human -defensins inhibit hemolysis mediated by cholesterol … · resonance assays revealed that...

INFECTION AND IMMUNITY, Sept. 2009, p. 4028–4040 Vol. 77, No. 90019-9567/09/$08.00�0 doi:10.1128/IAI.00232-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Human �-Defensins Inhibit Hemolysis Mediated byCholesterol-Dependent Cytolysins�

Robert I. Lehrer,1* Grace Jung,1 Piotr Ruchala,1 Wei Wang,1,2 Ewa D. Micewicz,1 Alan J. Waring,1Eugene J. Gillespie,3 Kenneth A. Bradley,3 Adam J. Ratner,4 Richard F. Rest,5 and Wuyuan Lu6

Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California 900951; Amgen, Thousand Oaks,California2; Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA,Los Angeles, California 900953; Departments of Pediatrics and Microbiology, Columbia University, New York, New York 100324;Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 191295; and

Institute for Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 212016

Received 26 February 2009/Returned for modification 7 April 2009/Accepted 24 June 2009

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependentcytolysins. Here, we report that human �-defensins HNP-1 to HNP-3 acted in a concentration-dependentmanner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO),listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against theother toxins. Human �-defensins HNP-4 and HD-6 and human �-defensin-1, -2, and -3 lacked protectiveability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog,in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced bindingand protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Aburesidues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmonresonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also thatsolution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibitedhemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or tocholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B ofClostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first timethese defensins have been shown to inhibit pore-forming toxins. An “ABCDE mechanism” that can account forthe ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.

Polymorphonuclear neutrophils (PMNs) contain three �-de-fensin peptides, called HNP-1, -2, and -3 (18, 59). They havealmost identical sequences (XCYCRIPACIAGERRYGTCIYQGRLWAFCC), where “X” is alanine in HNP-1, aspartic acidin HNP-3, and absent in HNP-2. Collectively, HNP-1 toHNP-3 comprise 30 to 50% of total protein in a human PMN’sprimary (“azurophil”) granules and 5 to 7% of the cell’s totalprotein (60). The concentration of HNP-1 to HNP-3 in azuro-phil granules approximates 50 mg/ml, ensuring that a PMN’sphagocytic vacuoles also contain high HNP concentrations(17). Human PMNs have small amounts of one additional�-defensin, HNP-4 (69), whose sequence differs substantiallyfrom HNP-1 to HNP-3 (Fig. 1; Table 1). The other human�-defensins, HD-5 and HD-6, are primarily expressed in smallintestinal Paneth cells and are believed to protect the intestinaltract against food- and waterborne pathogens, to modulate theintestinal flora, and to be key factors in the pathogenesis ofinflammatory bowel disease in genetically susceptible humans(3, 67). Although the studies described below focused on hu-man �-defensins, some studies also examined human �-defen-sin peptides hBD-1, hBD-2, and hBD-3. hBD-1 is expressedconstitutively by epithelial cells and keratinocytes throughout

the body (76). hBD-2 and hBD-3, which are typically inducible,are also widely expressed. hBD-3 differs from the other �- and�-defensins studied here in two important respects, as follows:its exceptionally high cationicity (net charge, �11) and therelative salt insensitivity of its antimicrobial activity (25, 49).

In addition to their antimicrobial, antiviral, and immunoen-hancing properties (4, 37, 60), HNP-1 to HNP-3 can inactivatemultiple bacterial exotoxins, including anthrax lethal factor(34), diphtheria toxin (36), exotoxin A of Pseudomonas aerugi-nosa (36), and toxin B of Clostridium difficile (21). Each ofthese exotoxins is an enzyme. Anthrax lethal factor is a zinc-dependent metalloprotease (14), diphtheria toxin (7) and exo-toxin A (10) mediate ADP-ribosylation, and C. difficile toxin Bglucosylates small, GTP-binding proteins (21).

This study examined the ability of defensins to inactivatethree homologous cholesterol-dependent cytolysins (CDCs)(22, 64), anthrolysin O (ALO) from Bacillus anthracis (42, 61),listeriolysin O (LLO) from Listeria monocytogenes (1, 50), andpneumolysin (PLY) from Streptococcus pneumoniae (28, 53).These exotoxins lack enzymatic activity and function by initiallybinding cell membrane cholesterol and then undergoing or-derly oligomerization and conformational changes that lead tothe formation of very large transmembrane pores (23, 64, 65).LLO, a major virulence determinant of L. monocytogenes, en-ables ingested bacteria to escape a macrophage’s phagosomeand enter its cytoplasm (50). ALO can be substituted for LLOin this activity (68). PLY contributes to virulence early inpneumococcal pneumonia (52, 53) and is a promising vaccine

* Corresponding author. Mailing address: Department of Medicine,David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue,Los Angeles, CA 90095. Phone: (310) 824-5340. Fax: (310) 206-8766.E-mail: [email protected].

� Published ahead of print on 6 July 2009.

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candidate. The contribution of ALO to the pathogenesis ofanthrax infection is uncertain; however, ALO is immunogenic(12), and antibody passively administered to ALO protectsmice challenged by an otherwise lethal dose of B. anthracisstrain Sterne (43).

MATERIALS AND METHODS

Cytotoxins. Recombinant LLO was purchased from bio-WORLD (Dublin,OH). We used a pTrcHis expression vector provided by Rodney Tweten of theUniversity of Oklahoma to prepare recombinant ALO with a six-histidine tag.This ALO was purified from 4� 500 ml cultures of Escherichia coli BL21(DE3)grown in “terrific broth” (Fisher) supplemented with 4 g/liter glycerol. ALOexpression was induced by adding 0.5 mM isopropyl-�-D-1-thiogalactopyranoside(IPTG) when the cultures reached an optical density at 600 nm of 1.0 to 1.2.After an additional 4-hour incubation at 37°C, the bacteria were pelleted bycentrifugation, resuspended in 40 ml of binding buffer (500 mM NaCl, 25 mMimidazole, 20 mM sodium phosphate [pH 7.4]), and lysed by one passage througha French press at 15,000 lb/in2. After the lysate was cleared by centrifugation, itwas loaded onto a 1-ml HiTrap chelating HP column (Amersham) that waswashed with binding buffer and eluted with a linear gradient of imidazole inbinding buffer. Fractions containing ALO were pooled and concentrated to 4mg/ml, followed by endotoxin removal using Detoxi-Gel, per the manufacturer’sinstructions (Pierce, Rockford, IL). Recombinant ALO, domain 4, containedresidues 403 to 512 of the holotoxin (GenPept accession no. YP_029366) and wasprepared as previously described (11). Recombinant ALO lacking domain 4(E35-Y402) but containing domains 1 to 3 was produced by amplification of B.anthracis 7702 chromosomal DNA with primer set 5�-GGTCTCCCATGGAAACACAAGCCGGT-3� (forward) and CTCGAGCTAATATTCTGTAGTTGTCGTCTC-3� (reverse) and cloned into expression vector pETHSu (K300-01; In-vitrogen) using the BsaI and XhoI sites. The protein, purified as previouslydescribed (11), was passed over polymyxin B-4% agarose columns (Sigma-Al-drich) to remove endotoxin, per the manufacturer’s instructions. Endotoxin wasundetectable by Limulus amebocyte lysate assay (Cambrex Bio Science, Walk-ersville, MD) in proteins eluted from the columns with 20 mM HEPES and 150mM NaCl, pH 7.2.

The coding sequence of the PLY gene was amplified by PCR with primersNdeI-Ply-up (5�-GGAATTCCATATGGCAAATAAAGCAG-3�) and Ply-down-XhoI (5�-CCGCTCGAGGTCATTTCTACCTTATC-3�), using genomicDNA of S. pneumoniae strain TIGR4 as a template. These primers added uniquerestriction sites (underlined) and led to amplification of the entire PLY se-quence, omitting the stop codon to allow for addition of a C-terminal six-histidine tag. The product was confirmed by sequencing, digested with NdeI andXhoI (New England Biolabs, Ipswich, MA), and cloned into pET29a (EMDChemicals, San Diego, CA) cut with NdeI and XhoI. The plasmid was main-tained in E. coli BL21-AI (Invitrogen, Carlsbad, CA), and protein expression wasinduced with 1 mM IPTG (Denville Scientific, Metuchen, NJ) and 0.02% L-arabinose (Sigma). Purification was done using Ni-NTA agarose (Qiagen, Va-lencia, CA), per the manufacturer’s instructions. The eluted PLY was dialyzedextensively against lipopolysaccharide-free phosphate-buffered saline (PBS).

Defensins. Human �- and �-defensins and analogs thereof were synthesized,as described previously (70–72). Their integrity was verified by analytical reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorptionionization mass spectrometry, and their concentrations were adjusted by absor-bance at 280 nm (A280) measurements.

Hemolysis assays. We measured CDC-mediated lysis in two ways, as follows:by a kinetic assay that provided real-time monitoring (40) and by a conventionalend-point assay. Briefly, red cells from normal human blood (2.5 ml) were

washed four times with PBS and suspended in PBS at 5% by volume. Defensinstock solutions, stored in 0.01% acetic acid at �20°C, were thawed and dilutedinto PBS before each experiment. Assays were done in 96-well plates, with a finalvolume of 0.1 ml/well. CDCs (25 �l) and defensins (25 �l) were added to thewells and preincubated for 15 min on ice before adding erythrocytes (RBC) (50�l). Final CDC concentrations varied from 10 to 250 ng/ml. After all componentswere mixed, the plate was placed in a SpectraMax 250 spectrophotometer (Mo-lecular Devices, Sunnyvale, CA) and incubated at room temperature for 30 min.During this time, the A700 of each well was recorded every 30 s, automaticallyshaking the plate before each reading. The A700 readings at 30 min were used tocalculate the percent protection for the “kinetic assay,” as described below.

For “end-point” analysis, after incubation of the plates for an additional 60min at room temperature, 200 �l of cold PBS was added to each well beforetransferring the contents to small, conical tubes. After these were centrifuged for3 min at 6000 � g, 150 �l of each supernatant was removed and transferred toa fresh 96-well microplate, and its A540 was read. Supernatants from PBS-treatedand Triton X-100-treated red cells were included as standards. End-point (90-min) assays were analyzed in a conventional manner. Since the kinetic assayswere performed with toxin concentrations that gave complete hemolysis by 30min, we calculated the protective effects of defensins using the following equa-tion, in which A700C is the untreated control (PBS plus red cells), A700T is redcells plus toxin, and A700TD is red cells plus toxin and defensin: (A700TD �A700T)/(A700C � A700T) � 100 � percent protection.

Surface plasmon resonance assays. Binding studies were performed on aBiacore 3000 instrument (Biacore AB, Uppsala, Sweden). CDCs and bovineserum albumin (BSA; a control) were immobilized on CM5 chips by aminecoupling, as described previously (66). Studies were done using HBS-EP buffer(0.15 M NaCl, 10 mM HEPES [pH 7.4], 3 mM EDTA, 0.005% polysorbate 20)at a flow rate of 50 �l/minute. We obtained useful information by performing“molar ratio analysis,” which is based on the principle that the magnitude of thebinding response, in resonance units (RU), is proportional to the total mass ofthe analyte bound to the biosensor surface (32, 33). In these experiments,defensins ranged in size from 0.1 to 100 �g/ml, and the maximal RU responsewas measured after 4 min of flow.

Briefly, molar ratio analysis takes into account the mass of the ligand immo-bilized on the biosensor relative to the mass of the analyte that is bound. Forexample, consider the binding of HNP-1 (3,442 Da) to full-length ALO (52,700Da). Each ALO molecule immobilized on the biosensor will give a signal, in RU,that is 15.32-fold higher (15.32 � 52,700/3,442) than the signal resulting frombinding of a single HNP-1 molecule. Consequently, to calculate a molar ratio(e.g., the average number of HNP-1 molecules bound per immobilized ALOmolecule), one first divides the RU of ALO immobilized on the biosensor by15.32 to obtain an adjusted RU (RUadj) and then next divides the amount ofbound HNP-1 (in RU) by this RUadj. For an experiment with HNP-1 and ALO,domains 1 to 3 (mass, 40.4 kDa), the RUadj factor would be 11.71 (40,400/3,442).For an experiment with HNP-1 and ALO, domain 4 (12.3 kDa), the RUadj factorwould be 3.57 (12,300/3,442). We also used surface plasmon resonance to exam-ine the binding of HNP-1 in solution to HNP-1 immobilized on a biosensor chip.For such experiments, the RUadj equals the observed RU, since the solution-phase and immobilized HNP-1 have the same mass.

ALO-HNP complexes. Nondenaturing (native) PAGE was done on a 4-to-20%Tris-glycine gradient gel (Novex, Invitrogen, Carlsbad, CA), using SDS-freeTris-glycine running buffer, pH 8.6. Samples were preincubated for 15 min inNovex Tris-glycine sample buffer SDS. Gels were electrophoresed toward theanode for 4 h at a constant voltage (150 V), stained with Coomassie blue,destained, and photographed. SDS-PAGE was performed on 10-to-20% Tris-glycine gradient gels (Invitrogen). Samples were treated with SDS but wereneither boiled nor reduced. The gels were electrophoresed for 90 min at aconstant voltage (125 V), stained with Coomassie blue, destained, and photo-graphed.

FIG. 1. The sequences of six human �-defensins are shown, withtheir conserved residues boxed.

TABLE 1. Properties of cytolysins used in this studya

Cytolysin Mass(kDa)

No. of residues(total no. of

residues/no. ofacidic residues)

pI

% Amino acid identity (% aminoacid similarity) of cytolysins to:

ALO LLO PLY

ALO 51.6 466/53 5.91 100 41 (64) 42 (66)LLO 52.7 473/55 6.18 41 (64) 100 43 (66)PLY 52.8 470/66 5.14 42 (66) 43 (66) 100

a The sequences of six human �-defensins are shown in Fig. 1.

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ELISAs. Rabbit polyclonal antibodies against full-length, recombinant ALO(48) or LLO (Abcam, Cambridge, MA) were used as primary antibodies. Affin-ity-purified, horseradish peroxidase-labeled goat anti-rabbit immunoglobulin(Dako, Carpinteria, CA) was the secondary antibody. Cholesterol-capture en-zyme-linked immunosorbent assays (ELISAs) were done in 96-well flat-bottomplates (Costar; Corning, Inc., NY). Wells were coated by applying 30 �l ofethanol containing 12 �g/ml of cholesterol (Sigma, St. Louis, MO). After theethanol evaporated under mild heating applied by a hair dryer, the wells wererinsed three times with a washing buffer containing 0.9% NaCl and 0.01% Tween20, pH 7.4. Thereafter, the wells were treated overnight at 4°C with a blockingbuffer containing 2.5% BSA (fraction V) in 0.8% NaCl, 8 mM Na2HPO4, 5 mMKH2PO4, and 2.5 mM KCl, pH 7.4. Before use, the wells were rinsed four timeswith the washing buffer.

Assay samples were prepared in blocking buffer supplemented with 0.01%Tween 20. After samples (100 �l/well) had incubated for 60 min at room tem-perature, the wells were washed four times with 200 �l of washing buffer. Suitablydiluted primary antibody, 100 �l/well, was added for 60 min at room tempera-ture, and the wells were washed four times again, as done previously. Afteraddition of the secondary antibody, wells were incubated for 60 min at roomtemperature in the dark, then washed six times with washing buffer, and incu-bated in the dark with 100 �l/well of tetramethylbenzidine solution (TMB singlesolution; Invitrogen, Carlsbad, CA). The reaction was monitored at an opticaldensity at 650 nm for 30 min on a SpectraMax 250 microplate spectrophotometer(Molecular Devices, Sunnyvale, CA), with readings obtained every 20 s.

With some ELISAs, wells were coated with glycophorin A (GpA) by incubat-ing the wells overnight at 4°C with 500 ng of GpA (type MN; Sigma) in buffer(0.8% NaCl, 0.41% Na2HPO4, 0.20% KH2PO4, 0.02% KCl [pH 7.4]). Subse-quent blocking, rinsing, and incubation steps were performed, as describedabove. To determine if bound HNP-1 interfered with the ability of the antitoxinantibodies to recognize their respective toxins, a 96-well flat-bottom plate wascoated with 25 ng per well of ALO or LLO. After the wells were washed, theywere blocked with 2.5% BSA at 4°C overnight, washed again, and then treatedwith 100 �l of solution containing 0 to 200 �g HNP-1/ml for 1 h at roomtemperature.

Binding of ALO and LLO to RBC. Following appropriate institutional ap-proval, 5 to 10 ml of blood was withdrawn from each healthy human volunteerinto a Vacutainer containing EDTA (Becton Dickinson, Franklin Lakes, NJ).The blood samples were washed four times with cold PBS, and the cells wereresuspended in PBS at 5% (vol/vol). Solutions containing 0, 20, and 40 �g ofHNP-1/ml in PBS were kept on ice. The toxins, at 1 �g/ml, were prepared inbinding buffer containing 0.1% Tween 20 and were also kept on ice. Equalvolumes of toxin and HNP-1 were mixed and incubated for 5 min on ice. Then,2 volumes of washed red blood cells were added and the incubation was contin-ued for an additional 15 min on ice. Finally, the mixtures were centrifuged topellet the red cells. No lysis occurred under these conditions. Supernatants weremixed with an equal volume of binding buffer-0.1% Tween 20, and their toxinconcentrations were measured in the cholesterol-capture ELISA. Samples con-taining toxin and defensins that were incubated without red blood cells wereincluded as controls.

Effect of cholesterol on ALO-mediated hemolysis. Pegylated cholesterol[poly(oxy-1,2-ethanediyl), alpha-(3beta)-cholest-5-en-3-yl-omega-hydroxy], witha molecular weight of 5,000, was purchased from the NOF America Corporation,

White Plains, NY. Assays were done in PBS, pH 7.4, using 96-well microplates.All assay components other than RBC were preincubated with ALO for 15 minbefore adding the RBC. Hemolysis was monitored every 30 s at 700 nm at roomtemperature.

RESULTS

Characterization of the CDCs. Table 1 shows selected prop-erties of the toxins examined in this study. Cytolysin purity wasassessed by running SDS-PAGE gels loaded with 3 �g toxin/lane. After being stained with Coomassie blue, LLO and PLYshowed a single band at approximately 50 to 55 kDa, with orwithout reduction by dithiothreitol. The ALO holotoxinshowed two major bands (monomer and dimer), with apparentmasses of 50 and 100 kDa, plus several faint bands represent-ing higher-order oligomers. After reduction with dithiothreitol,the ALO holotoxin showed a single band of 50 kDa. Recom-binant �LO, lacking domain 4 and containing only domains 1to 3, showed major bands of 40 and 80 kDa. RecombinantALO, domain 4, ran as a single band of 12 kDa (data notshown). All of the toxins were used without further purifica-tion.

Effect on hemolysis. Figure 2 shows representative kineticassays with human RBC and 250 ng/ml (50 nM) of each toxinand either 5 �g/ml (Fig. 2a and b) or 10 �g/ml (Fig. 2c) ofdefensin. Under these conditions, HNP-2 completely pre-vented hemolysis by all three CDCs, as did HNP-1 and HNP-3(data not shown). HD-5 also prevented hemolysis but withlower potency than HNP-1 to HNP-3. In addition to HNP-4(Fig. 2), HD-6 and hBD-1 to hBD-3 also lacked protectiveactivity (data not shown). Since hBD-3 is exceptionally cationic(net charge, �11), especially relative to HNP-1 or HNP-2 (netcharge, �3), defensin-mediated inhibition of hemolysis doesnot result simply from ionic interactions between a defensinand an anionic toxin molecule.

Table 2 compares the abilities of five human �-defensins toinhibit hemolysis induced by ALO, LLO, and PLY. Two 50%inhibitory concentrations (IC50s) are shown for each peptide,as follows: 30-min values from kinetic assays and 90-min valuesfrom classical hemoglobin release assays. The 30- and 90-minIC50s of HNP-1 to HNP-3 are similar for ALO and LLO.However, the 90-min IC50s for PLY are two- to threefoldhigher than the 30-min values, showing that HNP-1 to HNP-3

FIG. 2. Effects of human defensins on CDC-mediated hemolysis in kinetic assays. The symbols are identified in panel a. Each toxin was testedat 250 ng/ml. Defensins were tested at 5 �g/ml (a and b) and at 10 �g/ml (c). OD700, optical density at 700 nm.

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retarded rather than eliminated PLY-mediated hemolysis.HD-5, an intestinal �-defensin, protected against LLO-medi-ated hemolysis, with mean IC50s of 2.43 �g/ml (677 nM) at 30min and 5.44 �g/ml (1.52 �M) at 90 min. HD-5 was much lesseffective against ALO and was ineffective against PLY.

HNP-2 and HNP-3 were the most-potent inhibitors of LLOin our studies. At 30 min, HNP-3 inhibited the hemolytic ac-tivity of LLO with a mean IC50 of 0.57 �g/ml (164 nM), andHNP-2 inhibited it with a mean IC50 of 0.87 �g/ml (258 nM).Their IC50s were slightly higher in the 90-min assay, with meanIC50s of 0.99 �g/ml for HNP-3 and 1.55 �g/ml for HNP-2. Wedid the LLO studies at pH 5.5 for several reasons. First, LLOis more active at an acidic pH (24). Second, pH 5.5 bettersimulates the acidic environment of a macrophage phagosome.Third, while human macrophages do not produce HNP-1 toHNP-3, they may acquire them by importation of human PMNgranules (63) or by endocytosis of solution-phase HNPs.

ALO was an extremely potent hemolysin for human RBC.Under these conditions, as little as 10 ng/ml ALO causedvirtually complete hemolysis within a few minutes (21). Westudied ALO at various concentrations, most often at 250ng/ml (Fig. 2) or at 10 ng/ml (Table 2). At 10 ng ALO/ml, themean IC50s of HNP-2 and HNP-3 ranged from 1.37 to 1.66

�g/ml (406 to 476 nM), with no significant differences betweenthe 30- or 90-min values. At 250 ng ALO/ml, IC50s for HNP-2were slightly higher, as follows: 2.34 0.31 �g/ml at 30 minand 2.31 0.32 �g/ml at 90 min (mean standard error of themean [SEM]; n � 3). The almost-identical IC50s for HNP-2 at30 and 90 min indicated a durable inhibitory effect on hemo-lysis. HNP-3 had IC50s of 3.14 0.37 �g/ml and 3.42 0.49�g/ml (mean SEM; n � 3) at 30 and 90 min, respectively, inexperiments with 250 ng ALO/ml, and HNP-1 had IC50 valuesof 5.45 1.18 �g/ml and 5.75 1.10 �g/ml (mean SEM, n �4) at 30 and 90 min, respectively, in these experiments. HD-5had a considerably higher IC50, slightly above 100 �g/ml.

Effects of pH and serum. Whereas the contributions of LLOto pathogenesis have been widely studied, less is known aboutALO and PLY. When we determined their optimal pH forRBC lysis (Fig. 3), PLY, like LLO, functioned best in an acidicenvironment, suggesting that it might enable pneumococci toescape imprisonment in a macrophage’s phagosome. ALO hada broad pH optimum, with peak activity at pH 7.4. The broadpH optimum of ALO could allow it to act in serum-free ex-tracellular fluids, in the neutral pH environment of a humanPMN’s phagosome (8, 58), and in the more acidic phagosomeof a macrophage. Addition of 10% normal human serum (data

FIG. 3. Optimal pH for hemolysis of human RBC. Normal human RBC were exposed to PLY (a), LLO (b), or ALO (c) at pH 7.4, 6.5, 6.0,or 5.5. Untreated control RBC (open circles) and RBC treated with 0.1% Triton X-100 (open squares) served as controls.

TABLE 2. Ability of �-defensins to inhibit hemolysis by ALO, LLO, and PLY

�-Defensin

Inhibition (IC50) results of hemolysis with the indicated cytolysinc

ALO LLO PLY

30 min 90 min 30 min 90 min 30 min 90 min

HNP-1 2.56 0.61 2.58 0.59 1.12 0.21 2.22 0.36 13.9 1.39 29.2 3.13HNP-2 1.37 0.12 1.49 0.16 0.87 0.15 1.55 0.27 7.14 0.74 13.7 1.76HNP-3 1.63 0.17 1.66 0.21 0.57 0.08 0.99 0.09 8.90 0.23 23.4 2.77HNP-4 ��100a ��100a 90.4 5.13 �100b ��100a ��100a

HD-5 17.7 2.02 28.4 4.14 2.43 0.25 5.44 0.44 �100b ��100a

a No protection, even at 100 �g/ml.b A total of 100 �g/ml of HNP-4 and HD-5 reduced hemolysis by 32.8% 6.4% and 26.9% 5.5%, respectively, at 90 min. We tested LLO (100 ng/ml) at pH 5.5

because it is more potent at an acidic pH (39), but HNP-1 to HNP-3 also inhibited hemolysis effectively at pH 7.4.c IC50 data are in �g/ml and represent the means SEM. Experiments were performed three to five times for each entry. The 30-min results were from kinetic assays,

and the 90-min results are from classical, two-stage assays. The experimental conditions (toxin concentration, pH) were as follows: ALO, 10 ng/ml, pH 0.4; LLO, 100ng/ml, pH 5.5; PLY, 250 ng/ml, pH 7.4. We used these different toxin concentrations to compensate for their different intrinsic hemolytic potencies.



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not shown) or certain cholesterol derivatives (described below)to the medium completely prevented RBC lysis by ALO. Thisis consistent with previous reports that the direct cytotoxicactivity of ALO against human PMNs, monocytes and macro-phages, and RBC is abrogated by adding free cholesterol or10% serum (42, 61).

Binding studies. Figure 4 compares the abilities of sevenhuman defensins, each tested at 1 �g/ml, to bind ALO (Fig. 4a)and PLY (Fig. 4b). HNP-2 and HNP-3 bound best, followed inorder by HNP-1, HD-5, hBD-3, and HNP-4. hBD-1 and hBD-2were also tested but showed no binding. Overall, relative CDCbinding and neutralization were concordant. To learn if bind-ing was fully reversible, we extended the duration of the dis-sociation phase from 2 min, as shown in Fig. 4a and b, to 30min, as shown in Fig. 4c and d. Figure 4c shows that thecomplex between HNP-2 and PLY or ALO dissociates muchmore slowly than the complex between BSA and HNP-2, indi-cating the far greater stability of the defensin-toxin complexes.Addition of 10 mg/ml of fetuin, an extensively glycosylatedprotein found in fetal bovine serum, to the dissociation bufferenhanced the dissociation rate considerably, so that by 30 minonly about 10% of the HNP-2/toxin complexes remained in-

tact. Thus, the binding of HNP-2 to CDCs is predominantlyreversible, although a minor irreversible component, whichmight form via thiol-disulfide exchange (45), cannot be ex-cluded.

Binding to ALO domains. CDCs contain four domains. Do-main 4 contains a conserved undecapeptide (ECTGLAWEWWR) that is present in ALO, LLO, and PLY and whoseinteraction with target cell membrane cholesterol precedes theformation of a trans-membrane beta-barrel pore that is formedby domain 3 (27). To determine if defensins might bind thecholesterol-binding moiety of domain 4, we synthesized a 14-residue peptide (RECTGLAWEWWRTV-amide) and immo-bilized it on a CM5 biosensor chip. HNP-2 bound the 14-merin a biphasic manner. Maximal binding occurred after about70 s and then thereafter declined by about 25% over the next2 minutes. Thus, the maximum molar ratio achieved by 1 �g/mlof HNP-2 was 0.66, declining to 0.49 after 3 min (Table 3). Ifsimilar binding were to occur in defensin-treated CDC holo-toxins, it might interfere with the ability of the CDC toxins tointeract with their membrane cholesterol docking site. We willrevisit this possibility later.

Figure 5a shows binding isotherms, in RU, for 10 HNP-1

FIG. 4. Binding of human �- and �-defensins to immobilized ALO and PLY. (a, b) Binding of six �-defensins and one �-defensin (hBD-3) toimmobilized ALO (a) and PLY (b). Each defensin was tested at 1 �g/ml. hBD-1 and hBD-2 were also tested but showed no binding to ALO orPLY. These studies were performed by surface plasmon resonance. (c) Dissociation of HNP-2 complexed to ALO, PLY, and BSA over 30 minwhen the flow cells are perfused with HBS-EP buffer (0.15 M NaCl, 10 mM HEPES [pH 7.4], 3 mM EDTA, 0.005% polysorbate 20). Note thatthe ordinate scale is logarithmic. (d) Dissociation of HNP-2 from ALO, PLY, and LLO when the flow cells are perfused with HBS-EP buffersupplemented with 10 mg/ml fetuin.



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concentrations from 0.1 to 1.0 �g/ml. Its noteworthy featuresinclude clustered RU values between 0.5 and 0.9 �g HNP-1/mland high binding signals. Figure 5b shows binding of HNP-1 tothe following three ALO preparations: the 4-domain holo-toxin, a truncated ALO variant lacking domain 4, and domain

4 of ALO. Binding is expressed as the molar ratio, i.e., mol ofHNP-1 bound/mol of toxin. Values are the means from twoexperiments. At 0.8 �g HNP-1/ml, 4.5 defensin moleculesbound each 51.6-kDa ALO holotoxin molecule; approximatelythree HNP-1 molecules bound to domains 1 to 3, and oneHNP-1 molecule bound to domain 4. Figure 5c shows themolar binding ratios for higher HNP-1 concentrations. Mole-cules of ALO holotoxin exposed to 25 and 50 �g/ml of HNP-1bound, on average, about 18 and 25 defensin molecules, re-spectively. Figure 5d contains the binding data from Fig. 5c andb in a log-log plot, which allows them to be shown in a singlepanel.

All �-defensins contain three disulfide bonds and have acharacteristic disulfide pairing motif in which cysteine 1 (theamino-terminal cysteine) and cysteine 6 (the C-terminal cys-teine) are paired, as are cysteines 2 and 4 and cysteines 3 and5. To determine if these disulfide bonds were needed to allowHNP-1 to inactivate CDCs in hemolysis assays, we did theexperiment illustrated in Fig. 6a. Whereas normal HNP-1 pro-

FIG. 5. Binding of HNP-1 to ALO, ALO lacking domain 4, and domain 4 of ALO. (a, b) Binding of HNP-1 (0.1 to 1.0 �g/ml) to immobilizedALO holotoxin (a) and to ALO holotoxin (domains 1 to 4, open circles), ALO lacking domain 4 (domains 1 to 3, solid circles), and domain 4 ofALO (solid triangles) (b), each immobilized on the same CM5 biosensor. Every data point is a mean value derived from two separate experiments.HNP-1 binding to immobilized HNP-1 is depicted as open diamonds. When binding is shown as a molar ratio (molecules of HNP-1 per moleculeof ALO), this value was derived from the peak RU levels at each concentration. (c) Binding (molar ratios) for higher concentrations of HNP-1.(d) Log-log plot of the data shown in panels b and c.

TABLE 3. Binding to the cholesterol-binding domaina

�-Defensin ka (104/M/s) Kd (10�3/s) KD (nM)

HNP-1 14.8 21.6 145.9HNP-2 17.9 33.7 188.0HNP-3 19.3 37.5 194.3HNP-4 5.22 59.5 1,140HD-5 26.6 12.6 47.4HD-6 0.17 15 8,824

a The sequence of the immobilized, 14-residue peptide was RECTGLAWEWWRTV-amide. Its 11-residue cholesterol-binding domain is underlined. The14-residue sequence is identical to those found in the CDCs of alveolysin fromPaenibacillus alvei and ivanolysin from Listeria ivanovii. The first 13 residues areidentical to those found in PLY and LLO. Abbreviations: ka, rate of association;Kd, rate of dissociation; KD, equilibrium binding constant.



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tected red blood cells from ALO with an IC50 of 3 �g/ml, theIC50 of (Cys 9,29 �-aminobutyric acid [Abu])-HNP-1, a par-tially linearized analog lacking only the disulfide bond betweenits third and fifth cysteines, was 30 �g/ml. A fully linearizedHNP-1 analog, in which all six cysteines were replaced by Abu,showed no protective ability at 50 �g/ml, the highest concen-tration tested. Figure 6b shows the comparison of the bindingof HNP-1 and these two disulfide analogs to immobilizedALO. The fully linearized HNP-1 analog showed greatly re-duced binding to the holotoxin, but normal HNP-1 and (Cys9,29 Abu)-HNP-1 bound the toxin equally well. Figure 6cshows how these peptides (5 �g/lane) migrated on an SDS-PAGE gel. The fully linearized HNP-1 ran exclusively as amonomer, and HNP-1 ran as a mixture of monomers anddimers, with the dimers being more abundant. In contrast,monomers were considerably more abundant in the lane con-taining the (Cys 9,29 Abu)-HNP-1 variant. Thus, the lack ofthe disulfide bond between the third and fifth cysteine residuesof HNP-1 impedes the ability of solution-phase HNP-1 todimerize.

Figure 7 shows the comparison of the binding of HNP-1 andHNP-2 to ALO holotoxin and shows that they behave almostidentically. At the mean IC50 for ALO of HNP-1 or HNP-2(Fig. 1; Table 1), five to eight defensin molecules were boundto each ALO molecule, on average. The multiplicity rose toabout 15 at 10 �g/ml and to about 25 at 50 �g/ml. The inset inFig. 7 is relevant to these high defensin/toxin molar ratios. Itshows that relatively minor self-association of HNP-1 occurs atconcentrations up to 2.5 �g/ml. Thereafter, self-associationincreases progressively, reaching a 1:1 ratio at 20 �g/ml anda 2:1 ratio at 50 �g/ml. Thus, up to two-thirds of the 25HNP-1 molecules bound to ALO exposed to 50 �g HNP-1/mlmay be binding to ALO-bound defensins instead of bindingdirectly to ALO.

PAGE experiments. The formation of HNP/ALO complexeswas also shown by PAGE (Fig. 8). Because we used a setupthat allowed samples to migrate only toward the anode, posi-tively charged HNP-2 (net charge, �3) did not enter the gel. In

contrast, ALO holotoxin (net charge, �1) and ALO, domain 4(net charge, �2), did enter the gel and can be seen in Fig. 8,lanes 1, 3, 5, and 7. When ALO molecules bound HNP-2, theresulting complex acquired a net positive charge that pre-vented anodal migration and caused the cytolysins to disap-pear from the gel (Fig. 8, lanes 2 and 4). Addition of SDS tothe sample buffer gave toxin-HNP complexes a negativecharge that allowed them to migrate into the gel (Fig. 8,lanes 6 and 8).

We performed additional studies to see if the binding ofHNP-1 to ALO was representative of its binding to otherCDCs (Fig. 9a and b). Over a 500-fold concentration rangefrom 0.1 to 50 �g of defensin/ml, the binding of HNP-1 to

FIG. 6. Role of disulfide bonds in protection against hemolysis. Analogs of HNP-1 lacking one disulfide bond [(Cys 9,29 Abu)-HNP-1] orlacking all three disulfide bonds (linear HNP-1) were compared to HNP-1 for their ability to protect red cells from ALO-mediated lysis. In thelinearized molecule, all six cysteines were absent. In the partially linearized (Cys 9,29 Abu)-HNP-1 variant, only the disulfide bond between thethird and fifth cysteines (Cys 9 and 29) was absent. (a) The IC50 of the (Cys 9,29 Abu)-HNP-1 variant was approximately 30 �g/ml, i.e., about10-fold higher than the IC50 of HNP-1. The linearized molecule was completely nonprotective, even at 50 �g/ml. Three experiments wereperformed with each peptide. Symbols show means SEM of percent protection. (b) Comparison of the binding of HNP-1, (Cys 9,29Abu)-HNP-1, and linear HNP-1 to immobilized ALO holotoxin. (c) SDS-PAGE gel that was stained with Coomassie blue. One lane containsmolecular mass standards (SeeBlue Plus2; Invitrogen). The other lanes each contain 5 �g of the indicated peptides.

FIG. 7. Binding properties of HNP-1 and HNP-2. The main figuredemonstrates that HNP-1 (solid circles) and HNP-2 (open triangles)show virtually identical binding to immobilized ALO. Symbols repre-sent means SEM from five experiments with HNP-1 and threeexperiments with HNP-2. The inset shows the binding of HNP-1 toimmobilized HNP-1, a property called self-association in this report.



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ALO, LLO, and PLY was very similar. Note that, on average,each toxin molecule had bound approximately 10 HNP-1 mol-ecules when the analyte contained 10 �g/ml of HNP-1. Figure9c compares the binding of HNP-1 to ALO and to BSA. Al-though HNP-1 also bound BSA, this binding was 5- to 10-foldlower than its binding to ALO, whether expressed as molarratios or as RU. Hence, while binding by HNP-1 is not specific,it is somewhat selective.

Mechanistic studies. To this point, we have shown (i) thatcertain human �-defensins prevent lysis of human red bloodcells by CDCs (Fig. 2 and 3); (ii) that not all human defensinscan do this, including �-defensins HNP-4, HD-6, and �-de-fensins-1 to -3 (Fig. 1; Table 1); (iii) that effective humandefensins bound more extensively to CDCs than the ineffectiveones (Fig. 4); (iv) that multiple molecules of HNP-1 to HNP-3bind to a single CDC molecule (Fig. 9); and (v) that moleculesof HNP-1 to HNP-3 also bind to other molecules of HNP-1 toHNP-3, including those that have bound to CDCs (Fig. 7).

William of Ockham opined that “all other things beingequal, the simplest solution tends to be the best.” Keeping this700-year old precept (and the data in Table 3) in mind, wehypothesized that defensins interfered with the docking ofCDCs to cholesterol either by binding directly to the unde-capeptide cholesterol-binding motif in toxin domain 4 or bybinding close enough to this cholesterol-binding site to impairits function. To test this hypothesis, we performed sensitiveELISAs, using immobilized cholesterol as the capturing re-agent. Polyclonal antisera to ALO and LLO served as primaryantibodies, and horseradish peroxidase-labeled goat anti-rab-bit immunoglobulin was the secondary antibody. The assaysdetected 0.1 ng/100 �l of the corresponding CDC in theabsence of HNP-1. Concerned that defensins might interferewith the ELISA by masking CDC epitopes recognized by theprimary antibodies, we exposed ALO- or LLO-coated plates tovarious concentrations of HNP-1, washed them, and thenadded the respective primary and secondary antibodies, as perthe cholesterol-capture ELISA. As expected, the ability ofprimary antibodies to recognize HNP-1-treated CDCs was re-duced slightly for ALO and more substantially for LLO (Fig.10a). We applied these findings to correct the results of ourELISAs.

Figure 10b shows a cholesterol-capture ELISA that exam-ined the ability of HNP-1 to interfere with cholesterol bindingby ALO and LLO. At HNP-1 concentrations of up to 50 �g/ml,neither ALO nor LLO showed decreased binding to choles-terol. Indeed, when ALO was added in the presence of 25 �gHNP-1/ml, the amount of ALO detected by the ELISA was

FIG. 8. Demonstrating the formation of ALO–HNP-1 complexesby SDS-PAGE. Electrophoresis was done on a 4 to 20% polyacryl-amide gradient gel. Lanes 1, 2, 5, and 6 contained 2.5 �g of ALOholotoxin, and lanes 3, 4, 7, and 8 contained 2.5 �g of ALO, domain 4.HNP-2 (2.5 �g) was added to samples in lanes 2, 4, 6, and 8 (�) but notto samples in odd-numbered lanes (O). HNP-2 is positively chargedand, given the gel’s polarity, would not normally enter the gel. Samplesin lanes 1 to 4 were dissolved in conventional Tris-glycine samplebuffer. Samples in lanes 5 to 8 were dissolved in Tris-glycine buffercontaining SDS. No SDS was present in the pH 8.7 Tris-glycine run-ning buffer. Preincubation with HNP-2 caused the disappearance ofALO (lane 2) and domain 4 (lane 4) when samples were introduced innormal sample buffer. When the sample buffer contained SDS, thecomplex (arrows) between ALO and HNP-2 (lane 6) and ALO, do-main 4, and HNP-2 (lane 8) acquired sufficient negative charge tomigrate toward the anode.

FIG. 9. Comparative binding to CDCs and BSA. (a) Binding of 0.1 to 1.0 �g/ml of HNP-1 to ALO, LLO, and PLY. (b) Binding by higherHNP-1 concentrations. (c) Comparison of the binding of HNP-1 to three biosensors, one presenting ALO and two presenting BSA. All threebiosensors were immobilized on the same CM5 chip to similar densities (3,022 RU for BSA-1, 3,282 RU for BSA-2, and 3,704 RU for ALO).Binding is expressed as a molar ratio (mol HNP-1/mol toxin or BSA).



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threefold higher than in its absence. Consistent with this find-ing, neither 5 �g/ml of HNP-1 nor 10 �g/ml of HNP-1 pre-vented the binding of ALO to red blood cells (Fig. 10c), al-though both HNP-1 concentrations completely preventedALO-mediated hemolysis. These results suggested either thatRBC contained CDC-binding sites other than cholesterol orthat defensins could affix CDCs to other components of the redcell surface. Both of these possibilities proved to be correct.

Figure 10d shows results from an ELISA using GpA, insteadof cholesterol, as the capture reagent. GpA, an abundant (106

copies/cell) and extensively glycosylated surface protein of hu-man RBC, captured ALO 60% as well as cholesterol did.LLO also bound GpA but to a lesser extent. A mixture of ALOwith 25 �g HNP-1/ml resulted in an increase of threefold inALO capture, similar to our finding with cholesterol-coatedplates (Fig. 10b). The twice-noted binding increases of three-fold were likely caused by the binding of CDC-HNP complexescontaining multiple ALO and defensin molecules. In otherexperiments, washed red blood cells (2.5%, vol/vol) in PBSwere incubated for 30 min to 4 h at 0°C with ALO (3 to 250ng/ml), HNP-2 (100 �g/ml), or ALO (3 to 250 �g/ml) plus 100�g/ml of HNP-2. When examined directly or by microscopy, noevidence of aggregation or clumping was found.

We used red blood cell targets in our experiments with LLO,

PLY, and ALO, even though the bacteria that produce thesepore-forming toxins form nonhemolytic colonies on blood agarplates and do not cause significant hemolysis in vivo. While wecould reasonably attribute both behaviors to the protectiveeffects of serum, we began to wonder why serum was protec-tive. Since 1 ml of normal serum contains about 2 mg/ml oftotal cholesterol, we speculated that serum cholesterol couldbe responsible for preventing CDC-mediated hemolysis. How-ever, the minuscule solubility of free cholesterol in aqueousmedia made it difficult to test this hypothesis directly. Accord-ingly, we turned to more-soluble analogs of cholesterol, cho-lesterol sulfate, and pegylated cholesterol to test this notion.Figure 11a shows that cholesterol sulfate, which is slightlysoluble, can inhibit ALO-mediated hemolysis. Figure 11bshows that 250 �g/ml of the highly soluble pegylated choles-terol (corresponding to 19.3 �g/ml of cholesterol and 231.7�g/ml of polyethylene glycol [PEG]) was highly protective. Thesame PEG-cholesterol construct was only slightly protective at100 �g/ml and afforded no protection at 50 �g/ml (data notshown). To verify that the cholesterol moiety of PEG choles-terol was responsible for the effect shown in Fig. 11b, we tested1-mg/ml concentrations of cholesterol-free PEG preparationswith molecular weights of 8,000 (Fig. 11c) or 3,350 (data notshown) and found that neither provided any protection.

FIG. 10. Effect of HNP-1 on the binding properties of antibody and toxins. (a, b) Effect of HNP-1 on recognition of ALO and LLO by theircognate antibodies (a) and on binding of toxins to cholesterol (b). A total of 25 ng of ALO or LLO was added to cholesterol-capture ELISAplates 0 to 200 �g/ml HNP-1. Black bars show toxin concentrations, as determined by the ELISA. (a) The white (ALO) or gray (LLO) barscompensate for the effect of HNP-1 on the antibody’s ability to recognize the toxins. (c) HNP-1 does not decrease the binding of ALO to red bloodcells. (d) Effect of HNP-1 on binding of ALO and LLO to glycophorin A. A total of 25 ng ALO or LLO plus various concentrations of HNP-1(0 to 200 �g/ml) were added to ELISA plates coated with glycophorin A. The black bars show toxin concentrations, as determined by the ELISA.The white (ALO) or gray (LLO) bars stacked above them compensate for the decreased ability of the antibody to recognize the toxins in thepresence of HNP-1.



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Figure 12 illustrates the ABCDE mechanism, which is ourview of HNP-mediated toxin inactivation. In the cartoon rep-resentation, defensin molecules are portrayed as jigsaw puzzlepieces to show their ability to form dimers and to bind othermolecules. As shown in Fig. 4 to 6, when defensins accumulate(Fig. 12A) to concentrations beyond those found in normalserum, they can bind (Fig. 12B) toxin molecules and cross-link(Fig. 12C) them by virtue of their ability to dimerize and formhigher-order multimers (shown in Fig. 7). Furthermore, asshown in Fig. 10, adhering toxins to “irrelevant” molecules,such as GpA, diverts (Fig. 12D) the toxin molecules from its

normal receptor(s). Defensin-to-defensin binding will extend(Fig. 12E) the area of the toxin’s surface covered by defensins(deduced from Fig. 7) and can also cause additional cross-linking and aggregation.

DISCUSSION

The experiments described above add CDCs to the list ofbacterial exotoxins inhibited by human �-defensins, which al-ready includes anthrax lethal factor and lethal toxin (34), exo-toxin A of P. aeruginosa, diphtheria toxin (36), and toxin B ofC. difficile (21). All toxins previously shown to be inhibited bydefensins are enzymes that exert their toxic effects intracellu-larly after binding to the target cell and then gaining entranceto its cytoplasm. Kim et al. (34–36) concluded that HNPs werenoncompetitive inhibitors of lethal factor that bound to a re-gion remote from its active center (34–36) and that the inhi-bition of diphtheria toxin and exotoxin A by HNPs was com-petitive against elongation factor 2 and uncompetitive againstNAD�, the ADP-ribose donor (36). Uncompetitive inhibitionimplies that the inhibitor binds to the enzyme-substrate com-plex but not to the free enzyme. As CDCs lack enzymaticproperties, their inhibition by defensins cannot be analyzed inthis manner.

Recent studies of Clostridium difficile exotoxins provide use-ful counterparts to our findings with CDCs. C. difficile pro-duces two homologous toxins, A and B. Both are large (�250kDa), single-chain proteins whose sequences show 47% iden-tity and 68% similarity. Early studies in animals identified toxinA as the likely agent of pseudomembranous colitis and antibi-otic-associated diarrhea. However, toxin B is 100- to 1,000-foldmore toxic to cultured cells than toxin A (57) and can causesimilar effects in humans (38, 54, 56). Both toxins use UDP-glucose to catalyze the mono-O glycosylation of small, cytosolicGTP-binding proteins of the Rho and Ras families (reviewedin references 57 and 62), thereby disrupting functions regulat-ing cell shape, differentiation, and proliferation. Giesemann etal. (21) reported that HNP-1 and HNP-3 inactivated C. difficiletoxin B, with IC50s of 0.6 to 1.5 �M (about 2 to 5 �g/ml). HD-5also inhibited toxin B but less potently. Importantly, they notedthat high-molecular-mass aggregates, containing both toxin

FIG. 11. Effect of cholesterol on ALO-mediated hemolysis. (a) Cholesterol sulfate (nominal final concentration, 50 �g/ml) inhibited hemolysisinduced by 62.5 ng/ml of ALO. (b) Pegylated cholesterol (PEG-cholesterol), at a final concentration of 250 �g/ml, inhibited hemolysis induced by250 ng/ml of ALO. (c) PEG (1 mg/ml), with a molecular mass of approximately 8,000 Da (PEG 8000), did not prevent ALO-mediated hemolysis.

FIG. 12. ABCDE mechanism. This cartoon depiction represents�-defensins as pieces of a jigsaw puzzle that can accumulate (A) locallyto bind (B) toxin oligomers or cell surface glycoproteins, such as GpA,on the surface of RBC. HNPs self-associate to form dimers, allowingthem to cross-link (C) the molecules that they bind. By tethering toxinsto nonproductive bystander molecules, such as GpA, they can alsodivert (D) the toxins from their preferred surface receptors. Self-association—the defensin-to-defensin binding shown in the Fig. 7 in-set—extends (E) the space they occupy on the surface of the toxin andcan further sterically hinder the orderly toxin oligomerization requiredto form a pore.



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and HNP, formed in the presence of �2 �M HNP. By analyz-ing Coomassie blue-stained gels, they estimated that 18 pmolof toxin B coprecipitated about 250 pmol HNP-1, a molar ratioof about 14:1. HNP-1 formed similar aggregates with two B.anthracis toxins, protective antigen and lethal factor, but notwith C. difficile toxin A or Clostridium sordellii lethal toxin, bothof which have cytotoxic properties resistant to HNP-1.

Giesemann et al. (21) remarked that “complex formationseems to be a common feature” and that this “may representan additional mode of action for specific defensins (e.g., HD-5)at sites where high defensin concentrations are present, forexample, within specialized regions of the small intestine orphagocytic vacuoles of neutrophils.” Our data support thisinference. HNP-induced complex formation and precipitationcould occur by cross-linking due to dimerization (Fig. 12),which has been shown previously at the much higher HNPconcentrations (29) used for nuclear magnetic resonance orX-ray crystallography studies. Aggregation of the toxin-defen-sin complexes might also occur by a process akin to isoelectricprecipitation, since CDCs are acidic (Fig. 1; Table 1) andhuman defensins are modestly cationic. Thus, the binding ofdefensins to CDCs would cause local or global charge neutral-ization (Fig. 8) that might favor aggregation. In the previouslyreported studies, HNPs required an intact tri-disulfide struc-ture to inactivate anthrax lethal factor, diphtheria toxin, P.aeruginosa exotoxin A, and C. difficile toxin B. This is consistentwith our findings for ALO (Fig. 6).

Additional parallels exist to findings reported in studies withstaphylokinase, a 136-amino-acid protein produced by lyso-genic strains of Staphylococcus aureus. Staphylokinase forms a1:1 complex with plasmin(ogen) that activates other plasmin-ogen molecules to form plasmin, a serine protease that de-grades extracellular matrix proteins. Bokarewa and Tarkowski(5) reported that HNP-1 and HNP-2 bound staphylokinase,forming complexes with a greatly reduced ability to activateplasminogen (31). As the HNP-staphylokinase complexes con-tained up to a sixfold molar excess of HNP-1, the authorsconcluded that each staphylokinase molecule contained sev-eral binding sites for HNPs. Our studies also revealed thepresence of multiple binding sites for HNP-1 on CDCs (Fig. 7and 9).

We did not examine the ability of �-defensins to neutralizeCDCs in vivo but will speculate about where this would belikely to occur. Certainly, it would not occur in the circulation,because serum and plasma contain insufficient levels of �-de-fensins. Table 2 shows that IC50s for inhibiting ALO and LLOare higher than the concentrations of HNP-1 to HNP-3 ( 100ng/ml) found in carefully collected normal human sera (47).Even though higher HNP-1 to HNP-3 concentrations may oc-cur in patients with severe infection (46), the presence ofcholesterol may compete with the toxin’s binding to its cellsurface receptors. Defensin-mediated toxin neutralization ismore likely to take place within the phagosomes of a PMN, atextracellular sites of inflammation, or in milieus that containPMNs and stimuli that induce defensin secretion, such as leu-kotriene B4 (16, 19), the beta chemokines MIP1�, MIP1�, andRANTES (30), interleukin-8 (9), and tumor necrosis factoralpha (6). Other agents that induce the extracellular release ofthe azurophil granules of human PMNs include opsonizedzymosan, aggregated immunoglobulin G, C5a, N-formylated

peptides, and phorbol esters (2). The HNP-1 to HNP-3 contentof normal neutrophils is sufficiently high that the 4 millionneutrophils commonly found per milliliter of blood contain20 �g of HNP-1 to HNP-3 (46).

Although human monocytes and macrophages do not ex-press �-defensin mRNA (63), they can acquire �-defensins byingestion of PMNs or their azurophil granules (15, 63) or byendocytosis of fluid-phase defensin (our unpublished data).Importation of fluid-phase HNP-1 and/or HD-5 was shown forhuman T cells (74, 75), human smooth muscle cells, and hu-man cervical epithelial (CaSki) cells (26).

The likeliest site of action for HD-5 is the small intestine(44). Ghosh et al. reported that human terminal ileum con-tained 0.5 to 2.5 mg HD-5 per gram of tissue (20). BecausePaneth cells secrete �-defensins in response to lipopolysaccha-ride (51), bacteria (55), and cholinergic stimuli (51, 55), espe-cially high HD-5 concentrations will be present in the lumen ofthe intestinal crypts of Lieberkuhn. Just as cervical epithelialcells can acquire HD-5 from the external milieu (26), intestinalmucosal cells might acquire extracellular defensins and usethese peptides to influence the outcome of cytoplasmic orintravacuolar events.

Surface plasmon resonance allowed us to examine defensinself-association at the low micromolar concentrations com-mensurate with their IC50 levels for biological activities, withthe caveat that one defensin molecule was immobilized by anamide bond between its N-terminal amino group and the un-derlying dextran matrix. We do not know if the restrictedmotion of the immobilized defensin would allow its solution-phase defensin partner to access the dimer interfacial surfacedefined by nuclear magnetic resonance and crystallography.We do know that there must be at least two ways for defensinself-association to occur or else molar ratios greater than 1:1(Fig. 7, inset) could not occur.

The “ABCDE mechanism” shown in Fig. 12 illustrates fiveelements of �-defensin behavior that, in combination, allowthem to inhibit CDCs. It is likely that these same elementscontribute to the steadily growing list of activities attributed to�-defensins (13, 41, 73). Other factors may also contribute. Toform a pore, CDCs must diffuse freely in the target cell mem-brane, undergo conformational changes, and assemble up-wards of 60 toxin monomers (64, 65). �-Defensins might alsoprevent pore formation by hindering membrane diffusion, asnoted previously in photobleaching experiments with retrocy-clins (humanized �-defensins) (39), or by steric hindrance ofmembrane fusion, which was also previously noted for retro-cyclins (39). In addition, their accretion by self-association maydeform the occlusal surface(s) of CDC monomers in a mannerthat hinders the orderly oligomerization of toxin monomers.

We can view defensins as multipurpose molecular machines,composed of mutually adapted parts (amino acids) that oper-ate, as shown in Fig. 12, to accomplish many different kinds ofwork. The present study shows that certain �-defensins caninhibit CDCs, that they require an intact disulfide “skeleton”to do so, and that mere cationicity (e.g., HNP-4 or hBD-3) isinsufficient to impart this property. These observations indicatethat apart from the six conserved cysteines found in all �-de-fensins, one or more additional amino acid residues are crucialfor allowing some �-defensins but not others to inhibit CDCs.Identifying such critical residues would be a valuable step in



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cracking the defensin “code” and furthering our understandingof the mechanism(s) of inhibition.

REFERENCES

1. Andrews, N. W., and D. A. Portnoy. 1994. Cytolysins from intracellularpathogens. Trends Microbiol. 2:261–263.

2. Bentwood, B. J., and P. M. Henson. 1980. The sequential release of granuleconstitutents from human neutrophils. J. Immunol. 124:855–862.

3. Bevins, C. L. 2006. Paneth cell defensins: key effector molecules of innateimmunity. Biochem. Soc. Trans. 34:263–266.

4. Biragyn, A. 2005. Defensins—non-antibiotic use for vaccine development.Curr. Protein Pept. Sci. 6:53–60.

5. Bokarewa, M., and A. Tarkowski. 2004. Human alpha-defensins neutralizefibrinolytic activity exerted by staphylokinase. Thromb. Haemost. 91:991–999.

6. Bouma, M. G., T. M. Jeunhomme, D. L. Boyle, M. A. Dentener, N. N.Voitenok, F. A. van den Wildenberg, and W. A. Buurman. 1997. Adenosineinhibits neutrophil degranulation in activated human whole blood: involve-ment of adenosine A2 and A3 receptors. J. Immunol. 158:5400–5408.

7. Carroll, S. F., and R. J. Collier. 1984. NAD binding site of diphtheria toxin:identification of a residue within the nicotinamide subsite by photochemicalmodification with NAD. Proc. Natl. Acad. Sci. USA 81:3307–3311.

8. Cech, P., and R. I. Lehrer. 1984. Phagolysosomal pH of human neutrophils.Blood 63:88–95.

9. Chertov, O., D. F. Michiel, L. Xu, J. M. Wang, K. Tani, W. J. Murphy, D. L.Longo, D. D. Taub, and J. J. Oppenheim. 1996. Identification of defensin-1,defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins re-leased from interleukin-8-stimulated neutrophils. J. Biol. Chem. 271:2935–2940.

10. Chung, D. W., and R. J. Collier. 1977. Enzymatically active peptide from theadenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect.Immun. 16:832–841.

11. Cocklin, S., M. Jost, N. M. Robertson, S. D. Weeks, H. W. Weber, E. Young,S. Seal, C. Zhang, E. Mosser, P. J. Loll, A. J. Saunders, R. F. Rest, and I. M.Chaiken. 2006. Real-time monitoring of the membrane-binding and inser-tion properties of the cholesterol-dependent cytolysin anthrolysin O fromBacillus anthracis. J. Mol. Recognit. 19:354–362.

12. Cowan, G. J., H. S. Atkins, L. K. Johnson, R. W. Titball, and T. J. Mitchell.2007. Immunisation with anthrolysin O or a genetic toxoid protects againstchallenge with the toxin but not against Bacillus anthracis. Vaccine 25:7197–7205.

13. de Leeuw, E., and W. Lu. 2007. Human defensins: turning defense intooffense? Infect. Disord. Drug Targets 7:67–70.

14. Duesbery, N. S., C. P. Webb, S. H. Leppla, V. M. Gordon, K. R. Klimpel,T. D. Copeland, N. G. Ahn, M. K. Oskarsson, K. Fukasawa, K. D. Paull, andG. F. Vande Woude. 1998. Proteolytic inactivation of MAP-kinase-kinase byanthrax lethal factor. Science 280:734–737.

15. Estensen, R. D., M. E. Reusch, M. L. Epstein, and H. R. Hill. 1976. Role ofCa2� and Mg2� in some human neutrophil functions as indicated by iono-phore A23187. Infect. Immun. 13:146–151.

16. Flamand, L., P. Borgeat, R. Lalonde, and J. Gosselin. 2004. Release ofanti-HIV mediators after administration of leukotriene B4 to humans. J. In-fect. Dis. 189:2001–2009.

17. Ganz, T., M. E. Selsted, and R. I. Lehrer. 1986. Antimicrobial activity ofphagocyte granule proteins. Semin. Respir. Infect. 1:107–117.

18. Ganz, T., M. E. Selsted, D. Szklarek, S. S. Harwig, K. Daher, D. F. Bainton,and R. I. Lehrer. 1985. Defensins. Natural peptide antibiotics of humanneutrophils. J. Clin. Investig. 76:1427–1435.

19. Gaudreault, E., and J. Gosselin. 2007. Leukotriene B4-mediated release ofantimicrobial peptides against cytomegalovirus is BLT1 dependent. ViralImmunol. 20:407–420.

20. Ghosh, D., E. Porter, B. Shen, S. K. Lee, D. Wilk, J. Drazba, S. P. Yadav,J. W. Crabb, T. Ganz, and C. L. Bevins. 2002. Paneth cell trypsin is theprocessing enzyme for human defensin-5. Nat. Immunol. 3:583–590.

21. Giesemann, T., G. Guttenberg, and K. Aktories. 2008. Human alpha-de-fensins inhibit Clostridium difficile toxin B. Gastroenterology 134:2049–2058.

22. Gilbert, R. J. 2002. Pore-forming toxins. Cell. Mol. Life Sci. 59:832–844.23. Gilbert, R. J. 2005. Inactivation and activity of cholesterol-dependent cyto-

lysins: what structural studies tell us. Structure (Cambridge) 13:1097–1106.24. Glomski, I. J., M. M. Gedde, A. W. Tsang, J. A. Swanson, and D. A. Portnoy.

2002. The Listeria monocytogenes hemolysin has an acidic pH optimum tocompartmentalize activity and prevent damage to infected host cells. J. CellBiol. 156:1029–1038.

25. Harder, J., and J. M. Schroder. 2005. Antimicrobial peptides in human skin.Chem. Immunol. Allergy 86:22–41.

26. Hazrati, E., B. Galen, W. Lu, W. Wang, Y. Ouyang, M. J. Keller, R. I. Lehrer,and B. C. Herold. 2006. Human alpha- and beta-defensins block multiplesteps in herpes simplex virus infection. J. Immunol. 177:8658–8666.

27. Heuck, A. P., E. M. Hotze, R. K. Tweten, and A. E. Johnson. 2000. Mecha-nism of membrane insertion of a multimeric beta-barrel protein: perfringo-

lysin O creates a pore using ordered and coupled conformational changes.Mol. Cell 6:1233–1242.

28. Hirst, R. A., A. Kadioglu, C. O’Callaghan, and P. W. Andrew. 2004. The roleof pneumolysin in pneumococcal pneumonia and meningitis. Clin. Exp.Immunol. 138:195–201.

29. Hristova, K., M. E. Selsted, and S. H. White. 1996. Interactions of mono-meric rabbit neutrophil defensins with bilayers: comparison with dimerichuman defensin HNP-2. Biochemistry 35:11888–11894.

30. Jan, M. S., Y. H. Huang, B. Shieh, R. H. Teng, Y. P. Yan, Y. T. Lee, K. K.Liao, and C. Li. 2006. CC chemokines induce neutrophils to chemotaxis,degranulation, and alpha-defensin release. J. Acquir. Immune Defic. Syndr.41:6–16.

31. Jin, T., M. Bokarewa, T. Foster, J. Mitchell, J. Higgins, and A. Tarkowski.2004. Staphylococcus aureus resists human defensins by production of staphy-lokinase, a novel bacterial evasion mechanism. J. Immunol. 172:1169–1176.

32. Jonsson, U., L. Fagerstam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh,S. Lofas, B. Persson, H. Roos, I. Ronnberg, et al. 1991. Real-time biospecificinteraction analysis using surface plasmon resonance and a sensor chiptechnology. BioTechniques 11:620–627.

33. Karlsson, R. 1994. Real-time competitive kinetic analysis of interactionsbetween low-molecular-weight ligands in solution and surface-immobilizedreceptors. Anal. Biochem. 221:142–151.

34. Kim, C., N. Gajendran, H. W. Mittrucker, M. Weiwad, Y. H. Song, R.Hurwitz, M. Wilmanns, G. Fischer, and S. H. Kaufmann. 2005. Humanalpha-defensins neutralize anthrax lethal toxin and protect against its fatalconsequences. Proc. Natl. Acad. Sci. USA 102:4830–4835.

35. Kim, C., and S. H. Kaufmann. 2006. Defensin: a multifunctional moleculelives up to its versatile name. Trends Microbiol. 14:428–431.

36. Kim, C., Z. Slavinskaya, A. R. Merrill, and S. H. Kaufmann. 2006. Humanalpha-defensins neutralize toxins of the mono-ADP-ribosyltransferase fam-ily. Biochem. J. 399:225–229.

37. Klotman, M. E., and T. L. Chang. 2006. Defensins in innate antiviral immu-nity. Nat. Rev. Immunol. 6:447–456.

38. Komatsu, M., H. Kato, M. Aihara, K. Shimakawa, M. Iwasaki, Y. Nagasaka,S. Fukuda, S. Matsuo, Y. Arakawa, M. Watanabe, and Y. Iwatani. 2003. Highfrequency of antibiotic-associated diarrhea due to toxin A-negative, toxinB-positive Clostridium difficile in a hospital in Japan and risk factors forinfection. Eur. J. Clin. Microbiol. Infect. Dis. 22:525–529.

39. Leikina, E., H. Delanoe-Ayari, K. Melikov, M. S. Cho, A. Chen, A. J. Waring,W. Wang, Y. Xie, J. A. Loo, R. I. Lehrer, and L. V. Chernomordik. 2005.Carbohydrate-binding molecules inhibit viral fusion and entry by crosslink-ing membrane glycoproteins. Nat. Immunol. 6:995–1001.

40. Liu, C. C., and J. D. Young. 1988. A semi-automated microassay for com-plement activity. J. Immunol. Methods 114:33–39.

41. Menendez, A., and F. B. Brett. 2007. Defensins in the immunology of bac-terial infections. Curr. Opin. Immunol. 19:385–391.

42. Mosser, E. M., and R. F. Rest. 2006. The Bacillus anthracis cholesterol-dependent cytolysin, anthrolysin O, kills human neutrophils, monocytes andmacrophages. BMC Microbiol. 6:56.

43. Nakouzi, A., J. Rivera, R. F. Rest, and A. Casadevall. 2008. Passive admin-istration of monoclonal antibodies to anthrolysin O prolong survival in micelethally infected with Bacillus anthracis. BMC Microbiol. 8:159.

44. Ouellette, A. J. 2005. Paneth cell alpha-defensins: peptide mediators ofinnate immunity in the small intestine. Springer Semin. Immunopathol.27:133–146.

45. Panyutich, A., and T. Ganz. 1991. Activated alpha 2-macroglobulin is aprincipal defensin-binding protein. Am. J. Respir. Cell Mol. Biol. 5:101–106.

46. Panyutich, A. V., E. A. Panyutich, V. A. Krapivin, E. A. Baturevich, and T.Ganz. 1993. Plasma defensin concentrations are elevated in patients withsepticemia or bacterial meningitis. J. Lab. Clin. Med. 122:202–207.

47. Panyutich, A. V., N. N. Voitenok, R. I. Lehrer, and T. Ganz. 1991. An enzymeimmunoassay for human defensins. J. Immunol. Methods 141:149–155.

48. Park, J. M., V. H. Ng, S. Maeda, R. F. Rest, and M. Karin. 2004. AnthrolysinO and other gram-positive cytolysins are toll-like receptor 4 agonists. J. Exp.Med. 200:1647–1655.

49. Pazgier, M., D. M. Hoover, D. Yang, W. Lu, and J. Lubkowski. 2006. Humanbeta-defensins. Cell. Mol. Life Sci. 63:1294–1313.

50. Portnoy, D. A., V. Auerbuch, and I. J. Glomski. 2002. The cell biology ofListeria monocytogenes infection: the intersection of bacterial pathogenesisand cell-mediated immunity. J. Cell Biol. 158:409–414.

51. Qu, X. D., K. C. Lloyd, J. H. Walsh, and R. I. Lehrer. 1996. Secretion of typeII phospholipase A2 and cryptdin by rat small intestinal Paneth cells. Infect.Immun. 64:5161–5165.

52. Rubins, J. B., D. Charboneau, J. C. Paton, T. J. Mitchell, P. W. Andrew, andE. N. Janoff. 1995. Dual function of pneumolysin in the early pathogenesis ofmurine pneumococcal pneumonia. J. Clin. Investig. 95:142–150.

53. Rubins, J. B., and E. N. Janoff. 1998. Pneumolysin: a multifunctional pneu-mococcal virulence factor. J. Lab. Clin. Med. 131:21–27.

54. Samra, Z., S. Talmor, and J. Bahar. 2002. High prevalence of toxin A-neg-ative toxin B-positive Clostridium difficile in hospitalized patients with gas-trointestinal disease. Diagn. Microbiol. Infect. Dis. 43:189–192.



http://iai.asm.org/

Dow

nloaded from

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55. Satoh, Y. 1988. Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice. Cell Tissue Res. 253:397–402.

56. Savidge, T. C., W. H. Pan, P. Newman, M. O’brien, P. M. Anton, and C.Pothoulakis. 2003. Clostridium difficile toxin B is an inflammatory entero-toxin in human intestine. Gastroenterology 125:413–420.

57. Schirmer, J., and K. Aktories. 2004. Large clostridial cytotoxins: cellularbiology of Rho/Ras-glucosylating toxins. Biochim. Biophys. Acta 1673:66–74.

58. Segal, A. W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol.23:197–223.

59. Selsted, M. E., S. S. Harwig, T. Ganz, J. W. Schilling, and R. I. Lehrer. 1985.Primary structures of three human neutrophil defensins. J. Clin. Investig.76:1436–1439.

60. Selsted, M. E., and A. J. Ouellette. 2005. Mammalian defensins in theantimicrobial immune response. Nat. Immunol. 6:551–557.

61. Shannon, J. G., C. L. Ross, T. M. Koehler, and R. F. Rest. 2003. Charac-terization of anthrolysin O, the Bacillus anthracis cholesterol-dependentcytolysin. Infect. Immun. 71:3183–3189.

62. Takai, Y., T. Sasaki, and T. Matozaki. 2001. Small GTP-binding proteins.Physiol. Rev. 81:153–208.

63. Tan, B. H., C. Meinken, M. Bastian, H. Bruns, A. Legaspi, M. T. Ochoa, S. R.Krutzik, B. R. Bloom, T. Ganz, R. L. Modlin, and S. Stenger. 2006. Macro-phages acquire neutrophil granules for antimicrobial activity against intra-cellular pathogens. J. Immunol. 177:1864–1871.

64. Tweten, R. K. 2005. Cholesterol-dependent cytolysins, a family of versatilepore-forming toxins. Infect. Immun. 73:6199–6209.

65. Tweten, R. K., M. W. Parker, and A. E. Johnson. 2001. The cholesterol-dependent cytolysins. Curr. Top. Microbiol. Immunol. 257:15–33.

66. Wang, W., A. M. Cole, T. Hong, A. J. Waring, and R. I. Lehrer. 2003.Retrocyclin, an antiretroviral theta-defensin, is a lectin. J. Immunol. 170:4708–4716.

67. Wehkamp, J., G. Wang, I. Kubler, S. Nuding, A. Gregorieff, A. Schnabel,R. J. Kays, K. Fellermann, O. Burk, M. Schwab, H. Clevers, C. L. Bevins,and E. F. Stange. 2007. The Paneth cell alpha-defensin deficiency of ilealCrohn’s disease is linked to Wnt/Tcf-4. J. Immunol. 179:3109–3118.

68. Wei, Z., P. Schnupf, M. A. Poussin, L. A. Zenewicz, H. Shen, and H. Gold-fine. 2005. Characterization of Listeria monocytogenes expressing anthroly-sin O and phosphatidylinositol-specific phospholipase C from Bacillus an-thracis. Infect. Immun. 73:6639–6646.

69. Wilde, C. G., J. E. Griffith, M. N. Marra, J. L. Snable, and R. W. Scott. 1989.Purification and characterization of human neutrophil peptide 4, a novelmember of the defensin family. J. Biol. Chem. 264:11200–11203.

70. Wu, Z., B. Ericksen, K. Tucker, J. Lubkowski, and W. Lu. 2004. Synthesisand characterization of human alpha-defensins 4–6. J. Pept. Res. 64:118–125.

71. Wu, Z., D. M. Hoover, D. Yang, C. Boulegue, F. Santamaria, J. J. Oppen-heim, J. Lubkowski, and W. Lu. 2003. Engineering disulfide bridges todissect antimicrobial and chemotactic activities of human beta-defensin 3.Proc. Natl. Acad. Sci. USA 100:8880–8885.

72. Wu, Z., R. Powell, and W. Lu. 2003. Productive folding of human neutrophilalpha-defensins in vitro without the pro-peptide. J. Am. Chem. Soc. 125:2402–2403.

73. Yang, D., Z. H. Liu, P. Tewary, Q. Chen, G. de la Rosa, and J. J. Oppenheim.2007. Defensin participation in innate and adaptive immunity. Curr. Pharm.Des. 13:3131–3139.

74. Zaharatos, G. J., T. He, P. Lopez, W. Yu, J. Yu, and L. Zhang. 2004.alpha-Defensins released into stimulated CD8� T-cell supernatants arelikely derived from residual granulocytes within the irradiated allogeneicperipheral blood mononuclear cells used as feeders. J. Acquir. ImmuneDefic. Syndr. 36:993–1005.

75. Zhang, L., W. Yu, T. He, J. Yu, R. E. Caffrey, E. A. Dalmasso, S. Fu, T. Pham,J. Mei, J. J. Ho, W. Zhang, P. Lopez, and D. D. Ho. 2002. Contribution ofhuman alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviralfactor. Science 298:995–1000.

76. Zhao, C., I. Wang, and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett.396:319–322.

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