Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin


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Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin
  RetrocyclinsKillBacilliandGerminatingSporesof  Bacillusanthracis andInactivateAnthraxLethalToxin * Receivedforpublication,April14,2006,andinrevisedform,May30,2006  Published,JBCPapersinPress,June21,2006,DOI10.1074/jbc.M603614200 WeiWang ‡ ,ChandrikaMulakala §1 ,SabrinaC.Ward ¶ ,GraceJung ‡ ,HaiLuong ‡ ,DuyPham ‡ ,AlanJ.Waring ‡ ,YiannisKaznessis §1 ,WuyuanLu  ,KennethA.Bradley § ,andRobertI.Lehrer ‡2 FromtheDepartmentsof   ‡ Medicineand  ¶ Microbiology,Immunology,andMolecularGenetics,DavidGeffenSchoolofMedicine,UCLA,LosAngeles,California90095,the § DepartmentofChemicalEngineeringandMaterialsScience,UniversityofMinnesota,Minneapolis,Minnesota55455,andthe  InstituteforHumanVirology,BiotechnologyInstitute,UniversityofMaryland,Baltimore,Maryland21201   -defensins are cyclic octadecapeptides encoded by the mod-ified   -defensin genes of certain nonhuman primates. Therecent demonstration that human   -defensins could preventdeleteriouseffectsofanthraxlethaltoxin in vitro and in vivo ledus to examine the effects of     -defensins on  Bacillus anthracis (Sterne). We tested rhesus    -defensins 1–3, retrocyclins 1–3,and several analogues of RC-1. Low concentrations of     -de-fensins not only killed vegetative cells of   B. anthracis  (Sterne)and rendered their germinating spores nonviable, they alsoinactivated the enzymatic activity of anthrax lethal factor andprotected murine RAW-264.7 cells from lethal toxin, a mixtureoflethalfactorandprotectiveantigen.Structure-functionstud-ies indicated that the cyclic backbone, intramolecular tri-disul-fide ladder, and arginine residues of     -defensins contributedsubstantially to these protective effects. Surface plasmon reso-nance studies showed that retrocyclins bound the lethal factorrapidly and with high affinity. Retrocyclin-mediated inhibitionof the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhi-bition suggest that post-binding events, perhaps  in situ  oli-gomerization, contribute to the antitoxic properties of retrocy-clins. Overall, these findings suggest that    -defensins providemolecular templates that could be used to create novel agentseffective against  B. anthracis  and its toxins. Under normal circumstances  Bacillus anthracis  causeshuman infections only in individuals exposed to infected farmanimals or their spore-contaminated products. The virulenceof   B. anthracis  primarily derives from the hardiness of itsspores,ananti-phagocyticcapsulethatsurroundsitsvegetativecells (1), and two secreted binary toxins: lethal toxin (LeTx) 3 and edema toxin (EdTx). Both toxins contain protective anti-gen (PA, 83 kDa). LeTx also contains lethal factor (LF, 90 kDa),and EdTx contains edema factor (EF, 89 kDa). The genes for allthree toxin components, PA, LF, and EF, reside on the pXO1plasmid(2),andthoseresponsibleforcapsulesynthesisexistonthe pXO2 plasmid (3). Both of these plasmids are required for in vivo  virulence (3).EF is an adenylate cyclase (4) and LF is a zinc-dependentmetalloprotease that selectively attacks certain MAPK kinases(5, 6). PA is required to allow both of the other toxin compo-nents to enter host cells (7). When PA binds a cellular receptor(8), it is cleaved into PA63 (63 kDa) and PA20 (20 kDa). ThePA20 diffuses away, and the residual receptor-bound PA63molecules self-associate into ring-shaped heptamers (9) thatbind EF or LF with high affinity (10–12). Oligomerization of PA63 leads to endocytosis, which transports the complexes toanacidiccompartment(13–15).Here,theheptamericpre-porechanges into an integral-membrane pore (16, 17) that translo-catesEForLFintothecytosol(18).ImmunizationagainstPAisprotective (19).Defensins are small,   -sheet peptides that collectively pos-sess broad antibacterial, antifungal, and antiviral properties(20–23). They are believed to be especially important as “firstresponders” to microbial and viral incursions and to play criti-cal roles in defending the mucosal surfaces that line the respi-ratory, gastrointestinal, and genitourinary tracts. Humansexpress6different  -defensinsand30ormore  -defensins(24,25). Human   -defensins (HNPs) are potent noncompetitiveinhibitors of the metalloprotease activity of anthrax LF. They can protect murine macrophages from  B. anthracis  LeTx  invitro  and provide protection to mice when co-injected with alethal dose of LeTx (26).   -Defensins are cyclic octadecapeptides that are encoded by mutated   -defensin genes (27). They have been purified, aspeptides, only from the leukocytes and bone marrow of rhesus *  The work was also supported in part by National Institutes of Health (NIH)GrantsAI056921(toR. I. L.),AI057870(toK. B.),andAI061482(toW. L.).Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentof page charges. This article must therefore be hereby marked “ advertise-ment  ”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. 1 Supported by NIH Grant GM070989 and by the National ComputationalScience Alliance (under Grant TG-MCA04T033). 2  To whom correspondence should be addressed: Dept. of Medicine, DavidGeffen School of Medicine, UCLA, 10833 LeConte Ave., Los Angeles, CA90095. Tel.: 310-825-0133; Fax: 310-206-8766; E-mail: 3  Theabbreviationsusedare:LeTx,lethaltoxin;EdTx,edematoxin;EF,edemafactor; HNP-1, human neutrophil peptide-1 (an  -defensin); LF, lethal fac-tor;LGA,Lamarckiangeneticalgorithm;MAPKK-2,mitogen-activatedpro-tein kinase kinase-2; MEC, minimum effective concentration; PA, protec-tive antigen; RC100 IAA , reduced and alkylated RC-1; RC-1XX, variousretrocyclinanalogues;RC-1,retrocyclin-1;RTD,rhesus   -defensin;SPR,sur-face plasmon resonance; RU, resonance unit(s); BSA, bovine serum albu-min; r.m.s.d., root mean square deviation.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 43, pp. 32755–32764, October 27, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. OCTOBER 27, 2006• VOLUME 281•NUMBER 43  JOURNAL OF BIOLOGICAL CHEMISTRY   32755  macaques (28–30), whose three   -defensins are named rhesus   -defensins (RTDs) 1–3. Humans have multiple    -defensingenes, including some that are transcribed. However, humangenesandtheirtranscriptscontainaprematurestopcodonthataborts successful translation (27, 31). Retrocyclins 1–3 are syn-thetic    -defensin peptides, whose structures are based onhuman multiple    -defensin pseudogenes. Conceptually, they represent peptides last produced by apes whose eventual prog-enyincludedgorillas,chimps,andhumans.Thepurposeofthisstudy was to examine the effects of     -defensins on vegetativecells and spores of   B. anthracis  and on the enzymatic and cyto-toxic properties of anthrax LF. EXPERIMENTALPROCEDURES RetrocyclinandOtherPeptides Retrocyclins (31) and HNP-1, -2, and -3 (32) were preparedby solid-phase peptide synthesis as described previously. Pep-tide concentrations were established by quantitative aminoacid analysis (for    -defensins) or by   A 280  measurement (forHNPs). Table 1 contains the sequence of every peptide used inthis study. SporePreparation  B. anthracis  (Sterne strain 7702) spores were prepared asdescribed elsewhere (33). Briefly,  B. anthracis  was grown inTrypticasesoybrothmedium(T8907,Sigma)at30 °Cwithcon-stant shaking at 250 rpm for 5–7 days until sporulation. Theculture was centrifuged at 6000    g   for 20 min at 4 °C. Thepelletwasresuspendedinsterilewater,andculturedat30 °Cfortwo more days with constant shaking to promote further spor-ulation and bacterial lysis. Complete spore formation was con-firmed by light microscopy. Spores were centrifuged at 6000   g   for 20 min at 4 °C and washed five times with sterile water.Beforeuse,thesporeswereheatedat65 °Cfor30mintokillany germinated or germinating spores. No intact bacilli were pres-ent at this stage. Serial dilutions of the spore preparation wereplated on Trypticase soy agar plates to determine the concen-tration of colony forming units. RadialDiffusionAssay  Two-stage radial diffusion assays were used to test the anti-microbial activity of peptides against  B. anthracis  spores and vegetative cells (34). Stage1 —1–4  10 6 colonyformingunitsweredispersedinathin 1% agarose underlay gel containing 10 m M  phosphatebuffer (pH 7.4), 100 m M  NaCl, and 1% (v/v) Trypticase soy broth. A 6  6 array of wells, each with a 3-mm diameter and9-  l capacity, was punched. Serially diluted peptide solutionscontaining 250, 79, 25, 7.9, 2.5, and 0.79   g/ml peptides (8   leach)wereaddedtoeachsetof6wells.Theplatewasincubatedat37 °Cfor3htoallowthepeptidestodiffuseintotheunderlay gel. Stage 2 —An overlay gel containing 60 mg/ml Trypticase soy broth powdered medium plus 1% agarose was poured over theunderlay gel, and the plate was incubated overnight to allow surviving bacteria to form micro-colonies. The clear zonesaround each well were measured 18–24 h later. To determinethe minimal effective concentration (MEC), a linear regressionfunction relating the adjusted diameter (zone diameter minusthe well diameter) to the log 10  peptide concentration was cal-culated. The X-intercept defined by this function gives theMEC. Typically, the correlation coefficient ( r  2 ) was  0.98. EnzymaticAssay  The enzymatic activity of anthrax lethal factor (LF), a zincmetalloprotease, was measured by monitoring cleavage of aspecific substrate by fluorescence resonance energy transfer.The substrate, purchased from Calbiochem, was an internally quenched,  N  -acetylated, C-7-amino-4-methylcoumarin deriv-ative of a 14-mer mitogen-activated protein kinase/extracellu-lar signal-regulated-kinase-2 (MEK-2) peptide. Its cleavage by recombinant LF (Calbiochem) resulted in increased fluores-cencethatwasmonitoredkineticallywithanfmaxfluorescencemicroplate reader (Molecular Devices, Sunnyvale, CA), withexcitation set at 360 nm and emission at 460 nm. Unless other-wise noted, 100 n M  LF was incubated for 30 min at room tem-perature with the specified amount of     -defensin peptidebefore 50  M  substrate (final concentration) was added. MurineMacrophageIntoxication Murine macrophage-like RAW 264.7 cells were seeded in384-well plates at 4000 cells per well and incubated over-night at 37 °C. The medium was replaced by 20   l of freshDulbecco’s modified Eagle’s medium containing 25 m M HEPES, 2 m M  glutamine, 100   g (each) penicillin and strep-tomycin, and 1% fetal bovine serum. The   - and    -defensinpeptides were serially diluted into the same medium, and 20  l was added to appropriate wells. Peptide concentrations TABLE1 Sequencesofthepeptidesusedinthisstudy NoncyclicRC-100,thesyntheticoctadecapeptideprecursorofRC-1(RC-100),con-tains the three disulfide bonds found in RC-1. Protegrin PG-1, a potently antimi-crobial octadecapeptide srcinally isolated from porcine leukocytes (66), containstwo disulfide bonds. Name/laboratory ID SequenceHNP-1  ACYCRIPACIAGERRYGTCIYQGRLWAFCC RC-100  cyclic  GICRCICGRGICRCICGR  Chirality and order RC-110 cyclic  RGCICRCIGRGCICRCIG  (all-D) RC-111 cyclic  RGCICRCIGRGCICRCIG  RC-112 cyclic  GICRCICGRGICRCICGR  (all-D) Arginine substitutions RC-100 cyclic  GICRCICGRGICRCICGR  RC-107G cyclic  GICRCICG G GICRCICGR  RC-107GG cyclic  GICRCICG G GICRCICG G  RC-107G2H2 cyclic  GIC H CICG G GIC H CICG G  RC-107G2Ha cyclic  GICRCICG G GIC H CICG G  RC-107G2Hb cyclic  GIC H CICG G GICRCICG G  RC-101 cyclic  GICRCICG K GICRCICGR  Retrocyclins  versus  RTDs RC-100 cyclic  GICRCICGRGICRCICGR  RC-100b cyclic  GICRCICGR R ICRCICGR  RC-100c cyclic  RICRCICGR R ICRCICGR  RTD-1 cyclic  G F CRC L C R RG  V  CRCIC T R  RTD-2 cyclic  G  V  CRC L C R RG  V  CRCIC R R  RTD-3 cyclic  G F CRCIC T RG F CRCIC T R  Cyclic backbone and SS bonds Noncyclic RC-100  GICRCICGRGICRCICGR Reduced andalkylated RC-100cyclic  GICRCICGRGICRCICGR  Protegrin PG-1  RGGRLCYCRRRFCVCVGR -amide RetrocyclinsandtheAnthraxbacillus 32756  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 281•NUMBER 43• OCTOBER 27, 2006  ranged from 0 to 60   g/ml. LeTx (20   l) diluted in freshmedia was added to each well giving final concentrations of 100 ng/ml PA and 100 ng/ml LF. Cells were incubated over-night at 37 °C. Cell viability was assayed with the CellTiter-Glo   luminescent cell viability kit per the manufacturer’s(Promega, Madison, WI) instructions. IC 50  values wereobtained with GraphPad Prism Software. Ultracentrifugation Sedimentation equilibrium runs were performed at 25 °C in12-mm path length double sector cells on a Beckman OptimaXL-A analytical ultracentrifuge. Absorption was monitored at228 nm for 0.1 mg/ml samples and 260 nm for 1.0 mg/ml sam-ples.Peptidesampleswerein100m M NaCl,10m M Tris,pH7.4,and sedimentation equilibrium profiles were measured at40,000 and 50,000 rpm. The data were initially fitted with anonlinear least-squares exponential fit for a single ideal speciesusing the Beckman Origin-based software (version 3.01). Pre-liminary analysis of the association behavior used the globalanalysis software (the “multifit” option of the abovementionedsoftware) to analyze four scans simultaneously, correspondingtoproteinat0.1mg/mlat40,000and50,000rpmandproteinat1.0 mg/ml at 40,000 and 50,000 rpm. The partial specific vol-ume (0.711 of RC-1 was calculated from its amino acid compo-sition (35). SurfacePlasmonResonanceStudies SPR experiments were performed on a BIAcore 3000 system(BIAcoreAB,Uppsala,Sweden).Proteinswereimmobilizedona BIAcore CM5 sensor chip using the BIAcore amine-couplingprotocol. Analytes were introduced into the flow cells in a run-ning buffer containing 10 m M  HEPES, pH 7.4, 150 m M  NaCl, 3m M  EDTA. The running buffer also contained 0.005% polysor-bate-20 to reduce nonspecific binding. Values were correctedfor background binding to the CM5 chip. Data were analyzedwith BIAevaluation 3.1 software, and curve fitting was donewith an assumption of 1:1 binding.SPR results are expressed in resonance units (RUs). To cali-brate the instrument, we synthesized [ 14 C]RC-2, which con-tained [ 14 C]glycine (Sigma) and purchased  125 I-bovine serumalbumin (BSA) from MP Biomedicals (Irvine, CA). The[ 14 C]RC-2 had a specific activity of 21.6   Ci/mg, and the  125 I-BSA had a specific activity of 987   Ci/mg. To calibrate thesystemforBSA,weimmobilizedamouseanti-BSAmonoclonalantibody (U.S. Biological, Swampscott, MA) on a CM5 biosen-sor chip. After measuring the binding of   125 I-BSA (1  g/ml) tothe biosensor chip, the bound analyte was recovered using theBIAcore Analyte Recovery Wizard   program, and its radioac-tivitywasmeasuredinaBeckmanliquidscintillationspectrom-eter.Fromsevensuchmeasurements,wedeterminedthat1RUof BSA was equivalent to 13.35  0.55 pg (mean  S.E.). Weused this value to estimate the amount of immobilized LF onthe biosensor chip in experiments with retrocyclins. To obtaina conversion factor for RC-2, we used a biosensor that con-tained immobilized recombinant gp120 (BioDesign Interna-tional,Saco,ME).Fromtheseexperiments,wedeterminedthat1RUcorrespondedto4.41  0.15pgofRC-2(mean  S.E., n  3). We used these values to estimate the stoichiometry withwhich retrocyclins bound to LF. ComputationalMethods Docking was accomplished with the AutoDock 3.06 suite of programs (36) which assumes that the macromolecule is rigid,while the ligand is allowed torsional flexibility. A Lamarckiangenetic algorithm (LGA) searches the conformational space of the ligand in the vicinity of the macromolecule and ranks thedocked molecules on the basis of its binding energy. Also avail-able are two local search methods based on the method of Solisand Wets (37) and an empirical free energy function that esti-mates the binding free energy (36). In the present work all pro-tein and ligand hydrogen atoms were explicitly modeled, withpolar and nonpolar atoms being assigned Lennard-Jones 12-10and 12-6 parameters, respectively. They were added to thenative and ligand complexed forms of anthrax lethal factor(Protein Data Bank designations 1J7N (38), 1PWW (39), and1YQY) (40) and the ligand (L2) of 1PWW using the WHAT IFweb interface (41).The NMR structures of RTD-1 and retrocyclin (RC)-2(1HVZand2ATG)alreadyhadhydrogenatoms.RC-1wasgen-erated by mutating RC-2  in silico  with Pymol. 4 All water mole-cules were removed during docking. Partial charges wereassigned to the protein atoms using all-atom charges of theAMBER force field (43). Atomic solvation parameters andatomic fragmental volumes were added with the AddSol pro-gram of AutoDock 3.06. The Lennard-Jones parameters usedfor Zn 2  , taken form the work of Stote and Karplus (44), suc-cessfully reproduced the crystal structures of L1 and L2 (Table4). Hydrogen atoms for the hydroxamate ligand of 1YQY (L1)were added with BABEL, 5 and partial charges were generatedwith GAMESS (46). Ligand rotatable bonds for all dockedligandswere definedusing the AutoTorsmodule ofAutoDock.Van der Waals and electrostatic energy grid maps were pre-pared using AutoGrid (36). These grid maps define the cubicspace in the vicinity of receptor in which the search for theoptimally binding ligand conformer is focused. The grid pointswere spaced 0.375 Å apart and based on the centers of L1 andL2,withthegridsizedtoallowa5-Åclearanceoneithersideof the ligands in the  x ,  y , and  z   dimensions. For RC-2 and RTD-1two grid sizes were used. The first grid was based on the NterminusMAPPK-2peptideincomplexwithanthraxlethalfac-tor(1JKY)(38),whichspanstheentireactivesite.Thisgridwastherefore centered on the MAPKK-2 peptide, also with a 5-Åclearance on either side in the  x ,  y , and  z   dimensions and a gridspacingof0.375Å.Later,whenitwasfoundthattheRTD-1andRC-2 docked only in a cavity close to the C-terminal end of theMAPKK-2 peptide in active site, the grid size was reduced tocover only that volume of space to improve the search effi-ciency.RC-1wasthereforedockedusingthesmallergridalone.The force-field parameters of AutoDock 2.4 were used to eval-uate nonbonded interaction energies instead of using theparameters of AutoDock 3.0, which estimates free energies.This was because the presence of a large number of conforma- 4 W.L.Delano(2002)ThePymolMolecularGraphicsSystem, 5 P. Walters and M. Stahl (1992) BABEL, RetrocyclinsandtheAnthraxbacillus OCTOBER 27, 2006• VOLUME 281•NUMBER 43  JOURNAL OF BIOLOGICAL CHEMISTRY   32757  tionaldegreesoffreedominthedockedligandspresentedchal-lenges with the estimation of the torsional free energy term(36). The reported binding energies are therefore representa-tive of binding enthalpies and not binding free energies. Elec-trostatic interactions were evaluated using a distance-depend-ent dielectric constant to model solvent effects.For the global search using the LGA, the size of the initialrandom population was 200 individuals for the large grids and50 individuals otherwise, the maximal number of energy eval-uations was 2  10 7 , the maximal number of generations was500, the number of top individuals that survived into the nextgeneration,theelitism,was1,theprobabilitythatagenewouldundergo a random change was 0.02, the crossover probability was 0.80, and the average of the worst energy was calculatedover a window of ten generations.For a pure local search, the pseudo-Solis and Wets methodwas used, whereas the Solis and Wets method was used for theLGA part of the local search. The parameters used for localsearch in both cases were a maximum of 1000 iterations perlocal search, the probability of performing a local search on anindividual was 1.0, the maximal number of consecutive suc-cesses or failures before doubling or halving the step size of thelocal search was 4, and the lower bound on the step size, 0.01,wastheterminationcriteriaforthelocalsearch.ForRTD-1andRC-2, a total of 50 dockings was performed using the large gridbeforeswitchingtothesmallergrid.Fortheroot-meansquareddeviation (r.m.s.d.) calculation of docked of L1 and L2, theircrystal coordinates were used as reference. RESULTS  Activity against Spores and Bacilli —We used the capsule-deficient Sterne strain of   B. anthracis  to examine the antimi-crobial activity of retrocyclins. Radial diffusion and colony counting assays were performed in the presence of physiologi-cal NaCl concentrations. Table 2 summarizes the results of ourradial diffusion assays, which showed that RC-1 and HNP-1killed vegetative  B. anthracis  cells with an MEC    1   g/ml.Retrocyclin-1 also manifested this exceptional potency against  B. anthracis  spores, but HNP-1 did not (MEC 24.9    0.49  g/ml). Because RC-112, an all  D -amino acid version of RC-1,andRC-110, aretroenantio versionofRC-1 were aseffective asthe regular peptide, chiral interactions were apparently notrequired for antimicrobial activity. RC-111, the retro analog of RC-1, showed reduced activity against vegetative cells (MEC13.6    2.78   g/ml) but had excellent activity against spores(MEC 0.32  0.04  g/ml).Table 2 also contains structure-function data.    -Defensinspossess a cyclic peptide backbone and three intramoleculardisulfide bonds. RC-100ox, the immediate synthetic precursorof RC-1, contains the three disulfide bonds but has free aminoandCtermini.Thispeptidewas  3-foldlesspotentthancyclicRC-1againstvegetative  B.anthracis bacteria,and  36-foldlesseffective against  B. anthracis  spores. RC-100 IAA  was derivedfrom RC-1 by reducing its disulfide bonds with dithiothreitoland alkylating the liberated cysteine residues with iodoacet-amide. RC-100 IAA  thereby retained the net charge (  4) andcyclic peptide backbone of RC-1 but lacked its internal tri-di-sulfide scaffold. RC100 IAA  lacked activity (MEC  250   g/ml)against vegetative cells and spores of   B. anthracis  (Sterne).Thus,boththecyclicbackboneandthetri-disulfideladdercon-tributed to activity against  B. anthracis .Inadditiontoitssixcysteineresidues,Retrocyclin-1containsfourresidueseachofarginine,isoleucine,andglycine.Toassessthe contribution of the arginines to its activity against  B.anthracis  (Sterne), we synthesized analogs in which 1, 2, 3, or 4of these arginines were replaced by a glycine and/or a histidineresidue. Replacing one (RC-107G) or both (RC-107GG) argi- TABLE2 MECagainstvegetativecellsandsporesof  B.anthracis (Sterne) Values represent the mean  S.E. The number of determinations ( n ) appears in parentheses. Peptide name Short ID Vegetative cells Spores   g/ml  Human neutrophil peptide-1  HNP-1 0.85  0.04 (3) 24.9  0.49 (3) RC-1  RC-100 0.89  0.03 (5) 0.69  0.05 (8) Chirality and residue order Retroenantio-RC-1 RC-110 1.15  0.06 (4) 0.79  0.00 (3)Retro-RC-1 RC-111 13.6  2.78 (3) 0.32  0.04 (3)Enantio-RC-1 RC-112 0.99  0.05 (4) 0.51  0.02 (3) Arginine substitutions (R 9 G)-RC-1 RC107G 1.08  0.04 (3) 1.61  0.084 (4)(R 9,18 G)-RC-1 RC107GG 2.80  0.06 (3) 3.31  0.525 (4)(R 9,18 G, R 13 H)-RC-1 RC107G2Ha 7.91  1.02 (3)   250 (3)(R 9,18 G, R 4 H)-RC-1 RC107G2Hb 24.8  0.17 (3)   250 (3)(R 9,18 G, R 4,13 H)-RC-1 RC107G2H2   250 (3)   250 (3) Retrocyclins  versus  RTDsRC-2 RC-100b 0.47  0.05 (4) 0.28  0.025 (4)RC-3 RC-100c 0.88  0.03 (3) 7.70  0.64 (3)Rhesus   -defensin-1 RTD-1 0.98  0.05 (3) 0.39  0.018 (3)Rhesus   -defensin-2 RTD-2 0.72  0.05 (3) 0.25  0.02 (3)Rhesus   -defensin-3 RTD-3 1.62  0.24 (3) 0.68  0.03 (3)R9K-RC-1 RC-101 0.32  0.03 (4) 0.58  0.01 (4) Cyclic backbone and SS bonds Noncyclic RC-1 RC100ox 2.31  0.07 (3) 24.9  0.12 (3)Reduced and alkylated RC-1 RC100IAA   250 (3)   250 (3)Protegrin PG-1 PG-1 1.34  0.01 (3) 1.06  0.15 (3) RetrocyclinsandtheAnthraxbacillus 32758  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 281•NUMBER 43• OCTOBER 27, 2006  nineresiduesinthe  -turnregion(s)ofRC-1hadrelativelylittleeffect on activity against vegetative cells or spores (Table 2).However,replacingallfourarginines(RC-107G2H2)abolishedactivity(MEC  250  g/ml).Whenwereplacedthreearginines(RC-107G2Ha and RC-107G2Hb), this abolished activity against  B. anthracis  spores (MEC    250   g/ml), but activity against vegetative cells persisted (MEC 7.91  1.02 and 24.8  0.17   g/ml). Analogs of RC-2 containing a lysine instead of anarginineatposition4,9,10,13,or18ofRC-2killedvegetative  B.anthracis  cells with an MEC    1   g/ml, as did an analog inwhichbothArg-9andArg-10werereplacedbylysines(datanotshown).We also examined activity against vegetative cells of   B.anthracis  (Sterne) in colony count assays. Fig. 1 shows thatmid-logarithmic and stationary phase organisms showed simi-lar susceptibility to RC-2 and that its bactericidal activity wastime-andconcentration-dependent.Incontrast,whenweusedcolonycountassaystotestactivityagainst  B.anthracis (Sterne)spores,wesawnodecreaseincolonyformingunits/mlafter3hof incubation with 25  g/ml RC-2 (data not shown). This sug-geststhatthepotentactivityofRC-2against  B.anthracis sporesseeninourradialdiffusionassays(Table2)wasexertedafterthespores had commenced to germinate, rather than against qui-escent spores.  Effect on Enzymatic Activity —Anthrax LF is a highly sub-strate-specific Zn 2  -metalloprotease. Table 3 summarizes theabilityof16different   -defensinstoinhibittheenzymaticactiv-ityofLF.The tested peptides included RC-1 (RC-100) and itsretro (RC-111), enantio (RC-112), and retroenantio(RC-110) analogs. Although these peptides were similar incomposition, net charge, and sequence, the retro (RC-111)and enantio (RC-112) analogs were only half as potent asRC-1, and the retroenantio analog (RC-110) was   25% aseffective. Thus, chirality and polarity relative to the peptidebackbonealsocontributedtotheabilitytoinactivateLF.Thecyclic backbone was extremely important, because the IC 50 ofRC-100ox(the  -hairpin,noncyclicsyntheticprecursorof RC100) was increased 5-fold relative to RC-1.Replacingone  -turnarginineresidewithglycine(RC-107G)had slight effect on potency, but replacing two of them (RC-107GG) reduced potency 2-fold. Further replacement of asingle   -sheet arginine with histidine (RC-107G2Ha andRC-107G2Hb) caused little further impairment, but replacingboth of these arginines with histidines (RC-107G2H2), abol-ished it completely. These findings suggest that the argininesmay operate in a pairwise fashion with respect to inhibiting theenzymatic activity of LF. Placing a lysine instead of an arginineat position 4, 9, 10, 13, or 18 of RC-2 neither enhanced nordiminished inhibitory activity against LF (data not shown).Retrocyclins 1–3 and RTDs 1–3 had fairly similar potency (Table 3). Although RC-2 appeared somewhat more effectivethan RC-1, the difference was relatively small, and not statisti-callysignificant.Retrocyclin-3waslesseffectivethanRC-1(  p  0.05), despite having two additional arginine residues that gaveit a net charge of   6 instead of   4. The generally similar activ-ity of the retrocyclins 1–3 and RTD 1–3 suggests that theirability to inhibit LF likely resides in features they all share,namely a cyclic backbone, a conserved tri-disulfide ladder, andcertain arginine residues.  Effect of Preincubation —Fig. 2 a  shows that by preincubatingLF with retrocyclin, inhibition of the enzymatic activity of thetoxin was considerably enhanced. In the absence of preincuba-tion, 2.5   M  RC-1 inhibited enzymatic activity by   20%. Inhi-bition increased to 40% after a 5-min preincubation, to 60%after15-minpreincubation,andto80%aftera30-minpreincu-bation. In Table 3, inhibition was measured after a 30-min pre-incubationbetweenrecombinantLFandtheindicated   -defen-sin. Fig. 2 b  shows that increasing the substrate concentrationdid not reverse the inhibition of LF by RC-2, clearly indicating FIGURE1. Colonycountassays. Mid-logarithmic( a )andstationaryphase( b ) B. anthracis , Sterne strain bacteria were killed with similar kinetics by RC-2(RC-100b). TABLE3 InhibitionoftheenzymaticactivityofanthraxLF Data,shownas,aremean  S.E.valuesfromatleastthreeindependentexperimentswitheachpeptide.IC 50  valuesforeachpeptidewerecomparedtothoseobtainedforRC-1 by unpaired  t   test. Peptide Laboratory name Mean IC 50  S.E.   g/ml  RC-1  RC-100 4.23  0.82 Retroenantio-RC-1  RC-110 19.9  2.01 a Retro-RC-1  RC-111 7.73  0.29 b Enantio-RC-1  RC-112 9.37  1.21 b Retrocyclins  versus  RTDs RC-1 RC-100 4.98  0.88RC-2 RC-100b 3.60  0.23RC-3 RC-100c 11.1  2.15 b Rhesus   -defensin-1 RTD-1 5.13  0.48Rhesus   -defensin-2 RTD-2 7.93  1.41Rhesus   -defensin-3 RTD-3 4.70  0.60 Arginine substitutions RC-1 RC-100 4.23  0.82(R 9 G)-RC-1 RC107G 5.63  0.49(R 9,18 G)-RC-1 RC107GG 9.25  0.95 b (R 9,18 G, R 13 H)-RC-1 RC107G2Ha 9.43  0.43 a (R 9,18 G, R 4 H)-RC-1 RC107G2Hb 12.9  2.23 b (R 9,18 G, R 4,13 H)-RC-1 RC107G2H2   50 a Cyclic backbone and SS bonds Noncyclic RC-1 RC100ox 22.7  5.79 b Reduced and alkylated RC-1 RC100 IAA   50 a Protegrin PG-1 PG-1 14.8  2.41 b Human neutrophil peptide-1 HNP-1 6.58  1.48 a  p  0.01. b  p  0.05. RetrocyclinsandtheAnthraxbacillus OCTOBER 27, 2006• VOLUME 281•NUMBER 43  JOURNAL OF BIOLOGICAL CHEMISTRY   32759
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