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ARMENISE-HARVARDSYMPOSIUM2002
STRUCTURALBIOLOGYANDMICROBIALPATHOGENESIS
ANDTHEHOSTRESPONSE
6thAnnualSymposium
June27-29,2002,St.Thomas,U.S.VirginIslands
AbouttheSymposium
Thethreatofattack,theimportanceofdefense,andtheneedtoknowmoreabouteachhave
becomenearuniversalworriessinceSeptember11.Peoplewhoseldomthoughtabouttheseissues
inthewakeoftheColdWarnowavidlyfollowthedailynews.Incontrast,attack,defense,and
intelligencegatheringarelong-standingconcernsfortheselectgroupofresearcherswho
participatedin“StructuralBiologyandMicrobialPathogenesisandtheHostResponse,”the6th
AnnualSymposiumoftheGiovanniArmenise-HarvardFoundation.Althoughthesymposium’s
themewasdecidedbeforebioterrorismbecamearealitylastFall,thoseeventsgreatlymagnifiedits
relevance.
Anthrax,AIDS,tuberculosis,andmalariawereamongthegloballyimportantdiseasesexploredin
22invitedlecturesandanequalnumberofposterpresentations.InvestigatorsdescribedusingXraycrystallography,aswellasmicroarraysandotherpost-genomictools,tobetterunderstandthe
battlebetweenpathogensandtheirhosts.Thestakesarehigh:speakersemphasizedthatdespite
medicalprogress,infectiousdiseasesstillaccountforoneinthreedeathsworldwide–thesametoll
theytookonhumankind400yearsago.
ThesymposiumwasheldJune27-29atMarriottFrenchman’sReef,St.Thomas,U.S.VirginIslands.
AlthoughtheCaribbeanisjustlycelebratedforitsnaturalbeauty,thepeopleoftheseislandslive
withendemicmalariaandtuberculosis,andthisregionissecondonlytosub-SaharanAfricainits
rateofHIVinfection.
Inherclosingremarks,Dr.GiuliaDeLorenzowasoptimisticabouttheimpactofresearchpresented
duringthesymposium.Newtechnologiessuchasstructuralbiology,bioinformatics,andfunctional
genomicswillhelpgeneratenotonlynewtreatmentsandvaccinesforcombatinghumaninfectious
disease,butalsonewstrategiesforprotectingtheglobalfoodsupply.“Understandingthemolecular
logicofpathogenesisiscrucialforfutureprogress,”saidDr.DeLorenzo,aprofessorofplantbiology
attheUniversita’DiRomaLaSapienzaandamemberoftheprogramcommitteeforthe
symposium.
Lastyear,FoundationPresidentandCEODanielC.TostesoninauguratedaCareerDevelopment
programthatprovidestalentedyoungItalianinvestigatorswithsupportneededtoestablish
researchlaboratoriesintheirhomecountry.The2002CareerDevelopmentawardswerepresented
toGiampietroSchiavoandRosellaVisintin.Schiavo,whohasbeenstudyingmembranedynamicsat
thenerveterminalattheImperialCancerFundinLondon,willbejoiningtheDepartmentof
BiologicalChemistryattheUniversityofPadova.Visintinhasbeeninvestigatingkeyregulatorsof
mitosisattheMassachusettsInstituteofTechnology,andwillbereturningtoItalytoestablishalab
attheEuropeanInstituteofOncologyinMilano.Visintinwasunabletoattendthesymposium.
Schiavo,whogavealecture,wastheonlyparticipantaffiliatedwithaU.K.institution;28scientists
camefromfiveleadingItalianresearchinstitutionsand48representedthesixbasicscience
departmentslocatedontheQuadrangleatHarvardMedicalSchool.Italianparticipantscamefrom
theEuropeanInstituteofOncologyinMilano,theUniversityofPadova,theInstituteforCancer
ResearchandTreatmentattheUniversityofTorinoSchoolofMedicine,theDipartmentodiRicerca
BiologicaeTecnologica(DIBIT)atScientificInstituteSanRaffaeleinMilano,andUniversita’Di
RomaLaSapienza.
ReverseVaccinology:agenome-basedapproachtovaccinedevelopment
</em><strong>RinoRappuoli</strong>
IRIS,ChironSpA,Siena
<palign=""left"">Historically,thefirststepinvaccinedevelopmentwastogrowtheinfectious
agentinthelaboratory.Onceapathogenwasavailableinthelab,itwaskilledorattenuatedfor
testingasavaccine,thosebeingtheonlyapproachesknownpriortotheadventofrecombinant
DNAtechnology.Anewerabeganwhengeneticengineeringmadeitpossibleforvaccinologiststo
scrutinizelaboratory-grownorganismsforindividualantigensthatmightbeusedtoelicita
protectiveimmuneresponse–aprocessthatwaslabor-intensiveandoftenquiteslow.</p>
<palign=""left"">Now,theabilitytosequencetheentiregenomesofmicroorganismshascleareda
newpathtovaccinediscoverythatDr.Rappuolicalled“reversevaccinology.”Toillustratehowthis
newapproachworks,hedescribedChiron’sworktowardapreventivevaccineforgroupB
meningococcus,asevereandsometimesfataldiseaseofchildrenandadolescents.Insteadof
growingandmanipulatinglargequantitiesofthispathogen,companyresearchersfeditsgenetic
sequenceintoacomputerprogrammedtoscanforsequencesthatmightencodeantigenswith
potentialasvaccines.</p>
<palign=""left"">MeningococcusBchallengedreversevaccinologytodemonstratethatitisnot
merelyanewtool,butalsoabetterone.After40yearsoftrying,conventionalmethodshadenabled
vaccinologiststoidentifybetween15and20potentialgroupBmeningococcusantigens.
Unfortunately,testsshowedthatnoneoftheseprovideduniversalprotectionagainstdisease.When
Chironscientistsfedtheorganism’sfullsequenceintothecomputer,itpredictedthatsome600
antigensencodedbythebacteriummighthavepromiseforvaccinedevelopment.Thesewere
expressedasrecombinantproteinsin<em>Escherichiacoli</em>andnearly350weretestedin
mice;about90werenovelsurfaceproteinsand29oftheseappearedtostimulatedesirable
immuneresponses.Within18months,reversevaccinologyhadyieldedmorevaccinecandidates
thantraditionalapproachesgeneratedinfourdecades,Dr.Rappuolinoted.Thebestofthesenovel
antigenshavebeenincorporatedintowhatdevelopershopewillbeauniversallyprotective
vaccine,acandidatethatrecentlyenteredPhaseIclinicaltrials.</p>
<palign=""left"">Chironisnowapplyingreversevaccinologytothegenomesofotherbacterial
pathogens,includingpneumococcus,streptococcus,staphylococcusandtheorganismsresponsible
formalariaandtuberculosis.Genome-basedpredictivetechnologyisalsobeingusedtodesign
vaccinesagainstHIVandotherviruses.</p>
<palign=""left"">Dr.RappuolicharacterizedtheAIDSpandemicas“today’sBlackPlague,”and
warnedthatunlesstheeconomicsofvaccinedevelopmentcanbechanged,therewillbelittle
incentiveforcompaniestodevelopvaccinesagainstAIDSandotherdiseasesthatmostlykillpeople
inthedevelopingworld.Althoughvaccinesarethemostcosteffectivemedicalinterventionever
known,theyarelessfinanciallyattractivetocompaniesthantherapeuticdrugs.Heendedhis
lecturewithapleathatrichnationsshouldermoreresponsibilityformakingvaccinesthatare
desperatelyneededaroundtheworld.</p>
<hrnoshade=""noshade""size=""1""width=""400""/>
<palign=""left""><strong>OtherPresentations</strong></p>
<dl><dt></dt><dd><em>Anthraxtoxinandwaystoinhibitit
</em><strong>R.JohnCollier</strong>
DepartmentofMicrobiologyandMolecularGenetics
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Theeventsof
Fall2001galvanizedpublicinterestinbioterrorism,andaddedtheterm“weaponized”tothe
vocabularyofmillions.Althoughthereisnogenericdefenseagainstpathogensthatcanbeturned
intoweapons,Dr.Collierisconfidentthatbasicresearchwillyieldstrategiesforbluntingthelethal
powerofanthrax,themicrobethathislaboratoryhasstudiedfor15years.
AsmostnewspaperreadersbecameawarelastFall,peoplewhocomeintoskincontactwiththe
anthraxbacteriumareverylikelytosurviveinfection,whereasinfectionisnearlyalwaysfatalin
peoplewiththemisfortunetoinhaleit.Atpresent,theonlydefenseisalimitedsupplyofanotvery
goodvaccine;newandbettervaccineswon’tbeavailableforatleastseveralyears,andthereisas
yetnopublichealthstrategyformassimmunization.Asaresult,Dr.Colliersaid,thenationis
undeniablyvulnerabletoanthraxattack.Thisvulnerabilitycanbereduced,however,ifbetter
scientificinsightsintoanthraxvirulencecanbetranslatedintodefensivestratagems.
Bacillusanthracisproducesitstoxininthreeparts:ProtectiveAntigen(PA)bindstoareceptoron
thehostcellandfacilitatesentryoftwoenzymeeffectorsknownasEdemaFactor(EF)andLethal
Factor(LF).Astheirnamessuggest,EFcausesswellingwhereasLFcausesdeath.Originally
secretedfromthebacteriaasnontoxicmonomers,orsmallmolecularbuildingblocks,these
proteinsassembleonthesurfaceofreceptor-bearingcellstoformtoxicnoncovalentcomplexes.PA
bindstocells,coordinatesself-assemblyofthecomplexes,andultimatelydeliversEFandLFtothe
cytosol.EFisacalmodulin-dependentadenylatecyclase,andLFisazincmetalloproteasethat
cleavesmembersoftheMAPKKfamilyandpossiblyotherintracellularsubstrates.
NewfindingsfromDr.Collier’slabandotherresearchteamsindicatethatafterPAcomplexeswith
EFandLFonthecellsurface,theresultingstructureschangeshapeseveraltimesastheyinsinuate
themselvesthroughthemembraneandintothecell.Afullerunderstandingofthesestructurefunctionrelationshipssuggeststhreepossiblewaystoaborttheirdeadlymission,Dr.Collier
reported.Thefirstisadominant-negativeinhibitor,aslightlymutatedformofPAthatappearsto
protectinjectedratsagainstexposuretoanthraxtoxin.Anotherpossibilityisapolyvalentinhibitor,
asmallpeptidethatpartiallyblocksLFbindingwithPAonthecellsurface;itisalsobeingtestedin
animals.Finally,itmaybepossibletomakeasolubleformofthePAreceptorthatwouldattach
itselftotheanthraxtoxinbeforeitreachedhostcells.Allthreeareunderactivestudy.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Tetanustoxinanditsjourneyinmotorneurons
</em><strong>GiampetroSchiavo</strong>
MolecularNeuropathobiologyLaboratory,CancerResearchUK
LondonResearchInstitute
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></
dl>Infectionscausedbymembersofthe<em>Clostridia</em>familyhavelongfascinated
neuroscientists,inpartbecauseneurotoxinsfromrelatedbacteriausesharedcellularpathwaysto
producedramaticallydifferentclinicaleffects.<em>C.tetani</em>causestetanus,markedby
lockjaw,thefixedsmileknownas<em>risussardonicus</em>,andrigidspasticparalysisthatcan
befatal.<em>C.botulinum</em>,incontrast,causeswidespreadmuscleweaknessthatatworse
becomesaflaccidparalysisthatcankill.Neitherissomethingthatadoctorwantstodiscoverina
patient.Inthelaboratory,however,neurobiologistshaveusedbothtypesofneurotoxinsastools
forunderstandingtransportmechanismsinnervecells.
Dr.Schiavofocusesonaxonalretrogradetransport,amechanismnecessaryforneuronalsurvival,
inwhichneurotropinsaretransportedfromnerveterminalstothecellbody.Thedistances
involvedinaxonaltransportcanbeimpressive:inhumanterms,thedistancefromagiraffe’sbrain
toitsspinalmotorneuronswouldbetwicethedistanceoftheTourdeFrance.
Organelles,pathogens,neurotrophinsandothersynapticsignalingmoleculesusesimilar
mechanismstojourneyfromnerveendingtocellbody.Tetanustoxin(TeNT)isamongthe
virulencefactorsthatmakethistrip,whichbeginswhenitbindsattheneuromuscularjunctionand
eventuallyleadstothespinalcord.There,TeNT’sinterferencewiththenormalreleaseofinhibitory
neurotransmitterresultsinspasticitythatisthehallmarkoftetanusinfection.
AlthoughscientistsknowquiteabitabouttheintracellularcatalyticactivityofTeNT,controlofits
retrogradetransportislesswellunderstood.Dr.Schiavo’steaminventedatransportassaythat
usesanontoxicTeNTfragment(TeNTH<sub>C</sub>)asaprobe.Workingwithlivingmotor
neurons,theydeterminedthatTeNTbindstoastructurethatformsonlyinlipid-richmicrodomains
inthecellmembrane,andtheysoonrealizedthatsequesteringcholesterolcouldblockentryof
TeNT.WhentheypermittedTeNTH<sub>C</sub>tobind,itwasrapidlyendocytosedand
transportedintwotypesofvesicularcarriers,oneroundandonetubular,whichtravelatdifferent
speedsusingmicrotubulesandtheactincytoskeleton.Theorganellesarenotacidifiedduring
axonaltransportandlacktypicalendocyticmarkers.Dr.Schiavo’steamdiscoveredthatoneof
thesecompartmentsisthenormalonefortransportingnervegrowthfactor(NGF),whichis
apparentlyhijackedbyTeNTH<sub>C</sub>forretrogradeaxonaltransport.WhenDr.Schiavo
movestotheUniversityofPadova,heandcollaboratorstherewilluseaproteomicsapproachto
characterizethesetransportcompartmentsmoreprecisely.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Geneticdifferencesinthesusceptibilityofthehosttoanthraxlethaltoxin
</em><strong>WilliamF.Dietrich</strong>
DepartmentofGenetics
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd
></dl>Therearetwosidestothestoryofeveryinfectiousdisease,onetoldbythepathogenandthe
otherbythehost,andDr.Dietrichhasbeenlisteningcloselytothehost.TheLethalFactor(LF)
componentofanthraxtoxinkillsmacrophages,keyplayersininnateandacquiredimmunity,by
rupturingtheirplasmamembranesandcausingthecell’scontentstospill.AlthoughLFisknownto
beazincmetalloproteasethatcleavesmembersoftheMAP(membrane-associatedprotein)kinase
family,exactlyhowthesecleavageeventsleadtothedeathofmacrophagesisunclear.Dr.Dietrich’s
laboratoryhasbeenexploringthisquestionusinginbredmousestrainswhosemacrophagesexhibit
strikingdifferencesinsusceptibilitytotheeffectsofLF.Thesedifferenceshavebeentracedtoa
singlegene,locateddownstreamfromtoxinentry,called<em>Ltxs1</em>(lethaltoxinsensitivity
1).
<em>Ltxs1</em>hasbeenmappedtomousechromosome11,andDr.Dietrich’steamhas
determinedthatitisactuallythesamegeneas<em>Kif1C</em>,whichencodesakinesin-like
proteininvolvedinmicrotubuletransportofanunknowncargo.Severallinesofevidencesupport
theideathatthetwoareidentical.First,<em>Kif1C</em>istheonlygeneinthe<em>Ltxs1</em>
intervalexhibitingpolymorphismsbetweensusceptibleandresistantstrains.Second,multiple
allelesof<em>Kif1C</em>determinesusceptibilityorresistanceofculturedmousemacrophages
tointoxicationwithLF.Finally,treatmentofresistantmacrophageswithBrefeldin-A(which
disrupts<em>Kif1C</em>localization)inducessusceptibilitytoLFintoxication,whileectopic
expressionofaresistancealleleof<em>Kif1C</em>insusceptiblemacrophagescausesa4-fold
increaseinthenumberofcellssurvivingLFintoxication.Withtheseobservationsinhand,Dr.
Dietrichsaidthenextstepsinhisresearchwillinvolveworkingoutthecellbiologyof
<em>Kif1C</em>todiscoverwhereitislocalizedandexactlyhowitoperates.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>StudiesontheinvolvementofSNAREproteinsinendosomal
vacuolizationinducedby</em>Helicobacterpylori<em>VacAtoxin
</em><strong>MarinadeBernard</strong>
DipartimentodiScienzeBiomedicheSperimentali
UniversityofPadova
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl><em>Hel
icobacterpylori</em>,aGram-negativebacteriuminvolvedinthedevelopmentofchronicgastritis,
pepticulcerandgastriclymphoma,producesatoxin,calledVacA,whichinducesvacuoleformation
inepithelialcellsinculture.Thesevacuolesoriginatefromlateendocyticcompartments,as
demonstratedbythepresenceofthesmallGTPaseRab7intheirlimitingmembrane.Thepresence
ofaRabproteinsignifiesthataSNAREproteinontheendosomaltransportcompartmenthasbeen
properlymatchedwithacorrespondingSNAREproteinonthetargetmembrane.
RecentstudiesshowthatafterVacAbinds,itundergoesconformationalchangesandformsan
anion-specificchannelthatthelateendosomalcompartmentpassesthroughbeforeballooninginto
avacuolethatmaybe400timeslargerthantheoriginalcompartment.Thisdramaticsizeincrease
causedDr.deBernardtoaskwhetherthemembranefromasingleoriginalcompartmentprovides
enoughrawmaterialforsuchahugeexpansion,orwhetheradditionalmaterialmustbegathered
fromothervesicles.Ifothermaterialsarerequired,theymayderivefromseveralsources:
homotypicfusionoflateendosomes,fusionofendosomesandlysosomes,ordepositionintothe
membraneofcarriervesiclesderivedfromearlyendosomes.Eachofthesefusionshingesona
SNAREhook-upthatcannottakeplacewithoutacytosolicproteincalledα-SNAP.
Dr.deBernard’steamtransfectedcellswithadominantnegativemutantofα-SNAP,thenadded
VacAextracellularlyorexpresseditwithinthecell.IfvacuolizationwasdependentonSNAREmediatedfusion,thennovacuoleswereexpectedtoform.Infact,theinvestigatorsobservedthat
VacAhadnodifficultyinducinghuge,swollenvacuoleseventhoughSNARE-dependentfusionhad
beenblocked.ThisfindingledDr.deBernardtohypothesizethatrawmaterialsforvacuole
enlargementarealreadypresentintheoriginalendosomalcompartment,andthatRab7maybethe
keythatturnsinternalmembranevesicles(orinvaginations)intoanoutermembraneforthe
vacuole.SupportcomesfromrecentexperimentsshowingthatcellstransfectedwithadominantnegativeRab7mutantdidnotformvacuoleswhenexposedtoVacA,andfurtherinvestigationsare
underway.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Clostridialneurotoxins:mechanismofactionandtherapeuticuse
</em><strong>CesareMontecucco</strong>
CentroCNRBiomembraneandDipartimentodiScienzeBiomediche
UniversityofPadova
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Clostri
dialneurotoxinscompriseonetetanusneurotoxin(TeNT)andsevenbotulinumneurotoxins
(BoNT),whichcausetetanusandbotulism,thecontrastingneuroparalyticsyndromesthatDr.
Schiavodescribedinhislecture(above).Dr.Montecucco’slaboratoryconductsstructure-function
studiesoftheseneurotoxins,andhisfindingsrelatebothtobotulismasclinicaldiseaseandto
BoNT’sroleasanincreasinglypopulartherapeuticagent.
ThecrystallographicstructuresofBoNT/AandBoNT/Bshowthepresenceofafirstshellof
coordinatingresidues(2histidinesand2glutamicacids),andasecondshellofresiduescloseto
thecatalyticcenter,whicharedifferentfromanyknownmetalloproteaseandwhicharethoughtto
explainthestructures’uniquespectroscopicproperties.WhenDr.Montecucco’steammutated
severalsecondshellresiduesinTeNTandBoNT/A,theyfoundparallelsbetweenthespectroscopic
andcatalyticpropertiesofthemutantneurotoxins.Theyalsosawthatthemetalloproteolytic
activityoftheseneurotoxinsisstronglyactivatedbylipidsthatresemblethoseinsynapticvesicles,
afindingconsistentwithDr.Schiavo’sobservationthatTeNTneedscholesteroltoentercells.
Physicianshavelongobservedthatpatientswhosurvivetheacutephaseofbotulism,when
paralysisaffectsrespiratorymuscles,arelikelytomakeagoodrecovery.Similarly,clinicianswho
useBoNTstotreat“everythingfromstroketowriter’scramp”knowthatthebenefitsdon’tlast
forever.Inaseriesofexperimentswithrats,Dr.Montecuccofoundthatdamagecausedby
introducingBoNT/Aattheneuromuscularjunctiondiminishesasnerveterminalsgeneratenew
sproutsovertime.BoNT/Aisthetoxinapprovedforclinicaluse,andtheseanimalexperimentshelp
explainwhyrepeatedinjectionsarenecessary.Othertypesofbotulinumtoxinsmayworkbetter.
Dr.MontecuccoandhisclinicalcollaboratorshaveshownthatBoNT/Cprovidesmoredurablerelief
forseveraldystoniasoffacialmuscles,andtheyarenowevaluatingthetherapeuticpotentialof
otherBoNTserotypesaswell.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Comprehensiveidentificationof</em>Mycobacteriumtuberculosis<em>
genesrequiredforinfection
</em><strong>EricJ.Rubin</strong>
DepartmentofImmunologyandInfectiousDiseases
HarvardSchoolofPublicHealth
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Whenthe
firstwidespreadepidemicsoftuberculosisstruckEuropeinthe16thCentury,thediseaseswept
throughalllevelsofsociety.Aslivingconditionsimprovedforthemiddleandupperclasses,
however,thediseasesettledamongtheurbanpoorandthepeopleofunderdevelopedcountries.
Morerecently,theAIDSpandemichasgivenTBatremendousboost,manypeoplehavebeen
inadequatelymedicated,andferociousdrug-resistantstrainshavesurfacedinfar-flungpartsofthe
globe.DespiteTB’sstatusasoneoftheworld’sleadingcausesofdeath,lessisknownaboutitatthe
molecularlevelthanaboutotherpathogensthatkillfarfewerpeople.Onereasonforthisrelative
lackofinformation,Dr.Rubinsaid,isthatpharmaceuticalcompaniesdonotperceivethe
developingworldasaprofitablemarketplacefordrugs.
ThefunctionofthousandsofgenesintheM.tuberculosisgenomeremainsunknown.Toaddress
thisproblem,hislaboratoryhasinventedanoveltechnique,transposonsitehybridization(TraSH),
whichcanbecombinedwithmicroarraytechnologytoidentifyallthegenesexpressedinspecific
growthconditions.Dr.Rubin’steamisusingthisapproachtodeterminethefullsetofgenesneeded
by<em>M.tuberculosis</em>tocausedisease.
Transposonsarenaturallyoccurring,mobilegeneticelementswhosemovementscanbe
manipulatedinthelaboratory.TraSHcombineshigh-densitytransposonmutagenesiswithmicro
arraymappingofinsertionsitesinpoolsofmutants.Sofar,about100,000mycobacteriamutants
havebeencreatedusinga<em>mariner</em>-basedtransposonandefficientphagetransduction.
TraSHhasenabledDr.Rubinandhiscolleaguestodefinespecificsetsofgenesrequiredforthe
survivalofpathogenicmycobacteriagrownunderavarietyofdifferentconditions.Thistechnique
hasallowedthemtoidentifygenesthat<em>M.tuberculosis</em>needstoproceedthrough
progressivestagesofinfectioninamousemodel.Notsurprisingly,thegenesrequiredearlyinthe
infectiondifferfromthoseneededlateron.Atfirst,arelativelysmallnumberofgenesare
essential—mostofthemneededtoacquirenutrients.Butasinfectionprogresses,andthehost
fightsback,thepathogenrequiresanincreasingnumberofgenestosurvive.After8weeksof
infection,173essentialgeneswereidentified,andscientistshaveyettoidentifyexactlywhat107of
themdo.Havingsomanygenesinplayatonce,however,mayexplainwhytheTBpathogenisso
adeptatdevelopingresistancetotreatment.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Thelogicofbiosynthesisoftheglycopeptideantibioticvancomycin
</em><strong>ChristopherWalsh</strong>
DepartmentofBiologicalChemistryandMolecularPharmacology
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>V
ancomycinwasapprovedbytheFoodandDrugAdministrationin1958,butdidnotcomeinto
widespreaduseuntilthe1980s.Itsoongainedareputationamongphysiciansastheantibioticof
lastresortforsevereinfectionsinvolvingGram-positivebacteriasuchas<em>Staphylococcus
aureus</em>and<em>Enterococcusfaecalis</em>,anditsuseundoubtedlysavedmanylives.
Reportsofvancomycin-resistant<em>Enterococcus</em>begantosurfaceasearlyas1988,
however,andmorerecentlythesehaveskyrocketed.Scientistsnowknowthatasinglebiochemical
changeinthebacteriumcanconferasmuchasathousand-foldincreaseinresistance,Dr.Walsh
said.
Thereisapressingneedtore-engineervancomycinintoanewantibioticthatwillkilldrugresistantbacteria.Andthewaytodoso,accordingtoDr.Walsh,istoexaminehow
<em>Actinomycetes</em>,afamilyoffilamentousbacteria,originallydesignedvancomycinasa
weapontowieldagainsttheirenemies.Ifthecreator’soriginallogicisunderstood,thensciencewill
knowwheretogonext.Thescientistsarelikeateamofindustrialengineers,analyzingeverystep
inanautomobileassemblylinetolearnhowitcanberetooledfornextyear’smodel.
Earlierstudiesshowedthatpeptideantibioticssuchasvancomycin,penicillins,andpristinamycins
arebiosynthesizedbynonribosomalpeptidesynthetases(NRPS)actingasmultimodularprotein
assemblylines.Themanufacturinggenesforthesenonribosomalpeptidesareclusteredtogether,
presumablysotheycanregulateandcoordinateoneanother’sactivities.Dr.Walsh’steamhas
foundthatvancomycinproductionisdrivenbyaclusterof30genesintheorganism
<em>Amycolocaptosusorientalis</em>.Thesegenesencodeenzymesthatmakeaminoacid
monomers.Athree-subunitNRPSassemblesthesemonomers,alongwithdeoxyhexose
vancosamine,intotheheptapeptidebackboneoftheantibiotic.Thisacyclicheptapeptideisthen
oxidativelycrosslinkedbytandemactionofthreehemeproteins,thenglycosylatedbyspecific
glycosyltransferasestocreatevancomycin.NowthatDr.Walshandhiscolleagueshave
characterizedtheenzymaticstepsneededtobuildvancomycin,thenextstepistoreprogramthe
assemblylineandsynthesizenovelantibioticsthatwillside-stepthedefensesofresistant
pathogens.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Host-pathogenrecognition:thestructuralbasisoftheinteraction
betweenfungalpolygalacturonaseanditsplantinhibitorPGIP
</em><strong>BenedettaMattei</strong>
DipartimentodiBiologiaVegetale
UniversitàdiRoma“LaSapienza”
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Wh
enafungusinitiallyattacksaplant,itmustbatteritswaythroughthecellwallwhiletheplant
strugglestoresist.AseriesofstudiesattheUniversitadiRomaLaSapienzahavegradually
uncoveredthebattlethatragesbetweenfungalproteins,calledpolygalacturonases(PGs),and
correspondingplantrecognitionproteins,knownaspolygalacturonase-inhibitingproteins(PGIPs).
Aspartofthisongoingwork,Dr.MatteiandhercolleaguesuseX-raycrystallography,augmented
withavarietyofbiophysicalandbiochemicaltechniques,toperformstructuralanalysisof
interactionsbetweenspecificPGsandtheircorrespondingPGIPs.
Inthecellwall,oneofthefirststepstowarddiseaseisforPGstohydrolyzehomogalacturonan.
FightingbackarePGIPs,whichDr.Matteiandhercolleaguespreviouslyfoundtocomprise10
leucinerichrepeats(LRRs)—motifsspecializedforinteractionwithPG.Twoyearsago,hergroup
solvedthecrystalstructureofPGfromthephytopathogenicfungus<em>Fusarium
moniliforme</em>,describingitasaright-handedparallelβ-helixwith10coils,eachmadeupof
threeorfourparallelβ-helicalstrands.Overall,itresemblesasquared-offcoilofspring.These
findingsledtomorequestionsaboutwhathappenswhenPGandPGIPinteract.
Herrecentexperimentsusedsurfaceplasmonresonanceandmassspectrometrytocharacterize
thePG-PGIPinterface,aswellassite-directedmutagenesistolocatethePGresiduesinvolvedin
catalysisandsomeoftheresiduesrecognizedbyPGIP.BycloningandcharacterizingPGIPsfrom
Arabadopsisandbeanplants,theyhavelearnedthatasingleaminoaciddifferencecandetermine
whetherornotaPGIPinhibits<em>F.moniliforme</em>PG.Theyhavealsolearnedthat
indigenousplantPGs,neededfornormalprocesses,differslightlyfromfungalPGsandareableto
goabouttheirbusinesswithoutbeingrecognizedandattackedbyPGIPs.WhenaPGIPinhibitsthe
activityofitscorrespondingfungalPG,itappearstodosobycompetingwiththesubstratefor
bindingsitesandcoveringtheactivesitecleft,Dr.Matteisaid.Shepredictedthatstructure-function
studiesnowunderwayinherlaboratory,includingarecentcrystalstructureforPGIPfrom
<em>Phaseolusvulgaris</em>,willshedadditionallightonhowplantsprotectthemselvesagainst
enemies.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Mechanismsofdrugresistanceinprotozoanparasites:awindowinto
parasiteevolution
</em><strong>DyannWirth</strong>
DepartmentofImmunologyandInfectiousDiseases
HarvardSchoolofPublicHealth
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Four
hundredyearsago,infectiousdiseasesaccountedforonethirdofglobaldeaths,andthesameis
truetoday,Dr.Wirthsaid.Insomecases,decadesofmedicalprogresshavebeenreversedas
organismsdevelopedresistancetodrugs.Malariaisavexingexample:<em>Plasmodium
falciparum</em>needed16yearstodevelopstrainsresistanttochloroquine,butonly4yearsto
beginoutsmartingfansidarandanother4yearsformefloquineresistancetosurface.Strainsthat
foiledthenewestanti-malariandrug,atovaquone,showedupafteronly6months.
Drugresistanceinmalariaandotherpathogenshasbeentracedtoover-expressionofcarrier
proteinsfromtheABCtransportersuperfamily,whicharetypicallyseatedinthecellmembrane
andservetopumpdrugsoutoftheorganism.Dr.Wirth’steamhasbeeninvestigatingresistance
mechanismsmediatedbyABC-transportersintwoprotozoanparasites,<em>P.falciparum</em>
and<em>Leishmaniaenriettii</em>.Ina<em>Leishmaniaenriettii</em>model,vinblastin
resistanceiscontrolledbyasinglegene,LeMDRI1.Localizationexperimentsindicatethatthisgene
isexpressedintheflagellarpocketandothervesicularcompartments,ratherthanonthesurfaceof
theparasite.Dr.Wirthproposesamulti-stepmodelforresistancethatinvolvesdrugsequestration
andsubsequentvesiculartransport.Initially,expressionoftheABCtransporterincreasesin
responsetodrugtreatment.ParasiteswiththehighestlevelsofABCtransporterproteinshavea
survivaladvantage,andgoontodevelopspecificpointmutationswithinthetransportergenethat
conferresistance.WhenDr.Wirthandhercolleaguescomparedlaboratorystrainsofdrug-resistant
<em>P.falciparum</em>withisolatesfrompatientswhowerefailingtreatmentinthecommunity,
pointmutationsweremorecommonintheprimaryisolates.
Theseobservationsledthemtoabroaderinvestigationofspecificpointmutationsandsingle
nucleotidepolymorphismswithin<em>P.falciparum</em>.Sofar,mostofwhatisknownabout
thegeneticsofsurvivalinthisgloballyimportantparasitecomesfromexaminingonegeneata
time.The<em>P.falciparum</em>genomeisnearlycomplete,however,andwhenitisfinished
thenextstepwillbeusinggenomicstodiscoveralltheplayersindrugresistance.Sofar,Dr.Wirth’s
analysisshowsfarlessgeneticvariationin<em>P.falciparum</em>thanexpected,whichsuggests
thatitisarelativelyrecentscourge—perhapsonlyabout10,000yearsold.Resistanceand
pathogenicitygenesdisplaymorepolymorphismsthanotherpartsofthegenome,however,which
probablyhelpsthemevadeattackbydrugsorhostimmuneresponses.Ifmoreofthese
advantageouspolymorphismscanbeidentified,somemayturnouttobenewdrugandvaccine
targetsin<em>P.falciparum</em>.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Polygalacturonase-inhibitingproteinsinplantdefenseagainst
phytopathogenicfungi
</em><strong>GiuliaDeLorenzo</strong>
DipartimentodiBiologiaVegetale
UniversitàdegliStudi“LaSapienza”
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Like
aninvadingforcethatarrivesbyland,sea,andair,pathogensattackplantswithmanyweaponsat
once.Thatbeingthecase,it’sagoodthingthatplantshavewhatDr.DeLorenzocalls“impressive
redundancyintheirdefensivearsenal.”Centraltothebattlebetweenfungusandplantisthe
recognitionsystemintroducedearlierinthesymposiumbyDr.Mattei:polygalacturonase-inhibiting
proteins(PGIPs)intheplant’scellwallrecognizefungalendopolygalacturonases(PGs),moveto
limitlocalfungaldamage,andformpecticfragmentsthatinitiateachainofdefensiveresponses.
Dr.DeLorenzoandhercolleagueshavedoneextensiveresearchonPGIPs.Earlyon,theyrealized
thattheversatileleucine-richrepeat(LRR)structureofthesecellwallproteinssuggestedthat
plantsmightgenerateaPGIPforeveryPGsecretedbytheirfungalenemies.Thisechoesthehuman
immunesystem’scapacityforproducingantibodiesthatrecognizeavastarrayofpathogens.Justas
antibodiesareonlyonecomponentofthehumanimmuneresponse,Dr.Lorenzoandother
investigatorshavefoundthatrecognitionofpathogenicityfactorsisthefirstofseveralstepstoward
diseaseresistance,andthatplantsvaryintheircapacitytoidentifyandresistspecificinvaders.
Herlabisnowexploringhowbasicscienceinsightscanbeusedtoimprovetheglobalfoodsupply.
PossiblestrategiesforstrengtheningplantdiseaseresistanceincludemakingPGIPsmorealert
sentinels,reprogrammingdefensivepathways,ormodifyingthedefenseresponseitself.Thismight
involvetransformingcropplantswithdifferent<em>pgip</em>genes.InAfrica,forexample,
storedcropseedsareoftenpoisonedandmadeuselessbythefungus<em>Fusarium
moniliforme</em>.SomePGIPsfrombeanplantsareexquisitelysensitivetoPGsfrom<em>F.
Moniliforme</em>,andthegenesforthesewatchdogproteinsmightbetransferabletosusceptible
plants.Inadditiontosharpeningtheplant’sabilitytorecognizePGsdeployedbyitsenemies,Dr.De
Lorenzo’sgroupisalsoinvestigatinghowmultipledefensesmightbemobilizedwhenpathogens
strikewithseveralweaponsatonce.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Recognitionofbacterialeffectorproteinsbytheplantinnateimmune
system
</em><strong>BrianStaskawicz</strong>
DepartmentofPlantandMicrobialBiology
UniversityofCaliforniaatBerkeley
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Building
onthethemesofDr.DeLorenzo’slecture,Dr.Staskawiczdescribedhowpost-genometechnologyis
beingusedtocreatedisease-resistantplants.Inhislaboratory,experimentsaredoneusingamodel
systemwherethegenomesequencesofbothplantandpestareknown.<em>Pseudomonas
syringae</em>isaGram-negativeorganismthatcausesbacterialspeckdiseaseon
<em>Arabidopsisthaliana</em>,afavoritemodelplant.Becausebothgenomesequencesare
available,abioinformaticsapproachcanbeusedtoidentifyputativeeffectorproteinsin<em>P.
syringae</em>.
Likeotherpseudomonads,thisplantbacteriumisequippedwithaTypeIIIsecretionsystemthat
injectseffectorproteinsinsidehostcells,wheretheycausedisease.Theproductsofresistance
genescandefusethesetimebombsiftheyspotthemrightawayandkillcellsaroundtheinfection
site,whichkeepsdamagefromspreading.Dr.Staskawicz’teamhasidentifiedarabidopsis
resistancegenesthatappeartobepartofatoll-likereceptor(TLR)system.TLRsareahighly
conservedpartofinnateimmunesystemsinsimpleanimalmodels,suchasfruitflies,andin
mammalsandhumansaswell.Ifscientistsknowthegenesinvolvedinthesepathways,itshouldbe
possibletousethesetoblockproteinsencodedbyspecificgenesinthepathogen.Thevalueofthis
gene-generesistancestrategyisnowbeingtestedbycollaboratorsofDr.Staskawiczatthe
UniversityofFlorida.Agenethatmakespepperplantsresistanttobacterialspotdiseasehasbeen
insertedintotomatoplants,whicharenowbeinggrowninthefield.Sofar,Dr.Staskawiczsaysthat
thetransgenicplantsappeartosuppressthiscommondiseasequitewell.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Plantdefenseresponsesagainstnecrotrophicfungiareactivatedthrough
multiplesignalingpathways
</em><strong>SimoneFerrari</strong>
DepartmentofMolecularBiology,MassachusettsGeneralHospital
DepartmentofGenetics,HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl
>Dr.Ferraribeganhistalkbynotingthatabout15%ofallagriculturalcropsarelosttodisease,
whichputstheannualglobalcostofplantinfectionsatabout$500billionperyear.Fungiposea
majorthreattocrops,whichiswhylaboratorieslikehisareextremelyinterestedincharacterizing
defenseresponsesthatcanfendoffspecifictypesofthesepathogens.Fungicanbedividedintotwo
broadcategories:biotrophiconesthatgrowonlyonthelivingtissuesofspecificplants,and
necrotrophicpathogensthatkillhostcellsandcancolonizeawiderangeofhosts.Thenecrotrophic
fungimacerateplanttissueswithdegradingenzymessuchaspolygalacturonases(PGs),whichwere
thetopicofseveralotherpresentationsduringthissymposium.
Dr.Ferrari’slabstudiestheinteractionbetweenthemodelplant<em>Arabidopsisthaliana</em>
andthenecrotrophicfungus<em>Botrytiscinerea</em>,apre-andpost-harvestpathogenthatis
toblameformanyoftherottingvegetablesfoundinhomerefrigerators.Itinfectsmorethan200
plants,andisdifficulttocontrolbecausepesticidesobviouslycan’tbeusedonplantsharvestedas
foods.Asanalternativetoagriculturalpoisons,hislabstudieshowselectiveactivationofdefensive
responsesmightprotectagainstthispest.
Ingeneral,plantsusethreesignalingpathwaystodefendagainstfungi:ethylene(ET),jasmonate
(JA),andsalicylicacid(SA).Necrotrophicfungisuchas<em>B.cinerea</em>activatetwoofthese
paths,ETandJA,Dr.Ferrarisaid.HisteamhasrecentlycharacterizedtwoArabidopsisgenesthat
encodepolygalacturonaseinhibitors(PGIPs)effectiveagainstPGsfrom<em>B.cinerea</em>.
AlthoughoverexpressionofeithergenereducesdiseasesymptomsinArabidopsis,Dr.Ferrarihas
foundthatduringinfectiontheprotectivegenesAtPGIP1andAtPGIP2arecalledintobattleby
separatesignals–muchasArmyandNavyforcesrespondtodifferentcommanders.TheJA
pathwayusesonesetofsignalingmoleculestoactivateAtPGIP2,whereasdifferentintermediaries
turnonAtPGIP1.Thenextstepwillseewhetherinducingmultipledefenseresponsesthrough
separatepathwaysmaybeaneffectivewaytoprotectplantsagainstversatile,wide-rangingfungi
suchas<em>B.cinerea</em>.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Genomescienceinbacterialpathogenbiologyandevolution
</em><strong>JohnJ.Mekalanos</strong>
DepartmentofMicrobiologyandMolecularGenetics
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd>
</dl>Nearly70bacterialgenomeshavebeenfullysequencedandanother160areinprogress,a
developmentthatisrevolutionizingthestudyofmicrobialpathogens.Onceanorganism’scomplete
genomesequenceisavailable,researcherscanlearnmuchaboutitsbiologybyusingsequence
homologytoassignorguessatthefunctionsofitsgenes.Inaddition,variouspost-genome
technologiesenableresearcherstoanalyzeextremelylargenumbersofgenessimultaneously.InDr.
Mekalanos’lab,thesenewmethodshaveyieldedimportantinsightsintotheevolutionof<em>V.
cholerae</em>strainsresponsibleforaseriesofcholerapandemics,aswellasdetailsaboutthe
molecularbasisfor<em>V.cholerae</em>pathogenesis.
WorkingwithcollaboratorsatTheInstituteforGeneticsResearch,Dr.Mekalanos’teamsequenced
a<em>V.cholerae</em>straincalledN16961.Theydevelopedmicroarrayscontainingatleast
93%ofallopenpredictedreadingframes(potentialgenes)forthisstrain,andusedthesearraysto
comparethegenomesofseveral<em>V.cholerae</em>strains.Theyfoundasetofgenescommon
toallpandemicbiotypes,aswellasgenesthatsettheso-calledclassicalstrain,whichisblamedfor
sixearlierpandemics,apartfromitssuccessor,the7<sup>th</sup>pandemicorElTorbiotype.
Only22genesdifferentiateElTorfromtheclassicalstrainandfromotherstrainsthatfizzledbefore
causingglobaldisease;nowthechallengeistodetermineexactlywhichgeneproductsmaketheEl
Torstrainsuchathreattopublichealth.
AlreadyDr.Mekalanos’teamhasidentifiedseveralgenesthatmaketheElTorstrainwhatitisand
whichmightbegoodantibiotictargetsbecausetheirproteinsareessentialforbacterialreplication.
Anotherprojecthasfocusedonquorumsensing,themechanismthatenablesbacteriacolonizing
theintestinaltracttosenseandregulatetheirowndensity.Thispopulationcontrolmeasuremight
beusedagainstthemicrobes,ifadrugcouldsignal“enoughbacteriaalready”wheninfactthereare
onlyafew.Oneofthequorumsensinggenesalsoappearstohelpregulateexpressionofimportant
<em>V.cholerae</em>virulencegenes.Theinvestigatorsarenowprofiling<em>V.
cholerae</em>geneexpressioninarangeofanimalandhumanhostsaswellasdifferent
laboratorygrowthconditions,withthegoaloflearningmoreabouthow<em>V.cholerae</em>
sensesandrespondstohostsignals.
Eventually,Dr.Mekalanoshopestheseinvestigationswillcontributetobettertreatments,a
preventivevaccine,andenvironmentalcontrolstrategiesthatcanpreventthousandsofcasesat
once,ratherthancombatingcholeraonepatientatatime.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Searchingforthefunctionof</em>Neisseriameningitidis<em>B
proteinsidentifiedonagenomicbase
</em><strong>EmanuelePapini</strong>
DepartmentofBiomedicalSciences
UniversityofPadua
Email:<ahref=""mailto:[email protected]"">[email protected]</a></dd></dl>For
themostpart,theGram-negativebacteria<em>N.meningitidis</em>dwellswithoutincidentin
thehumannoseandthroat.Everysooften,however,itinvadestheepitheliumanddiffusesintothe
bloodstream.Whenthishappens,“nootherinfectionsoquicklyslays,”inthewordsofanearly20th
Centuryphysician.Meningitisorfatalmeningococcalsepsisisespeciallylikelyinchildren,and
althoughthesurvivalrateisabout90%forcasesidentifiedearly,thetreatmentwindowisvery
narrowandoutcomesareespeciallypoorwhendiagnosisisdelayed.
Althoughpreventivevaccinesareavailableforseveralpathogenicserotypesof<em>N.
meningitides</em>,nonehasbeendevelopedforserogroupB.Byanalyzingthegenomeofthe
MC58strainof<em>N.meningitidis</em>B,Dr.Papiniandhiscolleagueshaveidentifiedpotential
virulencefactorsincludingextracellularproteinsthatinteractwithhostmucosaandblood.They
haveused<em>E.coli</em>toexpresshighlypurifiedpreparationsoftheseproteins,someof
whichappearpromisingforvaccinedevelopment.Sofar,themostinterestingoftheseisalollypopshapedproteinonthe<em>N.meningitidis</em>surfacecalledNadA,whichisendocytosedby
humanmonocytesandcanactatthelevelofthebloodstreamaswellasatthemucosa.Inthe
laboratory,ithasahighaffinityformucosa-likeChangcells,suggestingthatitisanadhesionfactor.
NadAalsotriggersreleaseofcytokinesthatmightinitiateorworsensepticshockinpatients.But
themostinterestingpossibility,whichDr.Papini’slabiscurrentlypursuing,isthatitmightbea
novelvaccinecandidate.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Structuralandbiochemicalcharacterizationofthepolioviruscellentry
pathway
</em><strong>JamesM.Hogle</strong>
DepartmentofBiologicalChemistryandMolecularPharmacology
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>M
orethan15yearsago,Dr.Hogledeterminedtheatomicstructureofpoliovirus.Sincethen,his
researchhasrevealedthatwhenthevirusentersatargetcell,itresemblesaMarslandingmodule
thatsettlesintoplace,drillsintothesurface,thenpoursgeneticmaterialintothehole.Structural,
kinetic,biochemical,andgeneticstudiesallcontributedtothisunderstandingofhowthevirus
breachescells.
Thedramabeginsintheextracellularenvironment:asthematureviriontravelsfromcelltocellor
hosttohost,amaturationcleavageofacapsidprotein,VP0,locksitinameta-stablestate.Thevirus
remainsrigidlystableuntilitencountersVpr,itsmatchingreceptorontargetcells.Uponcontact,
thereceptoractsasacatalystthatovercomesanenergybarrierandfreesupthemeta-stablestate.
Aseriesofconformationalchanges,whichDr.Hogel’steamhasdocumentedovertheyears,allows
thevirustoattachtomembranes,formapore,andreleaseitsRNAgenomeintothecytoplasm.This
sequenceofeventsappearstobeaprototypeforthematurationandcellentryofmorecomplex
envelopedviruses,suchasinfluenzaandHIV.
Nowthattheyhavethebigpicture,Dr.Hogleandhiscolleagueshavedevelopedasimple,liposomebasedmodelsystemforcarryingoutdetailedstructuralandbiochemicalstudiesofpoliovirus’
entryintomembranes.Liposomesdecoratedwithpoliovirusreceptorsareusedtocapturethe
virus,thenpurifiedforelectronmicroscopy.Theresearchershavedemonstratedthatreceptordecoratedliposomesinduceconformationalchangesthatenablepoliovirustoattachdirectlyto
membranes,andpreliminarydatasuggestthatthePv-decoratedliposomesallowRNA
translocation.Thesestudiesareongoing.
Dr.Hogleintroducedhispresentationbynotingthathisscientificcareerhasbeendevotedto
studyingapathogenwhich,iftheWorldHealthOrganization’s2005eradicationgoalismet,will
soonbeeliminated.Heeagerlyanticipatesthismoment,andremarkedthathealwaysknewthat
eventuallytheurgencyforstudyingthisorganismwoulddiminish.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Underconstruction:buildingareovirusfactoryfromviralandcellular
components
</em><strong>MaxL.Nibert</strong>
DepartmentofMicrobiologyandMolecularGenetics
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Thefamily
<em>Reoviridae</em>includesgenerawhosemembersinfectplants,invertebrates,and
vertebratesandcausediseaseinnon-humanhostsrangingfromfishtohorses.Thereovirusesof
greatestclinicalimportanceinhumansarethegroupBrotaviruses,whichcausediarrhealdisease.
Reovirus-infectedcellsaredistinguishedbythepresenceofstructurescalledviralinclusions,which
insomecasesserveasfactorieswheretheviralRNAgenomeisreplicatedandlarge,non-enveloped
viralparticlesarebuilt.
Muchasengineersmightanalyzetheworkingsofanautomobilefactory,Dr.Nibertandhis
colleaguessetouttodeterminehowthesevirusfactoriesareformed,whererawmaterialscome
from,andhowtheyareorganized.Thepurposeofthisinvestigationistofigureouthowantiviral
drugsmightsomedaydisruptthisviralassemblyline.Theoutputofthesefactoriesisanonenvelopedviruswithtwocapsidproteins,10genomicsegments,adoublestrandoflinearRNAin
itscore,andawell-definedthree-dimensionalstructure.Areovirusispartlydisassembleduponcell
entry,genomicsegmentsaretranscribedinthecytoplasm,andtheresultingproteinsarefedinto
thefactory.
Dr.Niberthypothesizedthatsomethingasneatlyconstructedasareovirusmustbebuiltinahighly
orderedintracellularenvironment.Byusingimmunofluorescencemicroscopyandapanelof
antibodiestoviralandcellularproteins,histeamfoundthatreovirusfactoriescomeintwoshapes,
globularandfilamentous,withthefilamentousshapeprevailingin22of24strainstested.They
determinedthatthisshapeoccurswhenthefactoriesareassociatedwithcellularmicrotubules,a
linkageaccomplishedbyareoviruscoreproteincalledµ2,anNTPaseinvolvedinviralRNA
synthesis.Theroleofµ2wasconfirmedwhentheinvestigatorsintroducedapointmutationandthe
factoriesbecameglobular.Dr.Nibert’sgrouphassincestudiedhowrawmaterialsforvirion
constructionarerecruitedintothefactories.Experimentsshowthatµ2alsoplaysarolehere,by
recruitingamajorreovirusnonstructuralprotein,µNS,thoughttobindRNA,tofilamentous
factories.Currentstudiesareaddressingotherprotein–proteinandprotein–RNAinteractions,as
wellasconsideringhowcomponentsofthecellularubiquitin–proteasomesystemareassociated
withthefactories.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Down-regulationofMHCclassImoleculesbyhumancytomegalovirus
</em><strong>DomenicoTortorella</strong>
DepartmentofPathology
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Maj
orhistocompatibilitycomplex(MHC)moleculesplayanimportantroleinhumanimmunityby
restrictingT-cellresponsestoproteinantigens.AkeyfunctionofMHCclassImoleculesistobind
peptidesgeneratedbyinvadingvirusesorbacteria,andtopresentthemonthesurfaceofinfected
cellssothatCD8+Tcellswillattackanddestroythem.EverystepintheformationofMHC-antigen
complexesisvulnerabletointerference,andDr.Tortorellahasbeenstudyingastrategythathuman
cytomegalovirus(HCMV)usestothwartthisantigenpresentationsystem.Theevasionstrategy
usedbythisvirushasbroadmedicalimplicationsbecauseitparallelseventsthatoccurincystic
fibrosis,andcouldpointthewaytobettertreatmentsforthissevere,inheriteddisorder.
HCMVgeneratestwouniquegeneproducts,US2andUS11,thatsubvertnormalstepsinMHCclassI
antigenpresentation.Forstarters,US2andUS11speedthebreakdownofclassIheavychains,
whichinterfereswithantigenbindingbythetransmembranetailoftheMHCmolecule.Theheavy
chainsareextractedfromtheendoplasmicreticulum(ER)andrelocatedtothecytosol,wherethey
areshuttledtotheproteasome–thecellularequivalentofagarbagedisposal–wheretheyare
groundupandthrownaway.Thisisanormalwasteremovalmethodthatcellsusetorid
themselvesofmisfoldedproteinsandothertrash,Dr.Tortorellanoted,whichUS2exploitsby
taggingnormalMHCclassImoleculesforprematuredegradation.ThebreakdownofclassIheavy
chainsisstrikinglysimilartothedegradationofamutantformofcysticfibrosistransmembrane
conductanceregulator(CFTR)whichhasbeenimplicatedinprogressionofCF.Bothseemtobe
four-stepprocesses,Dr.Tortorellasaid,involvingrecognition,recruitment,relocation,and
degradation.
HisgroupisusingseveralmethodstoshedlightonadditionalfunctionsofUS2andUS11.A
mutagenesisstudyusingdeletionandchimericmutantsofUS2,US11andclassIheavychains
suggeststhatthecellemploysdifferentcomplexestoextractproteinsfromtheER.Aninvitro
degradationsystemwasusedtoidentifyERandcytosolicproteinsinvolvedmarkingMHCclassI
moleculesfordestruction.Morerecently,high-throughputscreeningwasusedtoexaminemore
than16,000chemicalstoseeifanywouldinterferewithUS2-andUS11-mediateddestructionof
classIheavychains.Fourinhibitorswereidentified,Dr.Tortorellasaid,andheiscertainthesewill
bevaluabletoolsforstudyingthedegradationreaction.Itisevenpossiblethatoneofthesemay
eventuallyturnouttobeaviabledrugforcysticfibrosisorotherdiseasesthatinvolvedegradation
ofmisfolded,butpotentiallyfunctionalproteins.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Three-dimensionalstructureofiron-bindingproteinsfrom
</em>Helicobacterpylori<em>and</em>Bacillusanthracis<em>
</em><strong>GiuseppeZanotti</strong>
DipartimentodiChmicaorganicaeCentroCNRBiopolimeri
UniversitàdiPadova
Email:<ahref=""mailto:[email protected]"">[email protected]</a></dd></dl>At
firstglance,onemightnotexpectahigh-profilebiologicalwarfareagentandtheleadingcauseof
ulcerstohavemuchincommon.Whattheyturnouttoshare,however,isthatbothneedironto
growproperly.<em>Bacillusanthracis</em>hasbeeninthenewssinceitwasdeployedasa
bioterrorismagentlastFall,andthereisintenseinterestindevelopingnovel,second-generation
preventivevaccinesthataresaferthanthecurrentone,whichisnotoriousforitsadverseeffects.
Thepathtoanewkindofvaccinemightbeilluminatedbycomparisonsofanthrax’siron-binding
proteinswiththosefoundin<em>Helicobacterpylori</em>.
Inthe<em>B.anthracis</em>genome,Dr.Zanottiandhiscolleagueshaveidentifiedtwogenes
thatencodeferritins,proteinsthatstoreironandreduceoxidativestress.CalledDlp-1and-2,these
aredodecamershomologoustotheDNAbindingproteinof<em>Escherichiacoli</em>,calledDps.
Dps-likeproteinsaregenerallyhighlyimmunogenic.<em>Helicobacterpylori</em>,whichisbest
knownforitsassociationwithulcerdisease,hasaniron-bindingproteinknownasHP-NAP,which
hastheunusualabilitytoactivateneutrophils.Becauseithasprovedhighlyimmunogenicin
humansandmice,ithasbeenincorporatedintoanexperimental<em>H.pylori</em>vaccine
beingdevelopedbyChiron,Dr.Zanottinoted.
HislabhasrecentlysolvedthecrystalstructuresofDlp-1,Dlp-2andHP-NAPgrownin<em>E.
coli</em>.Dr.Zanotti’steamshowedthateachhasaquaternarystructuresimilartothatofthe
dodecamericbacterialferritinsintheDps-likefamily:theyaresphere-likeproteinswith32-facets
andaninternalcavity.FunctionalstudiesconfirmthatDlp-1andDlp-2areinvolvedinironuptake
andregulation,fundamentalactivitiesduringbacterialgrowth.HP-NAPhasadifferentdistribution
ofsurface-potentialcharges,duetothepresenceofmanypositivelychargedresidues,andDr.
ZanottispeculatesthatthismayexplainwhyitcanactivatehumanleukocyteswhileDlp-1and-2
cannot.HP-NAPisapromisingvaccinecomponent,andalthoughithasfeaturesthatDlp-1and-2do
not,neverthelessthesimilaritiesamongtheseferritinsmayaugerwellfordevelopmentoffuture
anthraxvaccines.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Bacterial/cytolysin-mediateddeliveryofproteintothecytosolof
mammaliancells
</em><strong>DarrenHiggins</strong>
DepartmentofMicrobiologyandMolecularGenetics
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl><em>List
eriamonocytogenes</em>isacommonbacteriumthatpreysmainlyoninfants,elderlypeople,and
pregnantwomen.Itisamongthemostlethaloffood-bornepathogens,withacasefatalityrateof
25%to60%.Becauseithasingeniousmechanismsforgettingintomammaliancellsandspreading
fromonecelltothenext,Dr.Higginsandhiscolleaguesfind<em>L.monocytogenes</em>auseful
modelforstudyingintracellularbacterialpathogenesis,mechanismsofgrowthandcelltocell
spread,andbacterialdeterminantsofthehostT-cellresponse.Inworkpartlysupportedbya1999
ArmeniseFoundationFellowshipGrant,Dr.Higginsdevelopedaversatilesystemthatincorporates
abacterialcytolysinintoavectorfordeliveringproteinsofinteresttohostphagocytes.
Dr.Higgins’teamstartedwithanespeciallyharmlessstrainof<em>Escherichiacoli</em>,called
K-12,andexpressedinitcytoplasmicrecombinantlisteriolysinO(LLO),whichefficientlytargets
proteinstothecytosolofmacrophagesanddendriticcells,so-calledprofessionalphagocyticcells.
LLOisapore-formingcytolysinwhosenaturalbiologicalroleistoexplode<em>L.
monocytogenes</em>vacuoles,sothatbacteriacanescapeintothecytosolandreplicate.They
usedthisvectortodeliverseveraldifferentproteinstoprofessionalphagocyticcells.
Fortheirnextseriesofexperiments,theinvestigatorsusedanovelpackagingmethodcalleda
diaminopimelate(dap)auxotrophtoexpressLLOand<em>Yersiniapseudotuberculosis</em>
invasinin<em>E.coli</em>K-12.Theresultingbacterialvectorcoulddeliveraproteincargoto
thecytosolofnon-professionalphagocyticcells.Followinginvasin-mediatedentryintomammalian
cells,the<em>E.coli</em>remainedwithinvacuolesyetunderwentlysisbecausedapauxotrophs
arestampedwithakindofbiologicalexpirationdate.LLO-mediatedperforationofthevacuolethen
allowedreleaseofcargoproteinsintothecytosol.
UsingT-cellactivationassays,Dr.Higgins’teamdemonstratedthattheinvasive/autolyticE.
coli/LLOstraincoulddeliverantigenicproteintothecytosolicmajorhistocompatibilitycomplex
(MHC)classIprocessingpathway,whichsubsequentlypresentedproteinfragmentstocytotoxicTcells.Intheory,thissystemcouldbeusedtodeliveranyantigenicproteinforpresentationonMHC
classImolecules,whichcouldproveveryusefulforprimingprotectiveT-cellresponses,Dr.Higgins
said.Inthelongrun,thiscouldbeaplatformtechnologyforlaunchingnewvaccines.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Electronmicroscopicstudiesonintegrins:activationandligandbinding
</em><strong>ThomasWalz</strong>
DepartmentofCellBiology
HarvardMedicalSchool
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl>Integrinsare
celladhesionreceptorsthatmediatethebindinganddetachmentofligandsinvolvedincell-celland
cell-pathogeninteractions.In2001,Dr.Walzandcolleaguesdeterminedthecrystalstructureofthe
extracellulardomainofαVβ3integrin.Theirstudiesrevealedanextraordinaryheaddomain,which
theyobservedineithera“clasped”or“unclasped”position.Todeterminewhichpositionbound
ligandsmoreeffectively,theyusedmolecularelectronmicroscopytovisualizethestructural
changesinvolvedinactivationandligandbinding.
ThecrystalstructureofαVβ3’sextracellulardomainrevealedabentconformation,inwhichthe
ligand-bindingheadpieceisfoldedbackontothetailsofthemolecule,sothat<em>insitu</em>
theligand-bindingsitewouldbeclosetothemembranesurface.Thisconformationwasthoughtto
beanartifactofX-raycrystallographywithnophysiologicalsignificance,Dr.Walzsaid.Tofindout,
theyexposedtheintegrintocalciumormanganese,observedwhathappened,thenaddedligandto
themix.Acombinationofelectronmicroscopicstudiesandligand-bindingassaysrevealedthatthe
bentconformation,seeninthepresenceofCa<sup>2+</sup>,actuallyrepresentstheinactivestate
ofthemolecule.
Incontrast,whenintegrinαVβ3wasincubatedwithMn<sup>2+</sup>,boththeclaspedand
unclaspedformswereactivatedandtheirtailsfullyextended.Theseobservationssuggesta
“switchblade”-likemotionofαVβ3duringtheactivationprocessthatinvolvesthestraighteningof
thebentreceptor.Whenligandwasadded,bothassumedthebindingposition.Dr.Walz’steamhas
nowmovedontoanalyzingchangesofshapeinadifferentintegrin,α5β1,broughtaboutbyligand
binding.
<hrnoshade=""noshade""size=""1""width=""400""/>
<dl><dt></dt><dd><em>Requirementforap38MAPkinasesignalingpathwayin
</em>Caenorhabditiselegans<em>innateimmunity
</em><strong>FrederickM.Ausubel</strong>
DepartmentofGenetics,HarvardMedicalSchool
DepartmentofMolecularBiology,MassachusettsGeneralHospital
Email:<a
href=""mailto:[email protected]"">[email protected]</a></dd></dl
>Theinnateimmunesystemisthebody’sfirstlineofdefenseagainstthreatsfromtheoutside
world.Itreliesonphysicalandchemicalbarriersandtherapiddeploymentofspecializedcellsthat
torightoffinvadersuntiltheacquiredimmunesystemcangenerateantibodiesandTcells.
Importantinsightsintomammalianinnateimmunityhaveemergedfromstudiesin
<em>Drosophila</em>,wheregeneticistshaveidentifiedkeygenesthatareimportantnotonlyin
fliesbutinhumansaswell.Morerecently,developmentofexperimentalhost-pathogenmodels
involvingbacterialinfectionof<em>Caenorhabditiselegans</em>providestheopportunityto
exploreevolutionarilyconservedpathwaysofinnateimmunityinthegeneticallytractable
nematodehost.Dr.AusubelandhiscolleaguesatMassachusettsGeneralHospitalhaveputthis
modeltoespeciallygooduse.
IntheAusubellaboratory,a<em>C.elegans-Pseudomonasaeruginosa</em>pathogenesismodel
wasusedtoscreenfor<em>C.elegans</em>mutantswithenhancedsusceptibilitytopathogens.
Thescreenidentifiedtwogenesrequiredforhostdefense:<em>esp-2</em>and<em>esp8</em>,encodingMAPkinasekinaseandMAPkinasekinasekinasecomponentsofap38MAP
kinasesignalingcassettein<em>C.elegans</em>.Whentheesp8genewasknockedout,the
nematodeswereeasilykilledbypathogens,Dr.Ausubelsaid.ThedefensesystemwasalsoshortcircuitedwhenRNAinhibitionwasusedtotemporarilyturnoffthep38MAPkinasestepinthe
pathway.Inseparateexperimentsusing<em>Salmonellaenterica</em>asthepathogen,
programmedcelldeathinthe<em>C.elegans</em>gonad(previouslyshowntoprotectagainst
<em>S.enterica</em>-mediatedkilling)wasfoundtobedownstreamofthep38MAPcascadeand
dependentonlipopolysaccharide,acomponentoftheGram-negativebacterialcellwall.
Thesefindingsfitintoaglobalpictureofinnateimmunitythathasemergedoverthepastdecade.
Dr.AusubelsummarizedevidenceforaMAPkinasesignalingpathwayinplants,whichparallels
self-defensemechanismsinanimalsassimpleas<em>C.elegans</em>andascomplexashumans.
Recentfindingsin<em>C.elegans</em>validatetheuseofthisorganismasamodelforstudying
innateimmunity.Moreimportantly,theseexperimentsaddweighttotheideathattheMAPkinase
cascadeis“themostancientandconservedcomponentininnateimmunity,”Dr.Ausubel
said.";"Symposium2002:St.Thomas,U.S.VirginIslands";"";publish;open;open;"";symposium-2002st-thomas-u-s-virgin-islands;"";"";2013-02-1415:39:03;2013-02-14
15:39:03;"";0;http://armeniseharvard.sypdevelopment.com/?p=258;0;post;"";0