The ESAT-6 Protein of Mycobacterium tuberculosis

19 Pages·2014·3.73 MB·English

The ESAT-6 Protein of Mycobacteriumtuberculosis
Interacts with Beta-2-Microglobulin (b2M) Affecting
Antigen Presentation Function of Macrophage
Gopalkrishna Sreejit1., Asma Ahmed1., Nazia Parveen1, Vishwanath Jha1, Vijaya Lakshmi Valluri2,
Sudip Ghosh3, Sangita Mukhopadhyay1*
1LaboratoryofMolecularCellBiology,CentreforDNAFingerprintingandDiagnostics(CDFD),Nampally,Hyderabad,India,2DivisionofImmunologyandMolecular
Biology,LEPRASociety-BluePeterResearchCentre,Hyderabad,India,3MolecularBiologyUnit,NationalInstituteofNutrition(ICMR),Jamai-OsmaniaHyderabad,India
Abstract
ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor,
inactivationofwhichleadstoreducedvirulenceofM.tuberculosis.ESAT-6alone,orincomplexwithitschaperoneCFP-10
(ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well
understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding
activities.Therefore,wehypothesizedthatthecrucialroleplayedbyESAT-6inthevirulenceofmycobacteriacouldbedue
toitsinteractionwithsomehostcellularfactors.Usingayeasttwo-hybridscreening,weidentifiedthatESAT-6interactswith
the host protein beta-2-microglobulin (b2M), which was further confirmed by other assays, like GST pull down, co-
immunoprecipitationandsurfaceplasmonresonance.TheC-terminalsixaminoacidresidues(90–95)ofESAT-6werefound
to beessentialforthis interaction. ESAT-6,incomplex with CFP-10,alsointeracts with b2M. WefoundthatESAT-6/ESAT-
6:CFP-10canenterintotheendoplasmicreticulumwhereitsequestersb2MtoinhibitcellsurfaceexpressionofMHC-I-b2M
complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:b2M complex
could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel
mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful
infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective
vaccinedesign against tuberculosis.
Citation:SreejitG,AhmedA,ParveenN,JhaV,ValluriVL,etal.(2014)TheESAT-6ProteinofMycobacteriumtuberculosisInteractswithBeta-2-Microglobulin(b2M)
AffectingAntigenPresentationFunctionofMacrophage.PLoSPathog10(10):e1004446.doi:10.1371/journal.ppat.1004446
Editor:DavidM.Lewinsohn,PortlandVAMedicalCenter,OregonHealthandScienceUniversity,UnitedStatesofAmerica
ReceivedAugust1,2013;AcceptedSeptember4,2014;PublishedOctober30,2014
Copyright: (cid:2) 2014 Sreejit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisstudywasfundedbyDepartmentofBiotechnology,GovernmentofIndia(BT/01/COE/07/02andBT/PR12854/BRB/10/730/2009)andacoregrant
fromCDFD.GS,NPandVJarerecipientsofJuniorandSeniorResearchFellowshipsoftheCouncilofScientificandIndustrialResearch,GovtofIndiatowardthe
pursuitofaPh.D.degreeoftheManipalUniversity.AAissupportedbyaResearchAssociateFellowshipfromtheDepartmentofBiotechnology,GovtofIndia.The
fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected],[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction secretion pathway, M. tuberculosis possesses the ESX secretion
system,a Type VIIsecretion system(T7SS) whichisfoundtobe
Tuberculosis (TB) has remained a major cause of death non-essential for growth in vitro, but is required for bacterial
worldwide despite the availability of a vaccine and several virulence [4]. Mycobacteria possess at least five ESX secretion
chemotherapeutic agents [1]. Global emergence of multi-drug systems,namelyESX-1throughESX-5[5].Thefirstandthemost
resistantstrainsofMycobacteriumtuberculosisbesidessynergywith characterizedmemberoftheESXsecretionsystemfamily,ESX-1
HIV [1] warrants a better understanding of the host-M. is encoded by region of difference (RD) 1 which is absent in all
tuberculosis interaction in order to develop effective strategies for vaccine strains of avirulent M. bovis BCG, but present in the
containing the disease. Successful infection of macrophages by virulent laboratory and clinical strains of M. bovis and M.
pathogenic mycobacteria involves modulation of several immune tuberculosis[5].TheRD1regionisalsoabsentinM.microtiwhich
functions,whichallowsthemtosurviveandpersistinsidethehost seldom causes disease in immunocompetent individuals [6].
[2]. Crucial host cell functions required for development of anti- Interestingly, deletion of RD1 results in attenuation of M.
mycobacterialimmunitylikephagosome-lysosomefusion,autoph- tuberculosis and it is hence implicated to play an important role
agy, antigen presentation, apoptosis and bactericidal innate in mycobacterial pathogenicity [4,7]. RD1 complemented aviru-
immune responses are known to be inhibited by the pathogenic lent M. bovis BCG and M. microti strains were shown to grow
mycobacteria [2]. Particularly, the proteins secreted by virulent better in severe combined immunodeficient mice and persist
mycobacteria are found to play important roles in modulation of longerinorgansofimmunocompetentmicecorroboratingthefact
hostimmuneresponses[3].InadditiontotheclassicalSecandTat that deletion of RD1 resulted in attenuation of these strains [8].
PLOSPathogens | www.plospathogens.org 1 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
and lyse the phagosomal membrane [20]. Therefore, it appears
Author Summary
that the ESAT-6:CFP-10 complex provides the bacteria or its
M. tuberculosis is a dangerous and highly successful components the tools necessary to escape from the phagosomal
pathogen that has evolved several mechanisms to compartments to interact with the host cellular proteins and
manipulatethehostimmuneregulatorynetwork.Proteins modulatetheirfunctions[20].Basedonthesolutionstructure,no
secreted by M. tuberculosis play important roles in DNA binding or enzymatic function could be ascribed to the
virulence. One such protein is ESAT-6, which is secreted ESAT-6:CFP-10 complex[14] as the surface of the complex was
alongwithitschaperoneCFP-10.Despiteahostofstudies
foundtobedevoidofanyacidicorbasicpatchescharacteristicof
highlightingmodulationofimmuneresponsesbyESAT-6,
DNA-binding proteins and many transcription factors. Similarly,
there have not been many that identified host proteins
absence of significant clefts on the surface of the structure,
interactingwithESAT-6.Wehavenowfoundthatthehost
indicativeofanenzymeactivesite,suggestsanon-catalyticrolefor
proteinb2MinteractsveryspecificallywithESAT-6atitsC-
this complex. Therefore, we hypothesized that the crucial role
terminal region. The soluble ESAT-6:CFP-10 complex was
playedbyESAT-6inthevirulenceofmycobacteriacouldbedue
foundtobetraffickedintotheendoplasmicreticulum,and
toitsinteractionwithsomehostcellularfactors,theidentification
treatment with recombinant ESAT-6:CFP-10 or the over-
expression of ESAT-6 reduced cell surface expression of ofwhichislikelytoprovidebetterunderstandingoftheroleplayed
b2M and molecules which remain associated with it like byESAT-6inmodulationofhostimmuneresponses.Accordingly,
HLA-I. Recombinant ESAT-6:CFP-10 was also found to the aim of the present study was to identify ESAT-6 interacting
reduceclassicalandcrosspresentationofpeptideantigens partners of host origin by employing a yeast two hybrid assay
by MHC-I molecules. In summary, our data indicate that system.Here,wereportthatESAT-6canspecificallyinteractwith
interactionbetweenESAT-6andb2Mcanreducethelevels beta-2-microglobulin(b2M),asmall,13kDaproteinfoundtobe
of available free b2M that associate with HLA/MHC-I associated with major histocompatibility complex (MHC) class I
molecules. This could be an interesting mechanism by family of proteins that are involved in antigen presentation and
whichM.tuberculosisinhibitsclassicalandcrosspresenta- iron regulation. We found that ESAT-6 was able to enter the
tion of peptide antigens in order to prevent or delay the endoplasmic reticulum (ER), where it interacts with b2M and
onset ofanti-mycobacterial adaptive immuneresponses.
inhibits its association with MHC-I molecules leading to reduced
surface expression of MHC-I-b2M complex and consequently
MutantsdefectiveforESX-1secretionsystemarefoundtoexhibit inhibits loading of antigen-derived peptides to the MHC-I
a range of diverse phenotypes, including defects in immune complex. Data presented in this study highlight the existence of
modulation, tissue invasion, phagosomal trafficking and growth a novel mechanism by which M. tuberculosis ESAT-6 protein
insidethemacrophages [4,9,10]. exerts its virulence by undermining the host adaptive immune
InM.tuberculosis,theearlysecretoryantigenictarget(ESAT)-6 responsestoestablishasuccessfulinfectionandthesefindingsmay
orRv3875andtheculturefiltrateprotein(CFP)-10orRv3874are aid in the development of novel therapeutics against the deadly
abundantly produced and secreted in culture. They are also disease.
knowntobethemostimmunogenicmoleculesfoundintheculture
filtrate [11,12]. These proteins lack the classical Sec YEG Results
secretory signal sequences and their secretion was found to be
dependentonESX-1[4,8,10].ESAT-6andCFP-10togetherform ESAT-6 interacts with human b2M
a tight dimer and are dependent on each other for their stability Wecarriedoutyeasttwo-hybrid(Y2H)screeningtoidentifythe
and secretion [9,13,14]. Along with the ESX-1 secretion system, probable ESAT-6 interacting proteins from the host. ESAT-6
ESAT-6 and CFP-10 have been implicated in several virulence cloned in the bait vector pGBKT7 was used to screen a human
mechanisms of mycobacteria. They are capable of modulating leukocytecDNAlibraryclonedintothepreyvectorpACT2.The
both innate and adaptive immune responses and inactivation of suitabilityofESAT-6foruseasbaitwasconfirmedbymeasuring
ESAT-6 results in dramatically reduced virulence of M. tubercu- its expression as GAL4-ESAT-6 fusion protein and also by
losis [4,8,9]. Interestingly, specific mutations in ESAT-6 that did assessingthetoxicityofthefusionproteinaswellasauto-activation
notaffectfunctioningoftheESX-1secretionsystemcausedaloss of interaction markers. Yeast strain AH109 expressing the Gal4-
of virulence of mycobacteria [13,15]. This implies that ESAT-6 ESAT-6 fusion protein had normal growth kinetics and auto-
itselfiscrucialinmediatingthevirulencefunctionsassociatedwith activation of the reporter genes was not observed in these
the ESX-1 secretion system. ESAT-6 was found to induce transformants.ForY2Hscreening,Mat-astrainAH109harboring
apoptosis of macrophages by activating caspase expression [16]. the bait vector pGBKT7-ESAT-6 was mated with Mat-a strain
ESAT-6, CFP-10 and the ESAT6:CFP-10 complex can inhibit Y187 transformed with prey library plasmid and the mating
LPS-inducedNF-kappaBdependentgeneexpressionbysuppress- mixturewasplatedonQDOplates(SD/–Ade/–His/–Leu/–Trp)
ingproductionofreactiveoxygenspecies[17].ESAT-6aloneorin for high stringency of selection. The prey plasmids, rescued from
complex with CFP-10 has also been shown to interact with host the colonies that appeared on selection plates, were sequenced
proteins like laminin on the basolateral surface of pneumocytes using 39 AD Sequencing Primer and were identified by querying
leading to lysis of these cells that aid in the dissemination of M. these sequences against the NCBI GenBank database using the
tuberculosisinthehumanlung[18].Moreover,ESAT-6hasbeen MegaBlast program. One of these cDNA sequences in the prey
showntointeractdirectlywithtolllikereceptor(TLR)2resulting plasmidwasfoundtohaveveryhighsimilaritywithhumanbeta-2-
in reduced interleukin (IL)-12 p40 secretion in macrophages, microglobulin (Figure S1). The interaction was found to be truly
probablyfavouringaThelper2phenotypethathelpsintracellular positive as the prey plasmid carrying the b2M cDNA sequence
persistence and survival of M. tuberculosis [19]. However, the (pACT2-b2M)failedtogrowinthepresenceofemptybaitvector
exact mechanismof virulenceof ESAT-6isnot well understood. pGBKT7 alone (Figure 1A).
IthasbeenshownthatatlowerpH,characteristicoftheacidic The physical interaction between ESAT-6 and b2M was
phagosomal environment, the secreted ESAT-6:CFP-10 complex confirmed by a co-purification assay. ESAT-6 was cloned into
candissociateandoncedissociated,ESAT-6alonecandestabilize the first multiple cloning site (MCS) with an N-terminal His-tag
PLOSPathogens | www.plospathogens.org 2 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure1.ESAT-6proteininteractswithb2M.(A)InteractionbetweenESAT-6andb2Minayeasttwo-hybridsystemwasstudiedbymatingyeast
strainAH1109transformedwithbaitplasmidpGBKT7-ESAT-6withyeaststrainY187transformedwithahumanleukocytecDNApreylibraryona
synthetic dropout plate (–Ade/–His/–Leu/–Trp). Prey cDNA was amplified by PCR using primers encompassing the cDNA insert in pACT2 and
sequenced and identified to be b2M. The AH109 yeast strain transformed with pGBKT7-ESAT-6 and pACT2-b2M shows Ade and His interaction
reporteractivationonQDOplates.AH109transformedwithpGBKT7andpACT2-b2MwasusedasanegativecontrolwhileAH109transformedwith
pGBKT7-p53andpGADT7-Twasusedasapositivecontrolfortheyeasttwo-hybridscreening.(B)Untaggedb2MwasclonedalongwithHis-tagged
ESAT-6inthepETDuetvectorsystemandwastransformedintoE.coliBL21cells.ThetransformedcultureswereinducedwithIPTGandtheover-
expressed proteins were purified using TALON resin. Purified proteins were separated on a 16% Tris-Tricine SDS-PAGE and visualized by silver
staining.Lane1isamolecularweightmarker.(C)b2MwithnoN-terminalsignalsequence(ESAT-6:b2MNSS)andthefulllengthb2MwiththeN-
terminalsignalsequence(ESAT-6:b2MWSS)wereclonedinpETDuetvectoralongwith66His-taggedESAT-6.Cloneswereover-expressedinE.coli
BL21. Protein expression was induced by addition of IPTG and over-expressed proteins were purified using metal affinity TALON resin. Purified
proteinswereseparatedon16%Tris-TricineSDS-PAGEandtransferredontonitrocellulosemembranesandprobedwithanti-b2MAbtodetectb2M
andanti-HisAbtodetectESAT-6.Thebandscorrespondingtob2M(upperpanel)andESAT-6(lowerpanel)werevisualizedbychemiluminescence.
(D)GSTpre-clearedTHP-1macrophageextractwasmixedandincubatedwithGlutathione-agarosebeadbound-GSTorGST-ESAT-6andwashed.The
boundproteinswereelutedandresolvedona16%Tris-TricineSDS-PAGEgelandimmunoblottedforb2Mproteinusingrabbitanti-humanb2MAb
andHRPconjugatedanti-rabbitAb.Blotswerevisualizedbychemiluminescence.WholecellextractsfromTHP-1cellswasusedaspositivecontrolfor
b2M expression. (E) In another experiment, purified recombinant 66His-tagged ESAT-6 was incubated with THP-1 macrophage lysate and
immunoprecipitated(IP)withanti-HisAbboundtoproteinA/Gagarosebeads.Controlimmunoprecipitationwascarriedoutwithouttheadditionof
His-taggedESAT-6protein(IPcontrol).Theelutedproteinmixtureisresolvedona16%Tris-TricineSDS-PAGEgelandimmunoblotted(IB)forb2M
proteinusingarabbitanti-humanb2MAbandHRPconjugatedanti-rabbitsecondaryAbandtheblotswerevisualizedbychemiluminescence.Lane1
isinputcontrol.(F)Directinteractionofb2MwithESAT-6wasmonitoredusingaBIACORE3000Biosensorwhereb2Mwasimmobilizedonthesensor
chipandrecombinantESAT-6atdifferentconcentrationswasinjectedintherunningbuffer.Thechangesintherefractionindexatthesurfacedueto
interactionsbetweenimmobilisedb2MandfluidphaseESAT-6weredetectedandrecordedasRU(ResonanceUnits).CurvesgeneratedfromtheRU
tracewereevaluatedusingacurve-fittingalgorithm.ESAT-6wasfoundtobindspecificallytob2Mandnobindingwasobservedinacontrolcell
whichdidnothaveanyimmobilizedb2M.Resultsarerepresentativeofthreedifferentexperiments.
doi:10.1371/journal.ppat.1004446.g001
andb2McDNAfromthepreyplasmidwasclonedintothesecond and b2M (Figure 1B, Lane 5). b2M is known to possess a 20
MCS without any tag in a dual expression vector pETDuet-1. amino acid endoplasmic reticulum (ER) localization sequence at
Whentheover-expressedHis-taggedESAT-6proteinwaspurified its N-terminal which is removed from the mature protein during
from the transformed Escherichia coli using TALON affinity post-translational modifications. Since the prey plasmid cDNA
binding resin, b2M was found to be co-purified along with His- encoding b2M contained the signal peptide sequence, any
taggedESAT-6,indicatingapositiveinteractionbetweenESAT-6 interactionwithESAT-6throughthisregionwouldbemostlikely
PLOSPathogens | www.plospathogens.org 3 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
physiologically irrelevant. Therefore, we examined whether demonstrate that anti-b2M antibody can immunoprecipitate the
ESAT-6 could actually interact with the mature b2M devoid of full length ESAT-6 (Figure 2C, Lane 3), but not the mutant
the signal peptide. The N-terminal signal sequence (20 amino ESAT-6DC (Figure2C, Lane 4), indicating that the C-terminal
acids)ofb2Mwasthereforetruncatedandco-expressedalongwith (90–95) residuesof ESAT-6areimportantforitsinteractionwith
His-tagged ESAT-6. It was found that the mature b2M could be b2M.Lane5ofFigure2CisnegativecontrolwhereProteinA/G
co-purified along withHis-tagged ESAT-6 (Figure1C) indicating beads were used to rule out any non-specific adsorption to the
that the N-terminal signal peptide of b2M is not involved in the beads.Lanes1and2ofFigure 2Careinputcontrolsshowingthe
interaction withESAT-6protein. presenceofb2M(lowerpanel)andESAT-6(Lane1,upperpanel)
We also examined whether ESAT-6 could interact with the or ESAT-6DC (Lane 2, upper panel) in the reaction mixture
b2M naturally present within mammalian cells. For this, GST- containingTHP-1lysateandHis-taggedESAT-6orESAT-6DC.
tagged ESAT-6 was used to pull down b2M from the whole cell ESAT-6ispredominantlysecretedbyM.tuberculosisasatight
extractpreparedfromPMA-differentiatedTHP-1macrophages.It 1:1 heterodimeric complex with CFP-10 through the ESX-1
was found that the GST-ESAT-6 fusion protein was able to pull secretionsystem[22].EarlierwefoundthattheC-terminalendof
downb2MwhileGSTalonefailedtodoso(Figure 1D)indicating ESAT-6 (amino acid residues 90–95) is involved in interaction
that ESAT-6 can physically interact with mature b2M naturally withb2M(Figure 2,A–C).SincetheC-terminalendofESAT-6in
expressedinmacrophages.Tofurtherconfirmthisobservation,a the ESAT-6:CFP-10 complex is essentially exposed [14] (Figure
co-immunoprecipitation (Co-IP) assay was carried out where S3),itispossiblethatESAT-6whileincomplexwithCFP-10can
recombinant His-tagged ESAT-6 protein was incubated with stillinteractwithb2M.Therefore,theabilityoftheESAT-6:CFP-
THP-1wholecelllysateandthenanti-Hisantibody(Ab)coupled 10 complex to interact with b2M was examined by a co-
to protein A/G beads was added to the mixture to immunopre- immunoprecipitation assay. Affinity purified His-tagged ESAT-
cipitate the interacting complex. The immunoprecipitated com- 6:CFP-10orESAT-6DC:CFP-10orCFP-10alonewasincubated
plex was separated by SDS-PAGE followed by immunoblotting withTHP-1macrophagelysate.Mouseanti-humanb2MAbwas
usinganti-humanb2MAb.Ab2Mspecificbandwasdetectablein then added to these mixtures and detection of b2M-bound His-
the His-tagged ESAT-6-immunoprecipitated complex indicating taggedESAT-6proteinwascarriedoutusingrabbitanti-HisAb.
that ESAT-6 interacts with b2M present in the THP-1 macro- We were able to detect a band corresponding to the size of His-
phages(Figure 1E).PhysicalinteractionofESAT-6andb2Mwas taggedESAT-6inthereactionmixturecontainingESAT-6:CFP-
furtherconfirmedbyusingsurfaceplasmonresonance(Figure 1F). 10(Figure 2D,Lane6),however,ESAT-6DC:CFP-10orCFP-10
The Kd value of the ESAT-6:b2M complex as determined from does not interact with b2M present in the THP-1 extract
thesurface plasmon resonance data was1.0361026M. (Figure 2D, Lanes 7 and 8). Similarly, ESAT-6:CFP-10 was also
found to interact with mouse b2M (Figure S4). These results
b2M interacts with the C-terminal extreme of ESAT-6 suggestthatESAT-6aloneorincomplexwithCFP-10iscapable
of interacting with b2M and the C-terminal six amino acid
The extreme C-terminus ofESAT-6 isa floppy, structurally ill
residues (90–95)of ESAT-6are crucialforthisinteraction.
definedregion,awayfromthehelicalcoreandisnotknowntobe
So far we observed that the C-terminal end of ESAT-6 was
involved in the interaction with CFP-10 [14]. Mutant M.
involvedininteractionwithb2M.Sinceb2Misanintegralpartof
tuberculosis expressing a truncated ESAT-6 having residues
thefunctionalMHCclassImolecules,wenextexaminedwhether
deleted from the C-terminal end was found to be attenuated.
ESAT-6 can interact with HLA-bound b2M also. Therefore,
However, these mutant strains had a functional ESX-1 system
PMA-differentiated THP-1 macrophage lysate was mixed and
with normal secretion of the ESAT-6:CFP-10 complex [13,15]
incubated with either recombinant ESAT-6 or ESAT-6:CFP-10
indicating thattheresiduesat theC-terminal endofESAT-6are
andtheinteractingcomplexeswerepulleddownusingmouseanti-
notrequiredforastructuralroleintheformationofaheterodimer
humanHLAclassIandprobedwithanti-Hisaswellasanti-b2M
with CFP-10 but crucial for the virulence functions of ESAT-6.
antibodies. It was found that b2M but not ESAT-6 could be
Therefore, we speculated that the free C-terminus of ESAT-6 is
probably involved in the interaction with b2M and such an immunoprecipitated along with HLA-I (Figure S5). These results
indicatethatESAT-6bindsonlytothefreeb2M,butnottothat
interaction is possibly critical for M. tuberculosis virulence [21].
already incomplexwith HLA-Iheavy chain.
Once again using the yeast two hybrid assay, we observed that
deletion of the last 6 amino acids (valine, threonine, glycine,
methionine, phenylalanine and alanine) from the C-terminal end ESAT-6:b2M interaction is not affected by high salt
ofESAT-6(FigureS2)wassufficienttopreventtheinteractionof concentrations and pH
ESAT-6 and b2M (Figure 2A). To further validate the yeast two Interestingly, the interaction between ESAT-6 and b2M was
hybrid data, we used a C-terminally truncated ESAT-6 (ESAT- found to be significantly stable at high ionic concentrations. The
6DC) to carry out GST pull down assay. The GST-tagged full b2M in the ESAT-6:b2M complex immobilized to talon beads
lengthESAT-6protein(ESAT-6-GST)aswellastheGST-tagged failedtodissociatefromtheHis-taggedESAT-6proteinevenafter
ESAT-6DC(ESAT-6DC-GST)proteinwereincubatedwithTHP- washingwithbufferscontainingNaClashighas2 M(Figure 3A).
1 extract and the pulled down fractions were probed for the StabilityoftheESAT-6:b2Mcomplexinthepresenceofhighsalt
presence of b2M using anti-b2M Ab. It was found that only the andtheneutralnatureoftheamino-acidsindicatethattheESAT-
full length ESAT-6 protein was able to interact with b2M 6:b2M complex is stabilized by hydrophobic rather than ionic
(Figure 2B).Tofurtherconfirmourobservations,wecarriedouta interactions.
Co-IPassaywhereTHP-1lysateswereincubatedwithfulllength By changing the protonation state of the charged residues, the
His-tagged ESAT-6 or His-tagged ESAT-6DC and b2M-com- pH can significantly influence the nature of protein-protein
plexes were immunoprecipitated using anti-b2M Ab. The interactions. pH can considerably dictate the strength and
immunoprecipitated complexes were resolved on SDS-PAGE geometry of electrostatic interactions, conformation of the
andprobedwitheitheranti-HisAbtodetectESAT-6/ESAT-6DC interactingproteins,aswellasproteinhydrationwhicharecritical
(Figure 2C, upper panel) or anti-b2M Ab to detect b2M in stabilization of protein-protein interactions. Therefore, the
(Figure 2C, lower panel). The data shown in Figure 2C clearly effect of pH was examined on the stability of ESAT-6:b2M
PLOSPathogens | www.plospathogens.org 4 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure2.ESAT-6withadeletionofsixC-terminalaminoacids(ESAT-6DC)failstointeractwithb2M.(A)YeaststrainAH109transformed
withESAT-6/ESAT-6DCbaitalongwithb2MpreyplasmidwerestreakedoninteractionselectionQDOplates(SD/–Ade/–His/–Leu/–Trp)alongwith
thecontrolsandmonitoredthegrowthresultingfrompositiveinteractions.(B)ESAT-6orESAT-6DCexpressedasGSTfusionproteinwasincubated
withTHP-1lysateandprecipitatedusingglutathione-agarose.Presenceofb2MintheprecipitatedcomplexeswasdetectedbyWesternblotting
usinganti-b2MAb.(C)RecombinantHis-taggedESAT-6orHis-taggedESAT-6DCproteinwasmixedandincubatedwithTHP-1cellextractsandb2M
was immunoprecipitated using mouse anti-human b2M Ab and protein A/G beads. Presence of ESAT-6/ESAT-6DC and b2M in the
immunoprecipitatedcomplexesweredetectedbyWesternblottingusingrabbitanti-HisAbandrabbitanti-humanb2MAbrespectively.Lanes1
and 2 are inputcontrols. (D) THP-1macrophage wholecell extract was mixed and incubatedwith recombinant ESAT-6:CFP-10 or mutantESAT-
6DC:CFP-10orCFP-10protein.b2MwasimmunoprecipitatedusingproteinA/Gboundmouseanti-humanb2MAbandtheimmunecomplexeswere
subjected to Western blotting using rabbit anti-His Ab to detect ESAT-6:CFP-10 or mutant ESAT-6DC:CFP-10 or CFP-10. The lanes 1–3 are input
controlsfortheserecombinantproteins(upperpanel).Inthelowerpanel,thelanes1–3indicatethelevelsofb2Mintheinputcontrolsasdetermined
byWesternblottingusingrabbitanti-humanb2MAb.
doi:10.1371/journal.ppat.1004446.g002
complex.ThestabilityoftheESAT-6:b2McomplexatvariouspH iological role in the host-pathogen interaction, ESAT-6 and/or
was confirmed by glutaraldehyde cross-linking of ESAT-6 and ESAT-6:CFP-10 must find its way to the cellular compartments
b2M.Covalent cross-linkingoftwoproteinsisusuallyconsidered where b2M is present. b2M is known to be present in high
to be a convincing evidence of their physical proximity and concentration within endoplasmic reticulum (ER) where it is
interaction. Glutaraldehyde was found to covalently cross-link synthesized and undergoes necessary post-translational modifica-
ESAT-6 and b2M at pH 4.0, 5.0, 6.0 and 8.0. The covalently tionsandassociateswiththealphachainoftheMHC-I,aswellas
cross-linked ESAT-6:b2M protein complex was detectable as a otherclassIlikemoleculeslikeCD1andhemochromatosisprotein
,24kDa band on a denaturing SDS gel (Figure 3B). These (HFE) that are transported to the cell surface via the golgi
observations further confirm the stability of the ESAT-6:b2M apparatus [23,24].
complex acrossa wide pH range. Interestingly,ESAT-6and/orESAT-6:CFP-10complexsecret-
ed by the M. tuberculosis bacilli in the phagosome or cytoplasm
ESAT-6 or ESAT-6:CFP-10 is trafficked into the ER canmakeitswayoutbylysingthecellmembranes[25,26]which
The functional implications of protein-protein interactions can be taken up by the surrounding macrophages and dendritic
dependonthespatialandtemporallocalizationoftheinteracting cells. Another possible source of soluble ESAT-6 and/or ESAT-
partnerswithinthecell.Therefore,inordertohaveapathophys- 6:CFP-10 is mycobacteria surviving in the extracellular milieu of
PLOSPathogens | www.plospathogens.org 5 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure3.TheESAT-6:b2McomplexisstableathighsaltconcentrationsandlowpH.(A)Cobaltagarosebead-boundESAT-6:b2Mcomplex
waswashedwithincreasingconcentrationof0.5MNaCl(Lane2),1MNacl(Lane3)and2MNaCl(Lane4).Proteinsboundtothesebeadswere
elutedbyboilingin16PAGEloadingdyeandresolvedona16%Tris-TricineSDS-PAGEgelandvisualizedbyCoomassiebluestaining.Lane1shows
themolecularweightmarker.(B)PurifiedESAT-6:b2MproteincomplexwasdialyzedinacetatebuffersofvaryingpHlikepH4.0(Lanes,2,6and10),
pH5.0(Lanes,3,7and11),pH6(Lanes,4,8and12)andpH8.0(Lanes,5,9and13).Afterdialysis,theproteincomplexeswerecross-linkedby
glutaraldehydetreatmentfor15,20and30minutesat37uCandresolvedona16%Tris-TricineSDS-PAGEgelandvisualizedbysilverstaining.Protein
molecularweightmarkerwasloadedinLane1.Resultsarerepresentativeofthreedifferentexperiments.
doi:10.1371/journal.ppat.1004446.g003
caseating granulomas where the proteins secreted by the Similarly,FITC-labelledESAT-6alonewasfoundtobepresentin
extracellularbacilliareavailableforuptakebyantigenpresenting the ER-tracker dye-positive regions of KG-1 dendritic like cells
cells present in these granulomas [27]. Interestingly, studies have (Figure 4C). FITC-labelled ESAT-6:CFP-10 was incubated with
indicated that soluble proteins can gain direct access to the ER THP-1 macrophages at 4uC as a negative control forinternalisa-
lumen of dendritic cells [28]. Therefore, soluble ESAT-6 and/or tion(Figure 4A).TheseobservationssuggestthatsolubleESAT-6
ESAT-6:CFP-10takenupbymacrophagesanddendriticcellsmay andESAT-6:CFP-10cantranslocateintotheERlumenfromthe
beretrotranslocateddirectlytotheERofthesecellswhichwould extracellularmilieutointeractwiththeresidentb2MinKG-1cells
enable it to interact with b2M and modulate b2M-dependent andmacrophages.Fromtheconfocalmicroscopydata,itappears
responses. To test this, THP-1 macrophages were incubated for thatESAT-6:CFP-10isabletoenterthevesiclesofthephagocytic
2 hourswithFITC-labelledESAT-6orESAT-6:CFP-10andtheir pathwayaswellasothercellularcompartmentssincealltheFITC
localization was tracked along with the ER-specific marker signalemanatingfromlabelledESAT-6:CFP-10didnotsuperim-
calnexinbyconfocalmicroscopy.TheESAT-6:CFP-10wasfound pose with the signal emanating from ER-specific markers
to be present in the ER (Figure 4B). Similarly, FITC-labelled (Figure 4). However, as is evident from the data, sufficient
ESAT-6:CFP-10complexwasalsofoundtobepresentwithinthe quantities of ESAT-6/ESAT-6:CFP-10 are able to translocate to
ER-tracker dye-positive regions in THP-1 macrophages (Fig- theERwherethecomplexisavailableforpossibleinteractionwith
ure 4D) as well as in mouse peritoneal macrophages (Figure 4E). b2M.
PLOSPathogens | www.plospathogens.org 6 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
PLOSPathogens | www.plospathogens.org 7 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure 4. Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER. PMA-differentiated THP-1 macrophages were
incubatedwithFITC-labelledESAT-6:CFP-10(green)eitherat4uC(A)or37uC(B)forabout120minutes.Cellswerethenwashed,fixed,permeabilised
andstainedforanERmarkercalnexinusingrabbitanti-calnexinAbandAlexaFluor594conjugatedanti-rabbitsecondaryAb(red).Inanothersetof
experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal
macrophages(E)weretreatedwithFITC-labelledESAT-6orESAT-6:CFP-10(green)forabout100minutesat37uCandthenincubatedwithER-Tracker
dye (blue) for another 20minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different
experiments.
doi:10.1371/journal.ppat.1004446.g004
ESAT-6 or ESAT-6:CFP-10 inhibits surface expression of theER(Figure 5D).ThesedatasuggestthatintracellularESAT-6
b2M isnotonlyabletoentertheERbutalsocaninteractwiththeER-
IntheERlumen,b2MassociateswithMHCclassImolecules resident b2M. We next examined, whether such interaction of
ESAT-6withb2MinERhasanyeffectonthesurfaceb2Mlevels.
toformclassIcomplexeswhicharethenloadedwithpeptideand
transportedtothecellsurfaceforantigenpresentationtoCD8+T Therefore, THP-1 cells were nucleofected and HEK-293 cells
cells.ItispossiblethatoncetranslocatedtotheER,ESAT-6:CFP- were transfected with pEGFP-C1, pEGFP-C1-esat-6 or pEGFP-
10 can interact and sequester b2M and thereby reduce the C1-esat-6DC.NucleofectionefficiencyforTHP-1wasfoundtobe
availability of free b2M to form complex with HLA class I ,30% and transfection efficiency for HEK-293 was found to be
,50%. Surface b2M expression on GFP positive cells was
molecules.Insuchsituations,thesurfacelevelsofbothMHCand
b2Mmoleculesarelikelytobedecreased.Expectedly,whenTHP- measured by flow cytometry using PE conjugated anti-human
b2M Ab. It was observed that the intracellular expression of
1 macrophages were incubated with recombinant ESAT-6:CFP-
10 protein at two different concentrations [17,25], surface b2M ESAT-6 resulted in reduction of surface b2M levels in THP-1
(Figure 5E)andHEK-293(Figure 5F)cellswhencomparedtothe
levels were found to be decreased in a concentration dependent
cells transfected withpEGFP-C1 or pEGFP-esat-6DC.
manner (Figure 5A and Figure S6). Interestingly, no significant
changes in the surface expression of b2M were noticed in the
presenceofmutantESAT-6DC:CFP-10complexorCFP-10alone ESAT-6:CFP-10treatmentaffectsexpressionofHLAclassI
(Figure 5A),indicatingthatsuppressionofb2Msurfaceexpression molecules
was possibly due to its sequestration by the intact C-terminal Beta-2-microglobulinremainsassociatedwithHLAclassI,CD1
regionofESAT-6.Toruleoutthepossibilitythatthereductionin andHFEmoleculesonthecellsurface.Anychangeinthelevelsof
surface b2M levels was due to toxicity of the ESAT-6:CFP-10 b2Mwouldprobablyleadtochangesinthecellsurfaceexpression
complex, an MTT viability assay was performed with THP-1 of HLA class I, CD1 and HFE. As ESAT-6:CFP-10 treatment
macrophages treated with ESAT-6:CFP-10. We found that the leadstoreductioninsurfaceexpressionofb2M(Figure5),wenext
viability of the ESAT-6:CFP-10 treated cells was not affected examinedwhetherithasanyeffectonHLAclassIexpression.For
(Figure S7). Another possibility is that incubation with ESAT- this purpose, we utilized two different types of anti-HLA
6:CFP-10 may have caused general trafficking defects to reduce antibodies: HC-10 and W6/32. HC-10 is a monoclonal Ab that
thesurfaceexpressionofb2M.Toruleoutthis,weexaminedthe can detect only the free HLA-I heavy chain molecules not
surfaceexpressionofothercellsurfacemarkerslikeMac-1,TLR4, complexed with b2M [29]. Newly synthesized HLA molecules
MHC-II and CD14 using flow cytometry. Levels of all these form a tri-molecular complex with b2M and antigenic peptide
molecules were found to remain unchanged in ESAT-6:CFP-10 which is transported to the surface to present the peptide to its
treatedcells(FigureS8).ESAT-6:CFP-10alsodidnotaffectb2M cognate T cell receptor (TCR) [30]. In contrast, it has been
expressionattheproteinandmRNAlevel(FigureS9).Thesedata reportedthatheavychainsofMHCclassIallelescanalsoprogress
indicatethatthereductionofsurfaceb2Mlevelswaspossiblydue tothecellsurfacealonewithoutbeingassociatedwithb2M.Such
to physical sequestration of b2M by ESAT-6 inside the ER. M. free MHC class I heavy chains are present normally on cells,
tuberculosismayescapefromthephagosometothehostcytoplasm especially in activated lymphocytes [31], dendritic cells and
ofinfectedcellsinanRD1dependentmannerandsecreteESAT- macrophages and have been found to be upregulated in patients
6:CFP-10 directly into the cytoplasm of infected cells [26] which suffering from spondyloarthropathy [32]. Interestingly, we ob-
may find its way into the ER lumen to interact with the resident servedanincreasedbindingofHC-10AbinTHP-1macrophages
b2M.Totest this hypothesis,we over-expressed FLAG- orGFP- treated with ESAT-6:CFP-10 but not with ESAT-6DC:CFP-10
tagged ESAT-6 in cells in an attempt to mimic physiological (Figures 6A and 6B). Increased HC-10 staining upon addition of
conditionswhereESAT-6issecreteddirectlyintothecytosol.We ESAT-6:CFP-10wasobservedalsobyconfocalmicroscopywhere
transfected HEK-293 cells with pcDNA 3.1(+)-FLAG-esat-6 and intracellular levels of the free HLA molecules can also be
studiedlocalizationofESAT-6(stainedwithanti-FLAGAb)inER determined(Figure6C).Thesedataclearlysuggestthatthelevels
by confocal microscopy and was found to be present in the ofb2M-freeHLAclassImoleculeswereincreasedonthesurface
calnexin-positive ER compartments (Figure 5B) suggesting that as well as intracellularly after treatment with ESAT-6:CFP-10.
intracellular ESAT-6 can also find its way into the ER. We also Free HLA class I molecules are not only transported to the cell
performedco-immunoprecipitationstudiesincellsover-expressing surface but a portion may also undergo proteasomal degradation
full length ESAT-6 or ESAT-6DC cloned with an N-terminal since addition of MG-132 (a proteasomal inhibitor) to THP-1
EGFP tag in pEGFP-C1. ESAT-6 and b2M complexes could be macrophages resulted in increased HC-10 staining which was
pulled down from the whole cell lysates of HEK-293 cells over- furtherintensifiedinthepresenceofESAT-6:CFP-10(Figures6D
expressing pEGFP-C1-esat-6 but not from cells transfected with and6E).Therefore,itappearsthatonceESAT-6:CFP-10complex
pEGFP-C1 vector alone or pEGFP-C1-esat-6DC (Figure 5C). sequestersb2MinsidetheER,thenumberfreeHLAclassIheavy
Also,wewereabletopulldowntheESAT-6:b2Mcomplexfrom chainmoleculesareincreasedduetolessavailabilityoffreeb2M
the enriched ER fraction of HEK-293 cells transfected with moleculestoassociatewiththem.Thiswouldpossiblyexplainthe
pEGFP-C1-esat-6 but not from cells transfected with the vector increase in HC-10 staining observed in the presence of ESAT-
alone, indicating that ESAT-6:b2M complex was present inside 6:CFP-10.
PLOSPathogens | www.plospathogens.org 8 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure5.ExogenousadditionofESAT-6:CFP-10complexortransientexpressionofESAT-6downregulatesexpressionofsurface
b2M.(A)PMA-differentiatedTHP-1macrophagesweretreatedwithESAT-6:CFP-10orESAT-6DC:CFP-10orCFP-10proteinfor2hoursandstained
withPEconjugatedanti-humanb2MAbformeasuringsurfaceexpressionofb2Mbyflowcytometry.Medianfluorescenceintensitiesoftheb2M
levelsofvariousgroupswerecalculatedandtheresultsareshownasmean6SDof3differentexperiments.(B)HEK-293cellsweretransfectedwith
eitherpcDNA3.1(+)-FLAGcontrolplasmidorpcDNA3.1(+)-FLAG-esat-6andafter20–24hours,cellswerefixed,permeabilizedandstainedwithanti-
calnexinAbfollowedbyAlexaFluor594conjugatedanti-rabbitsecondaryAb(red)tovisualizetheendoplasmicreticulumandanti-FLAGAbfollowed
byAlexaFluor488conjugatedsecondaryanti-mouseAb(green)tovisualizeintracellularESAT-6.NucleuswasvisualizedbyDAPIstaining(blue).Cells
were observedunder confocal microscope. (C) Lysatespreparedfrom the HEK-293cells transfected witheitherpEGFP-C1or pEGFP-C1-esat-6 or
pEGFP-CI-esat-6DC were incubated with anti-GFP Ab bound to protein A/G agarose beads. Immunoprecipitated complexes (Lanes 4–6) were
separatedona15%SDS-PAGEandtransferredtoanitrocellulosemembranewhichwasprobedwithanti-b2MAb.About10%ofthelysateswere
loadedasinputcontrol(Lanes1–3).(D)HEK-293cellsweretransfectedwitheitherpEGFP-C1orpEGFP-C1-esat-6andafter20–24hours,cellswere
usedtoprepareenrichedroughendoplasmicreticulumfractions(RER).EqualamountsofproteinsextractedfromtheenrichedRERfractionswere
incubatedwithanti-GFPAbboundtoproteinA/Gagarosebeads(Lanes3and4).Immunoprecipitatedcomplexeswereseparatedona15%SDS-
PAGEandtransferredtoanitrocellulosemembraneandwasprobedwithanti-b2MAb.About10%ofthelysatesofenrichedRERfractions(Lanes1
and2)wereloadedasinputcontrols.(E)THP-1cellswerenucleofectedand(F)HEK-293cellsweretransfectedwitheitherpEGFP-C1orpEGFP-C1-
esat-6orpEGFP-CI-esat-6DCandafter20–24hours,cellswerestainedwithPEconjugatedanti-humanb2MAbandb2Mexpressiononthecellsurface
wasmeasuredbyflowcytometryforEGFP-positivecells.Resultsarerepresentativeofthreedifferentexperiments.
doi:10.1371/journal.ppat.1004446.g005
On the other hand, when the surface expression of b2M- resulting in reduced HLA class I-b2M complex formation and
complexedHLAclassImoleculeswasmeasuredwiththehelpof consequentlyincreasingthelevelsoffreeHLAclassIheavychain
W6/32,(amonoclonalAbthatrecognizesaconformationspecific molecules.
epitope on the HLA class I molecules only when associated with
b2M [33,34]) we observed a significant decrease in staining in ESAT-6:CFP-10 inhibits MHC-I presentation of SIINFEKL
ESAT-6:CFP-10-treated but not in ESAT-6DC:CFP-10-treated peptidederivedfromcytoplasmicandsolubleovalbumin
cells (Figures7A and 7B). Also, a pull down assay with W6/32 Most cytoplasmic antigens are processed by the proteasomal
yielded lesser amount of b2M in ESAT-6:CFP-10 treated cells, machineryandthederivedrestrictedpeptidesarepresentedbythe
indicatingthatlessamountofb2MwascomplexedwithHLAclass MHC-ImoleculestoCD8+Tcells.Wehaveobservedearlierthat
I molecules in these cells compared to untreated as well as those surface expression of b2M and MHC-I molecules is reduced in
treatedwithESAT-6DC:CFP-10(Figure 7C).DatafromFigures 6 macrophages treated with ESAT-6:CFP-10 (Figures5A, 7Aand
and7indicatethatESAT6:CFP-10cansequesterfreeb2MinER 7B).WealsofoundthatESAT-6:CFP-10complexisabletoenter
PLOSPathogens | www.plospathogens.org 9 October2014 | Volume 10 | Issue 10 | e1004446
M.tuberculosisESAT-6Interactswithb2M
Figure 6. Soluble ESAT-6:CFP-10 increases the levels of free HLA class I heavy chain molecules. (A) PMA-differentiated THP-1
macrophagesweretreatedwitheitherESAT-6:CFP-10orESAT-6DC:CFP-10protein(12.5mMeach).After2hours,cellswerewashedandincubated
withanti-HLAclassIheavychainmAbHC-10followedbyFITCconjugatedanti-mousesecondaryAb.SurfaceexpressionoffreeHLAclassIheavy
chainmoleculeswerestudiedbyflowcytometry.CellsstainedwithappropriateisotypeAbwereusedascontrol.(B)Medianfluorescenceintensities
ofdifferentexperimentalgroupsdescribedinFigure6Awerecalculatedandtheresultsareshownasmean6SDof3differentexperiments.(C)THP-
1macrophagespre-treatedwitheitherESAT-6:CFP-10orESAT-6DC:CFP-10protein(12.5mMeach)werefixed,permeabilizedandstainedwithHC-10
AbfollowedbyAlexaFluor594conjugatedanti-mousesecondaryAb(red).MatchingisotypeAbwasusedascontrol.Thenucleuswasvisualizedby
DAPIstaining(blue).Thestainedcellswereobservedunderaconfocalmicroscope.(D)Inanothersetofexperiments,THP-1macrophageswerepre-
treatedfor30minuteswith5mMMG-132followedbyincubationwitheitherESAT-6:CFP-10orESAT-6DC:CFP-10protein(12.5mMeach)for2hours.
FreeHLAclassImoleculesonthecellsurfacewerestainedwithHC-10AbfollowedbystainingwithFITCconjugatedanti-mousesecondaryAband
studiedbyflowcytometry.Isotype-matchedAbwasusedascontrol.(E)THP-1macrophagestreatedwitheitherESAT-6:CFP-10orESAT-6DC:CFP-10
protein (12.5mM each) in the absence or presence MG-132 werefixed, permeabilized and stained with HC-10 Ab followed by Alexa Fluor 594
conjugatedsecondaryanti-mouseAb(red).NucleuswasstainedwithDAPI(blue)andcellswerevisualizedunderaconfocalmicroscope.Datashown
isrepresentativeofthreeindependentexperiments.
doi:10.1371/journal.ppat.1004446.g006
vesicles of the phagocytic system and ER network (Figure 4 and treated with either medium or ESAT-6DC:CFP-10 protein
Figure 5B) and by interacting and sequestering b2M can reduce (Figure 8A). We also confirmed the staining experiment with an
b2M-MHC-I complex formation (Figure 7C). Therefore, we IL-2assayasareadoutforTcellfunction.Theresultsshowthat
expected a reduction in the amount of peptides derived from the the decreased SIINFEKL staining in cells treated with ESAT-
cytoplasmicantigenstobepresentedonMHC-I-b2Mcomplexat 6:CFP-10 actually correlates with a functional defect in presen-
the cell surface leading to inefficient presentation of antigen- tation ofovalbumin antigen (Figure8B).
derivedpeptidestoCD8+Tcells.Therefore,thioglycolate-elicited Cross-presentation is a process by which endocytosed antigens
mouseperitonealmacrophagesfromC57BL/6mice(H-2Kb)were are processed and presented by the MHC-I system. Cross-
cytosolicallyloadedwithnativeovalbumin(OVA)andthelevelsof presentationisthoughttobehighlyrelevanttothedevelopmentof
ovalbumin-derivedSIINFEKLpeptide(OVA )presentedby adaptive immunity against M. tuberculosis infection [35]. There-
257–264
H-2Kb was examined by flow cytometry in the absence or fore, presence of ESAT-6 in the ER can also affect cross-
presence of ESAT-6:CFP-10 or ESAT-6DC:CFP-10 complex. presentation of exogenously added antigens. When peritoneal
OurdataindicatethatthesurfacelevelsofSIINFEKLincomplex macrophages from C57BL/6 mice were pre-treated with soluble
withH-2KbweresignificantlyreducedincellstreatedwithESAT- ESAT-6:CFP-10, washed and incubated with soluble ovalbumin
6:CFP-10 protein when compared with ovalbumin-pulsed cells antigen,cross-presentationofSIINFEKLpeptidederivedfromthe
PLOSPathogens | www.plospathogens.org 10 October2014 | Volume 10 | Issue 10 | e1004446