels of prokaryote, fungal, and mammalian organisms. Potential interactions between peptides and Nav channels were not studied, as no NMR- or X-ray-validated structure models for these channels were available. Relative docking affinities may reflect the relative electrostatic attractions of cationic crotamine and hBD-2 peptides to anionic Kv channel surfaces (most electronegative to least: Kv1.2 > CAKv > KcsA) or larger molecular areas of eukaryotic versus prokaryotic channels. However, the finding that the peptides were distinct in TTX antagonism suggests that specific stereogeometric interactions influence relative targeting of hBD-2 and crotamine for specific ion channels.

Computational docking analyses suggested a preferential reciprocity of hBD-2 and crotamine for interactions with prokaryotic versus eukaryotic Kv channels, respectively. In these analyses, crotamine localizes to the inner-pore domain through a classical cationic-aromatic (e.g., Lys27 and Trp32) functional dyad. Consistent with this theme is the fact that Lys27 occurs at the “X” position of the GXnC element of the γ-core domain in many defensin-like toxins and is present within crotamine (GKMDC). However, a Lys residue is absent from the GXnC element of the hBD-2 γ-core motif (GTC). The current results also indicate that crotamine perturbs eukaryotic Kv channels as do the charybdotoxin (23), kaliotoxin (24), and cobatoxin (25) family of scorpion toxins (26). Basic residues in such toxins form a cationic facet integrating Lys or Arg residues of their γ-core GXnC motif (2, 3). In turn, this cationic facet binds to 4 highly conserved Asp452 residues symmetrically distributed on the extracellular P-loops of Kv tetramers (27). In addition, a highly-conserved aromatic residue, typically Phe or Tyr, also participates in this pore-occluding complex, forming the classical cationic-aromatic residue dyad (25). Collectively, the current data support the hypothesis that residue-specific differences in peptides that have conserved overall 3D homology may contribute to target ion-channel preferences.

Many defensins exert considerable cytotoxicity in cell and animal models (21, 22). The present data suggest that such toxicities may be attributable in-part to structure-mechanism correlates paralleling those of crotamine-like toxins. Crotamine causes rapid paralysis and myonecrosis following prey envenomation. Early studies suggested an interaction with voltage-dependent Nav channels predominant in fast-twitch muscles of mammals (28). However, recent studies by Rizzi et al. (29), using expressed Nav1.1–1.6 α-subunits, did not find an interaction between crotamine and Nav channels. Further support for the interaction of defensin-like toxins with Kv ion channels derives from recent structural mapping of the Kv channel surface and, in some cases, toxin/channel complexes (2325). Together, these facts support the concept that hBD-2 and crotamine may preferentially but not exclusively target respective Nav, NaKv, or other TTX-sensitive ion channels—versus Kv channels—contributing to their net cytotoxic effects. Ongoing studies are designed to assess the potential direct and/or indirect interactions through which such peptides may differentially target and antagonize these or other channels structurally or mechanistically.

The present findings may have significant implications for development of polypeptide anti-infectives or other therapeutics. In the past decade, a sizeable investment has been made in exploiting microbicidal activities of CS-stabilized peptides such as defensins to address the mounting resistance of many important human pathogens to conventional antibiotics. However, for systemic use, toxicity not unlike that induced by cytotoxins has been a significant barrier to such advances (2022). For example, the current data suggest that crotamine and hBD-2 initiate apoptotic pathways in eukaryotic target cells (Candida, HUVECS). Such events have been linked to peptide perturbation of ion channel functions in fungal as well as mammalian cells (11). Identification of molecular determinants that differentiate cytotoxicity from antimicrobial activity may enable optimization of molecules for therapeutic efficacy without concomitant host toxicity. Alternatively, engineering peptides to have selective host cell toxicity may offer approaches to prevent or treat cancer, autoimmune, or other diseases. Thus, a clearer understanding of the ancient molecular features that both unite and distinguish host defense, toxin, and venom peptides may aid in development of anti-infectives or other therapeutics to address 21st century medical challenges.

Materials and Methods

Microorganisms.

Microorganisms representing Gram-positive (S. aureus; ATCC 27217; and Bacillus subtilis; ATCC 6633); Gram-negative (E. coli; ML-35); and fungal (C. albicans; ATCC 36082) human pathogens were studied. Microorganisms were cultured overnight in brain heart infusion (BHI) broth (Difco) at 37 °C (bacteria) or 30 °C (fungi). Cells were sonicated and adjusted to 106 CFU/mL.

Endothelial Cells.

Studies using HUVECs were conducted in accordance with National Institutes of Health (NIH) and institutional guidelines for human subjects. HUVECs were harvested as described previously (30). Cells were cultured to confluency (M-199 medium; Invitrogen; 10% FBS; Gemini Bio-Products; 2 mM L-glutamine, penicillin, and streptomycin; Irvine Scientific), detached with 0.1% trypsin EDTA, washed, and enumerated (30).

Molecules.

TTX (Sigma). hBD-2 (Peptides International). Crotamine was enriched from yellow venom of Crotalus durissus terrificus as described previously (5). Crotamine purification was achieved by RP-HPLC on a C18 column (Vydac) equilibrated with 0.01% triflouroacetic acid and eluted with a 0–40% gradient of water:acetonitrile. Crotamine identity and purity were authenticated using MALDI-TOF spectrometry.

Radial Diffusion Antimicrobial Assay.

Antimicrobial assays were performed using a radial diffusion method (3). As pH can influence peptide antimicrobial efficacy, the assays were conducted at pH 5.5 or 7.5 (31, 32). These conditions reflect the relevant contexts in which antimicrobial peptides often function (e.g., intracellular phagolysosome, pH 5.5, or extracellular milieu, pH 7.5) and which can influence peptide activities. Organisms were inoculated (106 CFU/mL) into buffered agarose (10 mM Pipes, pH 7.5, or 10 mM MES, pH 5.5); peptides (10 μg/well) were aliquoted into wells in the seeded matrix and incubated for 3 h at 37 °C. Zones of inhibition were measured 24 h later. Independent experiments were repeated a minimum of 2 times.

Flow Cytometry.

Multicolor flow cytometry was used to assess the comparative mechanisms of hBD-2 and crotamine versus prokaryotic (E. coli, S. aureus) or eukaryotic (C. albicans, HUVECs) target cells. The fluorophores used were as follows: Membrane permeabilization, PI (Ex535nm/Em620nm; Sigma); transmembrane potential, DiOC5 (Ex484nm/Em500nm; Invitrogen); phosphatidylserine accessibility, annexin V (allophycocyanin conjugate; Ex650nm/Em660nm; Invitrogen). For experiments, 105 cells were incubated with peptide (20 μg/mL) or peptide with TTX (50 nM; subinhibitory concentration) in 100 μL 10 mM Pipes, pH 7.5, for 15 min or 1 h with shaking at 30 °C (C. albicans) or 37 °C (bacteria, HUVECs). Pipes is a zwitterionic organic-based buffer that is not absorbed through cell membranes and is nontoxic to study cells as assayed (3133). Cells were stained for 10 min at room temperature by adding 900 μL stain buffer (PI, 5.0 μg/mL; DiOC5, 0.5 μM; annexin V, 2.5 μL/mL in 50 mM K+ MEM). Control cells were exposed to SDS (0.5%; Sigma), CCCP (100 μM; Sigma), or K+ MEM buffer (Sigma) alone. Flow cytometry was performed using a FACSCalibur instrument (Becton Dickinson) in 10 mM K+ MEM, pH 7.2. Fluorescence of a minimum of 5 × 103 cells was acquired for statistical analysis.

Bioinformatics.

Structural superimpositions and root mean squared deviation (RMSD) calculations were carried out using combinatorial extension [http://cl.sdsc.edu./ce; (34)]. Sequences for phylogenetic analyses were identified in iterative BLASTp searches using β-defensin and toxin sequences. Sequences were aligned with CLUSTALW (35), and phylogenetic trees were constructed using the neighbor-joining method (36).

Computational Modeling and Protein Docking.

A Kv channel model from C. albicans was generated using homology modeling [Phyre; 3D-PSSM folding server; (37)]. The highest scoring template was the mammalian shaker-family Kv1.2 channel (95% estimated precision; E-value, 4.5 × 10−5; PDB code, 2A79). Channel regions spanning S2 and S4–S6 were derived from C. albicans [residues 271–293 and 365–494, respectively (gi:68486701)], which includes all of the P-loop domains used for docking studies. The monomeric C. albicans Kv channel was then assembled using MUSTANG (38) implemented in YASARA (39). The resulting tetramer (CAK) was energetically minimized with conjugant gradients for 5,000 steps using NAMD (40). The structural coordinates for mammalian Kv1.2–2.1 (2R9R), S. lividans KcsA (1BL8), crotamine (1H5O), and HBD-2 (1FD3) were obtained from the Protein Data Bank (www.pdb.org).

Computational models for Kv channel-peptide complexes were generated using RosettaDock (www.rosettacommons.org) implemented in CAPRI (41). In brief, the docking method used a 2-step process: (i) rigid-body Monte Carlo searches and (ii) parallel optimization of backbone displacement and side-chain conformations using Monte Carlo minimization. The Kv channel domains available for docking were restricted to extracellular regions. The initial search yielded ≈2 × 104 decoys for each ligand (crotamine or hBD-2). For each of the top 50 ranked conformers, the α-carbon RMSD of the decoy was compared against each member of the conformational set in the second search. Iterative refinement resulted in 8 top-scoring conformations per ligand. Interaction sites were ranked by binding energy, and the energy contributions per residue (5 Å radius) tabulated. Ligand-protein residue pairs were then ranked based on total energy contribution and orientation-dependent hydrogen bonding. The 8 most favorable docking complexes were evaluated as potential binding sites.

Statistical Analyses.

Experiments were performed a minimum of 2 independent times on different days. Unpaired Student's t test was used to compare differences in data exhibiting normal distributions; data exhibiting discontinuous distributions were analyzed using the standard nonparametric Kolmogorov-Smirnoff methodology. P values ≤ 0.05 were considered significant.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Trang Phan and Scott G. Filler (Division of Infectious Diseases, Harbor–UCLA Medical Center, and the General Clinical Research Center at Harbor–UCLA Medical Center) for providing HUVECs for these studies and H. Ronald Kaback, Terry J. Smith, Robert I. Lehrer, Eric P. Brass, and John E. Edwards, Jr., for helpful discussions. This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases Grants 5R01AI39001 and 5R01AI48031 to M.R.Y.

Footnotes

Conflict of interest statement: M.R.Y. is a shareholder of NovaDigm Therapeutics, Inc., and has received research funding from Pfizer, Inc., Amgen, Inc., Cubist Pharmaceuticals, and Novozymes Pharmaceuticals. None of these entities provided support for the current studies. This article is a PNAS Direct Submission.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0904465106/DCSupplemental.

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