Quantitative proteomics for identifying biomarkers for tuberculous meningitis

Kumar, Venugopal, Mahadevan Anita, Renuse Santosh, Harsha, Sahasrabuddhe, Pawar Harsh, Sharma Rakesh, Kumar Praveen, Rajagopalan Sudha, Waddell Keith, Ramachandra, Satishchandra Parthasarathy, Chaerkady Raghothama, Prasad, Shankar K, Pandey Akhilesh

Description

Tuberculous meningitis is a frequent extrapulmonary disease caused by Mycobacterium tuberculosis and is associated with high mortality rates and severe neurological sequelae. In an earlier study employing DNA microarrays, we had identified genes that were differentially expressed at the transcript level in human brain tissue from cases of tuberculous meningitis. In the current study, we used a quantitative proteomics approach to discover protein biomarkers for tuberculous meningitis. Methods To compare brain tissues from confirmed cased of tuberculous meningitis with uninfected brain tissue, we carried out quantitative protein expression profiling using iTRAQ labeling and LC-MS/MS analysis of SCX fractionated peptides on Agilent’s accurate mass QTOF mass spectrometer. Results and conclusions Through this approach, we identified both known and novel differentially regulated molecules. Those described previously included signal-regulatory protein alpha (SIRPA) and protein disulfide isomerase family A, member 6 (PDIA6), which have been shown to be overexpressed at the mRNA level in tuberculous meningitis. The novel overexpressed proteins identified in our study included amphiphysin (AMPH) and neurofascin (NFASC) while ferritin light chain (FTL) was found to be downregulated in TBM. We validated amphiphysin, neurofascin and ferritin light chain using immunohistochemistry which confirmed their differential expression in tuberculous meningitis. Overall, our data provides insights into the host response in tuberculous meningitis at the molecular level in addition to providing candidate diagnostic biomarkers for tuberculous meningitis.

Sujets

• Cerebrospinal fluid
• Histopathology
• Tuberculosis

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Kumar
etal.ClinicalProteomics
2012,
9
:12
http://www.clinicalproteomicsjournal.com/content/9/1/12

RESEARCH

CPLIRNOICTAELMOICSOpenAccess

Quantitativeproteomicsforidentifying
biomarkersfortuberculousmeningitis
GhantasalaSSameerKumar
1,2
,AbhilashKVenugopal
1,2,3,4
,AnitaMahadevan
5
,SantoshRenuse
1,3,4,6
,HCHarsha
1
,
NandiniASahasrabuddhe
1,7
,HarshPawar
1,8
,RakeshSharma
9
,PraveenKumar
1
,SudhaRajagopalan
10
,
KeithWaddell
11
,YarappaLRamachandra
2
,ParthasarathySatishchandra
12
,RaghothamaChaerkady
1,2,3,4
,
TSKeshavaPrasad
1,6,7,13
,KShankar
5*
andAkhileshPandey
1,3,4,14,15*

Abstract
Introduction:
Tuberculousmeningitisisafrequentextrapulmonarydiseasecausedby
Mycobacteriumtuberculosis
andisassociatedwithhighmortalityratesandsevereneurologicalsequelae.InanearlierstudyemployingDNA
microarrays,wehadidentifiedgenesthatweredifferentiallyexpressedatthetranscriptlevelinhumanbraintissue
fromcasesoftuberculousmeningitis.Inthecurrentstudy,weusedaquantitativeproteomicsapproachtodiscover
proteinbiomarkersfortuberculousmeningitis.
Methods:
Tocomparebraintissuesfromconfirmedcasedoftuberculousmeningitiswithuninfectedbraintissue,
wecarriedoutquantitativeproteinexpressionprofilingusingiTRAQlabelingandLC-MS/MSanalysisofSCX
fractionatedpeptidesonAgilent
’
saccuratemassQTOFmassspectrometer.
Resultsandconclusions:
Throughthisapproach,weidentifiedbothknownandnoveldifferentiallyregulated
molecules.Thosedescribedpreviouslyincludedsignal-regulatoryproteinalpha(SIRPA)andproteindisulfide
isomerasefamilyA,member6(PDIA6),whichhavebeenshowntobeoverexpressedatthemRNAlevelin
tuberculousmeningitis.Thenoveloverexpressedproteinsidentifiedinourstudyincludedamphiphysin(AMPH)and
neurofascin(NFASC)whileferritinlightchain(FTL)wasfoundtobedownregulatedinTBM.Wevalidated
amphiphysin,neurofascinandferritinlightchainusingimmunohistochemistrywhichconfirmedtheirdifferential
expressionintuberculousmeningitis.Overall,ourdataprovidesinsightsintothehostresponseintuberculous
meningitisatthemolecularlevelinadditiontoprovidingcandidatediagnosticbiomarkersfortuberculous
meningitis.
Keywords:
Relativequantitation,Cerebrospinalfluid,Histopathology,Earlydiagnosis,Tuberculosis

Introduction
HealthOrganization(
WHO
)hasestimatedthatone
Tuberculosis(TB)isacommonandsometimesfatalmillionchildrendevelopTBannuallyworldwidewhich
transmissibledisease,especiallyindevelopingcountries.accountsforabout11%ofallTBcases[1].Tuberculous
Approximatelythirtypercentoftheglobalpopulationisbacillimostcommonlyinfectlungs.
Mycobacterium
exposedtotheacid-fastbacillicausingTB.Ofthosewho
tuberculosis
(MTB)mayalsospreadtoextrapulmonary
areinfectedwithtuberculosis,~10%percentdevelopasitesincludingthemeninges,lymphnodes,genitourinary
clinicalmanifestationofthediseaseduringtheirlifetime.tract,skeletalsystemandskin[2].Tuberculousmeningi-
Fromaglobalperspective,approximatelytwentypercenttis(TBM)istheinfectionofmeningescausedbyMTB,
ofTBinfectedpopulationliveinIndia.TheWorldwithamortalityrateof~30%.Further,thosewhosurvive
TBMareusuallyleftwithsevereneurologicaldefects
*Correspondence:[e-mail];[e-mail]
[3-5].ThereisanincreasedriskofTBMinHIV-infected
5
DepartmentofNeuropathology,NationalInstituteofMentalHealthand
patientsascomparedtonon-HIVinfectedcasesalthough
Neurosciences,Bangalore560029,India
1
InstituteofBioinformatics,InternationalTechnologyPark,Bangalore560066,
theclinicalmanifestationsofthediseasedonotdiffer
India
betweenthetwogroups[6,7].
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Kumaretal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

Kumar
etal.ClinicalProteomics
2012,
9
:12
http://www.clinicalproteomicsjournal.com/content/9/1/12

Culturingmycobacteriaandsubsequentmicrobio-
logicalexaminationisconsideredagoldstandardforthe
diagnosisofTBM.However,thismethodistimecon-
sumingandinsensitive,withapositiveoutcomeachieved
onlyin25
–
70%ofclinicallydiagnosedcases[8].Al-
thoughPCRassayscanbeanalternativerapidapproach
fordiagnosis,theycannotdifferentiatebetweenlatentor
activeformsofthedisease.Althoughnucleicacidampli-
ficationtest(NAAT)hasahighspecificitywhentested
inbodyfluids,itlacksadequatesensitivityincasesof
meningitisandpleuritis[9].
Amongsttheexistingmolecularmarkers,Adenosine
deaminaseisoenzyme-2(ADA2)hasasensitivityof100%
andaspecificityof86.4%forthedetectionofTBMin
cerebrospinalfluid(CSF)[10].Adenosinedeaminase
(ADA)activityintheCSFofTBMpatientshasbeensug-
gestedtobeusefulforearlydifferentialdiagnosisofTBM
[11].TheADAactivityofCSFandplasmahavebeen
evaluatedasadiagnosticaidinTBM[12]andADA
activityinCSFwasconsideredtobeasimple,usefuland
rapiddiagnostictestforearlyrecognitionofTBMin
children[13].However,overexpressionofADAwasalso
oftenoverexpressedinotherformsofmeningitisinclud-
inginfectionswithpyogenicbacteria[14].IntheCSFof
TBMpatients,thepresenceof65kDaheatshockprotein
antigenmightbeamarkerforearlydiagnosisofthedis-
ease[15].HighlevelsofCSFlactateandlactatedehydro-
genaselevelshavealsobeensuggestedfordiagnosing
TBM[16].
EarlydiagnosisofTBMisconsideredakeytoeffective
treatmentandprognosis.Approximately90%ofthe
patientsarediagnosedinstageIIorIII[17].Overall,the
diagnosisofTBMstillremainsamajorchallengedueto
inadequatecurrentdiagnosticmethodsandpoorsensi-
tivityand/orspecificityofexistingmarkers.Although
corticosteroidsareusedextensivelytoreducemortality
andneurologicaldisability,itmaynotbetheonlysolu-
tiontoreducethemortalityandmorbidity[18].InTBM,
anumberofpathologicalchangesincludingmeningeal
adhesion,infarction,tuberculomaandhydrocephalusmay
occurleadingtoneurologicalsequelae[4].Thesesequelae
areknowntocorrelatewiththestageofmeningitis
atadmission.Patientstreatedatanearlystagehavea
fivetimeshigherchanceofrecoverythanthosewith
advanceddiseasestages[19].Therefore,patient
’
sclinical
conditionatadmissionanddelayinstartingthetreat-
mentareimportantfactorsfordeterminingtheirsurvival
[20].Thesefindingsemphasizetheneedtofocusoniden-
tifyingcandidatemolecularmarkerswhichcanbedevel-
opedasdiagnostictoolsinthemanagementofTBM.
Massspectrometry-basedquantitativeproteomicshas
emergedasapowerfulapproachforidentifyingand
studyingdiseasebiomarkersandhasbecomeoneofthe
essentialtoolsinbiomarkerdiscovery[21,22].Advances

Page2of13

inquantitativemassspectrometryhaveledtoidentifica-
tionandquantitationofbiomarkerswhichserveas
indicatorsofdiseaseprogression,prognosis,drugsafety
andhelptoelucidatethemechanismofdrugtreatment
[23].Therearevariouslabelingapproachesthatone
canemploytocarryoutquantitativeproteomicmea-
surements.
Invitro
labelingmethodsincludeIsobaric
TagsforRelativeandAbsoluteQuantitation(iTRAQ),
Isotope-CodedAffinityTags(ICAT),
18
Olabelingand
invivo
methodsincludeStableIsotopeLabelingby
15AminoacidsinCellculture(SILAC)andNlabeling
[24,25].iTRAQlabelingisaneffectivemethodfor
studyingdifferentialproteinexpressionlevelsintissue
samples.Ithasbeenextensivelyusedforbiomarker
discoveryinvariousdiseasecontexts[26-34].
Inthisstudy,weusedaniTRAQ-basedquantitative
proteomicapproachtoidentifydifferentiallyexpressed
proteinsfrombraintissuesoftuberculousmeningitis
casesascomparedtocontrols.Weidentifiedseveralpro-
teinswhicharedifferentiallyexpressedinTBM.These
proteinsincludebothnovelandpreviouslyreportedcan-
didateproteinmarkers.Wevalidatedsomeofthesecan-
didatebiomarkersusingimmunohistochemicallabeling.
Materialsandmethods
Samplecollection
Thestudywasapprovedbyscientificethicscommittee
ofNationalInstituteofMentalHealthandNeuro
Sciences(NIMHANS),Bangalore,India.Samplesfrom
frontalcortexwithoverlyingmeningesfromcasesoftu-
berculousmeningitis(n=6)andsimilar,butuninfected,
controlbraintissues(n=6)fromvictimsofroadtraffic
accidentswerecollectedatthetimeofautopsy.The
autopsywasconductedwithin8
–
16hpostmortemwith
thebodykeptat4°Cafterdeath.Thesampleswere
obtainedfromtheHumanBrainTissueRepository,
DepartmentofNeuropathology,NIMHANS,Bangalore.
Sampledetailsincludingtheselectioncriteriaarepro-
videdinAdditionalfile1:TableS1.
SamplepreparationandiTRAQlabeling
Braintissuesampleswerelysedin0.5%SDS,sonicated,
homogenizedandcentrifugedat13,000rpmfor10min
at4°C.Supernatantwascollectedandproteinquantita-
tionwascarriedoutbyLowry
’
sassay(Bio-RadHercules,
CA;USA).Foreachcondition,160
μ
gofproteinsample
wasutilizedfortheexperiment.Eachsamplewastreated
with4
μ
Lofreducingagent(tris(2-carboxyethyl)phos-
phine(TCEP))at60°Cfor1handalkylatedwith2
μ
Lof
cysteineblockingreagent,methylmethanethiosulfonate
(MMTS)for10minatroomtemperature.Afteralkyl-
ation,thesamplesweresubjectedtotrypsindigestion
(SequencingGradeModifiedTrypsin,PromegaCat#:

Kumar