Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Short

communication

Mapping

of

the

continuous

epitopes

displayed

on

the

Clostridium

perfringens

type

D

epsilon-toxin

Guilherme

Guerra

Alves

_,

_Ricardo

_Andrez

_{Machado-de-Ávila}

_,

Carlos

Delfin

Chávez-Olórtegui

_,

_Rodrigo

_Otávio

_Silveira

_Silva

_,

Francisco

Carlos

Faria

Lobato

a_{UniversidadeFederaldeMinasGerais(UFMG),EscoladeVeterinária,BeloHorizonte,MG,Brazil}

b_{UniversidadeFederaldeMinasGerais(UFMG),InstitutodeCiênciasBiológicas,BeloHorizonte,MG,Brazil} c_{UniversidadedoExtremoSulCatarinense(UNESC),Criciúma,SC,Brazil}

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Articlehistory:

Received21February2016 Accepted17October2016 Availableonline3February2017 AssociateEditor:RoxanePiazza

Keywords: Epsilon Toxin Enterotoxemia Epitope Mapping

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Theepsilontoxin,producedbyClostridiumperfringens,isresponsibleforenterotoxemiain ruminantsandisapotentialbioterrorismagent.Inthepresentstudy,15regionsofthe toxinwererecognizedbyantibodiespresentintheserum,withdifferentimmunodominance scales,andmaybeantigendeterminantsthatcanbeusedtoformulatesubunitvaccines.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Theepsilontoxin(ETX), producedbyClostridium perfringens

typesBandD,isresponsibleforenterotoxemiasindomestic ruminants,leadingtogreateconomicloss.1_{Inaddition,ETXis} consideredapotentialbioterrorismagent.2,3_Despitethe rec-ognizedimportanceofthistoxin,epitopesmappingstudies ofETXarescarce.4_{Thesestudiescancontributeto} identify-inginteraction sitesbetweenthe toxinand itsneutralizing antibodies,leadingtoabetterunderstandingofits neutral-izingmechanisms,thusfacilitatingthedevelopmentofnew vaccines and treatments against ETX.4 _{The present study} aimedtomapcontinuousepitopesatthe ETXbyusingthe

∗ _{Correspondingauthor.}

E-mail:guilhermeguerra.vet@gmail.com(G.G.Alves).

SPOT-synthesis technique followed by enzyme immunoas-says using hyperimmune serum against the purified toxin.5

ETXwasproducedandpurifiedaccordingtothemethod describedbyParreirasetal.6_{BeingthemainproducerofETX} andmaincauserofdisease,C.perfringenstypeDstrainwas fermentedinabenchbioreactor(BioFlo110,NewBrunswick ScientificC.O.,UK)andconcentrated10timesusingan ultra-filtrationsystemwitha10kDamembrane(Amicon,Beverly, USA).Thetoxinwaspurifiedbyionexchangechromatography (DEAE-SepharoseCL6B).

http://dx.doi.org/10.1016/j.bjm.2016.10.023

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ThepurifiedETXwassubjectedtoinactivationbythe addi-tionof0.5%formalin(v/v).Aluminumhydroxide[Al(OH)3]was

addedasanadjuvanttoobtainafinalconcentrationof2.83% offreealuminumoxide(Al2O3).7ThreeNewZealandrabbits

weighingbetween1.5and2.5kgweregivenfivesubcutaneous inoculationswith100␮goftheepsilontoxoid. Theanimals wereimmunizedondays0,28,49,70and91,andtotalblood collection wasperformed on day105. The serum obtained

was titratedbyan indirectEnzyme-Linked Immunosorbent Assay(ELISA)accordingtothemethoddescribedby Chavez-Olortegui et al.8 _{Atotal of 100␮L ofa solution containing} 2␮g/mLofthepreviouslyobtainedrecombinantETX toxin9 wasusedtocoattheplates.

Theparallelsynthesisofpeptidesonacellulosemembrane wasperformedusingtheSPOTtechnique.10_Overlapping 15-aminoacidpeptidesweresynthesized,withchangesinthe

1 130 Peptide sequence P eptide n umber Reactivity Peptide sequence P eptide n umber Reactivity Peptide sequence P eptide n umber Reactivity

Fig.1–ETXSPOTmembraneimmunochemicalassayagainstanti-ETXhyperimmuneserum.Alistofthesynthesized peptidesandthemeanreactivityofpositivespotswithnumerical(0–5)andcolorintensity(noreactivity–nocolor,more reactive–darker)scalesisshown.Mutatedresiduesmarked.

Table1–ReactivepeptidesinClostridiumperfringenstypeDETXandeachpeptide’soriginatingspot,sequence,number ofaminoacids(aa),molecularweight,isoelectricpoint(pI)andgrandaverageofhydropathicityindex(GRAVY).

Epitope Spots Sequence Numberofaa Mass(Da) pI GRAVYa

1 7 YSIVNIVSPTNVIAK 15 1617.9 8.59 0.787 2 11 IAKEISNTVSNEMSK 15 1650.8 6.14 −0.533 3 16–26 KASYDNVDTLIEKGR YNTKYNYLKRMEKYY PNAMAYFDKVTINPQ 45 5459.1 9.26 −1.100 4 37–38 MNYLEDVYVGKALLT NDT 18 2059.3 4.03 −0.128 5 40 GKALLTNDTQQEQKL 15 1686.8 6.07 −1.160 6 43 QQEQKLKSQSFTSKN 15 1780.9 9.70 −1.947 7 45 KSQSFTSKNTDTVTA 15 1614.7 8.59 −0.980 8 47 SKNTDTVTATTTHTV 15 1576.6 6.46 −0.640 9 50–52 TTTHTVGTSIQATAK FTVPFN 21 2222.4 8.44 0.014 10 68–73 LVPANTTVEVIAYLK KVNVKGNVKLVGQVS 30 3181.8 9.83 0.470 11 78–79 GSEWGEIPSYLAFPR DGY 18 2044.2 4.14 −0.644 12 86–88 TVNKSDLNEDGTINI NGKGNY 21 2266.4 4.56 −1.119 13 93–98 SAVMGDELIVKVRNL NTNNVQEYVIPVDKK 30 3386.9 5.94 −0.233 14 102 DKKEKSNDSNIVKYR 15 1824.0 9.40 −2.120 15 104–106 NDSNIVKYRSLSIKA PGIK 19 2103.4 10.00 −0.479

a _{GRAVYindicatesthesolubilityofthepeptideanditiscalculatedbyaddingthehydropathyvalueforeachresidueanddividingbythelength}

ofthesequence.

threeinitialresidues,coveringtheentireprimarystructureof ETX.ThepeptidesweresynthesizedusingtheFmoc-synthesis technique11_{adaptedforthecellulosemembrane,and} approx-imately40nanomolesofpeptideperpointinthemembrane wasobtained.10_{Thepeptidesynthesiswasperformedinan} automaticsynthesizer(ResPepSL/AutomaticSpotSynthesizer, IntavisGmbH,Koln,Germany).

Intotal,130peptidesweresynthesized(Fig.1).Peptides107 through130werederivedfrom regionsthatarepart ofthe originalstructureofthetoxinandincludedmutationsinone ortwoaminoacidresidues.Thesepeptideswereusedinthe presentstudytoevaluatetheantigenicimportanceofmutated aminoacidresidues.

The membrane with synthesized peptides was blocked overnight in a solution containing 3% bovine serum albu-min (BSA) (ID Bio, France) and 5% sucrose (Dinâmica,São Paulo,Brazil)in0.1%Tris-bufferedsaline(TBS).Subsequently, themembranewasincubatedwiththehyperimmuneserum dilutedinblockingsolution.Adilutionwasusedforwhichthe absorbanceintheindirectELISAwascloseto1.0.5

Bindingbetweenantibodiespresentintheserumandthe peptides was detected by incubating the membrane with rabbitanti-IgGconjugatedwithalkalinephosphataseatthe dilutionrecommendedbythemanufacturer(Sigma–Aldrich, St. Louis, USA). The substrates used consisted of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and MgCl2 (Sigma). After 20min, the reaction was stopped by

discardingthereagentsandwashingthemembranewith dis-tilledwater.Theprotocoldescribedabovewasrepeatedtwice

with the hyperimmune serum and once with preimmune rabbitserum.

Duringtheimmunochemicalassays,theanti-ETX hyper-immune serum was able to bind to somepeptides and to detecttheantigenicdeterminantsofETX(Fig.1).Theassays performedwiththepreimmuneserumdidnotdetectany reac-tivityofthespotswiththeantibodiespresentintheserumof animalsunimmunizedagainstETX(resultsnotshown).This resultsuggeststhatinassayswithhyperimmuneserum, reac-tivepeptidesarepresentinepitopicregionsofC.perfringens

typeDETX.

Fig.1listsallofthereactivespotsinthe immunochemi-calassaysperformed.Changesintheaminoacidsofpeptides 107–130didnotresultinsubstantialchangestothebinding patternofantibodiestopeptides;removingorinserting spe-cificaminoacids intheETX sequencedidnotsignificantly decreaseorincreaserecognitionbyanti-ETXantibodies.Thus, wemayinferthatmodifiedaminoacidsarenotessentialto theantigenicityoftheirrespectiveETXregions.

Basedonthereactivityanalysisofspotsthatwere recog-nizedbyanti-ETXhyperimmuneserumantibodies,15epitopic regionswereidentifiedinC.perfringenstypeDETX(Table1).

Someoftheprobabledeterminantsmappedwerenotable for their strong reactivity in immunochemical assays or duetoexistingdataintheliterature(describedbelow) that corroborate the existenceofthese ETX epitopes. Epitope3 (23_{LIEKGRYNTKYNYLKRMEKYY}43_{)wasthesecondmost}

reac-tiveepitopeinimmunochemicalassays,whichsuggeststhat therearemanyantibodiesagainstthisepitopeintheanti-ETX serum,whichinturn,suggeststhatitisanimmunodominant

A

B

C

E

D

Fig.2–Viewoftheepitopesinthe3DstructureofClostridiumperfringenstypeDETX.(A)Epitope3.Theyellow,greenand redcolorsrepresentlow,mediumandhighreactivityofregions,respectively.(B)Epitope9isshowninyellow;the

determinantmappedbyMcClainandCover15_{isshowninblack.(C)Epitope9isshowninyellow;theessentialdomainfor} ETXactivityisshowninred;theoverlappingregionbetweenthisdomainandepitope9isshowninorange.(D)Epitope12 isshowninyellow;thenegativeelectrostaticpotentialisshowninred;thepositiveelectrostaticpotentialisshowninblue. (E)Epitope15isshowninyellow.

epitope.Inaddition,thisepitopeislocatedindomainIofC. per-fringenstypeDETXandislocatedintheamino-terminalregion ofthetoxin(Fig.2A).Thisregion,whichismostlycomposedof alongalpha-helix,isbelievedtocontainthetoxininteraction sitethatbindstoreceptorspresentintarget-cells.3,12_Thus,it ispossiblethatneutralizingantibodiesproducedagainstETX bindprimarilytotheamino-terminalregionandpreventthis toxinfromadheringtocellreceptors.

IvieandMcClain13_{introducedmutationsinETXby} replac-ing seven accessible aromatic amino acids on its surface. Subsequently,theyevaluatedtheabilityofthesemutantsto interactwithcells and tocause cell death.Asaresult,the authorsobservedthatmutationsintyrosinesY42_,Y43_,Y49_and

Y209_{dramaticallyreducedproteinbindingtotarget-cellsand}

thattheseresiduesarethusimportantfortoxincytotoxicity.

Threetyrosinesarepresentintheprimarystructureofepitope 3(Y42_,Y43_,Y49_{),whereasthelatter(Y}209_{)isspatiallycloseto}

theepitope(Fig.2A),whichthereforecorroboratesourresults. Determinant 9 (116_{TTTHTVGTSIQATAKFTVPFN}136₎

con-tainstheaminoacidhistidine(H)atposition119oftheprimary ETXstructure(Fig.2B).AccordingtoOystonetal.,14_whenthis residueisreplacedbyaproline,thetoxinisinactivated.Thus, H119_{andtheregioncontainingitare essentialforthetoxic}

activity oftheprotein,andanti-ETXantibodiesagainstthis epitopemustneutralizethisregion.

Indeed, using two anti-ETX neutralizing monoclonal antibodies,McClainandCover15_{mappedanantigenic} deter-minantclosetoepitope9;thisproximityoccursbothlinearly andspatially,asshowninFig.2B.Accordingtotheauthors, whenthemonoclonalantibodiesusedboundtotheidentified

determinant, they neutralized ETX by an allosteric mech-anism, sterically preventing the loop containing H119 _from

exertingitsfunction.However,accordingtotheseauthors,the regionbetweenaminoacids119and158isimportantfortoxin activity,likelybecauseitistheinsertionregionoftheprotein intotheplasmamembraneandparticipatesintheformation ofthetransmembranepore.

Knapp et al.16 _{also reported the importance of an ETX} domainextendingovertheregionfromH119_toA149_.Epitope

9overlapsthisdomainat18aminoacids,fromH119_toN136

(Fig.2C).Accordingtotheauthorsandcorroboratingthe pre-viouslydiscussedfindings,thisregionisinvolvedinbothtoxin insertioninto the membraneand cell pore formation, and mutationsinsomeaminoacidsleadtochanges inprotein cytotoxicityandthecharacteristicsoftheformedpores.

Among the positive spots in the immunochem-ical assays, those corresponding to epitope 12 (224_{TVNKSDLNEDGTININGKGNY}244_{) showed the highest}

reactivity, which suggests that many immunoglobulins against this antigenic domain are present in the anti-ETX sera.TheGRAVYindexof−1.119ofthisepitopeindicateshigh hydrophilicity,whichinturn,predictsthatthisdeterminant isexceptionallyaccessibleinETX.Thisfactiscorroborated by the position of epitope 12 in the 3D structure of the protein(Fig.2D).Moreover,whencalculatingtheelectrostatic potential of the toxin, a large area with high potential is observed around a large portion of antigenic determinant 12 (Fig. 2D). Regionswith electrostatic potential may form protein–protein or protein–ligand electrostatic interactions among themselves.17 _{In fact, these are major interactions} that occur during the binding of an antibodyto a protein antigen.18

Determinant15(282_{IVKYRSLSIKAPGIK}296_{)ispartofdomain}

IIIinthecarboxy-terminalportionofETX(Fig.2E),andthis domainisbelievedtobeinvolvedintheinteractionbetween thetoxin chainsduringoligomerizationtoformthe hepta-mericcellpore.Inthe nativeprotein,the carboxy-terminal peptide seems to block the oligomerization of monomers andpreventtheactivationoftheprotoxin.19_{Antibodiesthat} bind to this region may competitively inhibit its cleavage by enzymes and then prevent the activation of the toxin. There are several examples described in the literature of antigensthat have determinantsin their carboxy-terminal portions,20–24_{includingC.botulinumtoxinA}4_{andC.perfringens}

typeAalphatoxin.2,25,26

Thepresentstudymapped15probableepitopes,with dif-ferent immunodominance scales, consisting of part ofthe primary structureof C. perfringens type D ETX.Due tothe strongreactivityinimmunochemicalassaysandtoexisting knowledgeintheliterature,antigenicdeterminants3,9,12 and15haveagreaterpossibilityofbeingimmunodominant agents.Therefore,thesedeterminantsmaybethebasisfor studiesthataimtodevelopandformulatesubunitvaccines againstC.perfringensETX.

Conflicts

of

interest

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgements

TheauthorswouldliketothanktheMinasGeraisResearch Support Foundation (Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG), the Brazilian Federal Agency fortheSupportand Evaluation ofGraduate Educa-tion(Coordenac¸ãodeAperfeic¸oamentodePessoaldeEnsino Superior–CAPES)andtheNationalCouncilforScientificand Technological Development (Conselho Nacional de Desen-volvimentoCientíficoeTecnológico–CNPq).

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