Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Evaluation

of

the

role

of

Mycobacterium

tuberculosis

pili

(MTP)

as

an

adhesin,

invasin,

and

cytokine

inducer

of

epithelial

cells

Saiyur

Ramsugit

_,

_Balakrishna

_Pillay

_,

_Manormoney

_Pillay

a_{MedicalMicrobiologyandInfectionControl,UniversityofKwaZulu-Natal,Durban,SouthAfrica} b_{SchoolofLifeSciences,UniversityofKwaZulu-Natal,Durban,SouthAfrica}

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Articlehistory:

Received17August2015 Accepted26November2015 Availableonline31December2015

Keywords: Mycobacteriumtuberculosis Curlipili Adhesin Invasin

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ThisstudywasundertakeninordertoassesstheinvolvementofMycobacteriumtuberculosis

pili(MTP)asanadhesin,invasin,andcytokineinducerintheM.tuberculosis-epithelialcell interaction.AMTP-deficientstrainofM.tuberculosisdemonstratedasignificantreductionof 69.39%(p=0.047)and56.20%(p=0.033)initsabilitytoadheretoandinvadeA549pulmonary epithelialcells,respectively,incomparisonwiththewild-typestrain.Complementationof theMTP-deficientmutantrestoreditsadhesion andinvasioncapacitybacktothe wild-typelevels.Overall,itwasfoundthatsimilarconcentrationsofIL-1␤,IL-4,IL-6,IL-8,G-CSF, IFN-␥,MCP-1,andTNF-␣wereinducedinA549cellsinfectedwiththeMTP-proficientand MTP-deficientstrains.However,at48hpost-infection,theMTP-deficientmutantinduced significantlylowerlevelsofTNF-␣thanthewild-typestrain(p=0.033).Furthermore,at72h post-infection,themutantinducedsignificantlyhigherlevelsofIL-8thanthewild-type (p=0.005).WeconcludethatMTPisanadhesin/invasinofepithelialcellsand,whileplaying aroleinM. tuberculosisentry,theydonotappeartolargelyinfluencetheepithelialcell cytokineresponse.

©2016PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Mycobacterium tuberculosis, thenotorious causativeagent of tuberculosis(TB),remainsoneofthemaincausesofhuman mortality by an infectious disease. The lack of effective treatment, diagnostic,and preventative strategies continue

∗ _{Correspondingauthorat:MedicalMicrobiologyandInfectionControl,SchoolofLaboratoryMedicineandMedicalSciences,Collegeof}

HealthSciences,UniversityofKwaZulu-Natal,1stFloorDorisDukeMedicalResearchInstitute,PrivateBag7,Congella,4013Durban, SouthAfrica.

E-mailaddress:Pillayc@ukzn.ac.za(M.Pillay).

to impede TB control, which is further compounded by the increasingprevalenceofmulti-,extensively,and totally drug-resistantstrainsofthepathogenandtheHIV/AIDS co-epidemic.Abetterunderstandingofthevirulencefactorsthat areassociatedwiththepathogenesisofthisorganismis cru-cial to improve strategies to control and reduce global TB burdens.

http://dx.doi.org/10.1016/j.bjid.2015.11.002

1413-8670/© 2016 Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Adhesins are molecules or structures that are present extracellularly on the bacterial cell surface. They play an important role in the initial host-pathogen interaction by mediatingadherence,aprecursortohostcellentry.AmajorM. tuberculosisadhesinofepithelialcellsistheheparin-binding hemagglutininadhesin(HBHA),whichhasalsobeen impli-cated in extrapulmonary dissemination of the pathogen.1

Bacterialspeciesareknowntoproducemultipleadhesinsto effectively infecthostcells. Therefore, other adhesinsmay contributetoM.tuberculosisadhesiontoepithelialcells.2

Piliare awell-characterizedfamily ofbacterialadhesins. TwopilitypeshavebeendescribedforM.tuberculosis,namely curliandtypeIVpili.3_{Thecurliorcoiledpilusmorphotype,}

firstidentifiedinEscherichiacoliandSalmonellaspp.,areknown tobemediatorsinbiofilmformation,adherencetoand col-onizationofthehost,andinductionofhostinflammation.4

Thecurli-likepiliofM.tuberculosis,MTP,identifiedbyAlteri etal.,5_{resemblethepilifibersofE.coliandSalmonellaenterica}

andareencodedbythemtp(Rv3312A)gene.Theseresearchers alsoreportedontheabilityofMTPtobindtotheextracellular matrixproteinlaminininvitro,whichsuggestedthatMTPmay functionasanadherencefactor.5

Acentralfocusofourresearchgroupcurrentlyis elucidat-ingthefunctionofMTP.Wehavepreviouslyidentifiedtherole ofMTPininvitrobiofilmformation6_{andintheadherenceto}

andinvasionofmacrophages.7_{Wehavealsoshownthatthe}

mtpgenesequenceisuniquetoM.tuberculosiscomplexstrains andhighlyconservedamongstclinicalisolatesand,thus,MTP maybeasuitablebiomarkerforaTBdiagnostictest.8

Inthis study, we comparedadhesion and invasionof a wild-type,mtpmutant,andmtp-complementedM. tubercu-losisstrains tothe A549 pulmonaryepithelial cell line. We alsocomparedcytokineproductionbyA549cellsinfectedwith thesestrains.

Materials

and

methods

Bacterialstrainsandgrowthconditions

M.tuberculosisclinicalisolateV91249_{andthepreviously}

con-structedMTP-deficientmtpmutantandMTP-overexpressing

mtp-complementedstrains6_{wereusedinthisstudy.Cultures}

weregrownat37◦_{CinMiddlebrook7H9broth(Difco),}

supple-mentedwith10%(v/v)oleicacid-albumin-dextrose-catalase (OADC; Becton Dickinson), 0.5% (v/v) glycerol (Sigma), and 0.05%(v/v)Tween80(Sigma).Hygromycin(75␮gmL−1_;Roche

Applied Sciences) or kanamycin (30␮gmL−1_{; Sigma) were}

includedforthemutantandcomplementedstraincultures, respectively.

Epithelialcellpreparation

TheA549humantypeIIalveolarepithelialcellline(ATCC CCL-185)wasmaintainedinEagle’sMinimumEssential Medium with Earle’s Balanced Salt Solution and 2mM l-glutamine (Lonza), supplemented with 10% (v/v) fetal bovine serum (Biowest).Followingtrypsinization,2×105cellswereseeded into 24-wellplates(NEST Biotechnology)and incubated for 24hat37◦_Cin5%CO

2.

Infectionofepithelialcells

Logarithmicphase mycobacterialcultures were pelletedby centrifugation (2000×g for10min; Heraeus Multifuge 3S-R Centrifuge,ThermoScientific)andresuspendedinfreshcell culturemedia.Theepithelialcellswereinfectedwiththe bac-terialstrainsatamultiplicityofinfection(MOI)of1:1forthe adhesion and invasionassays. AMOI of10:1 was usedfor thecytokineassaytoensuresufficientcytokineproduction.10

Plateswereincubatedat37◦_Cand5%CO

2atthedesiredtime

intervals.Theinoculawerealsoseriallydilutedandplatedin triplicateontoMiddlebrook7H11(Difco)agarplates, contain-ing10%(v/v)OADCand0.5%(v/v)glycerol,toenumerateviable bacilliusedforinfection.Trypanblueexclusionwasusedto determinethenumberofviableepithelialcellsatthetimeof infection,toconfirmtheMOI.

Adhesionassay

Theadhesionassaywasperformedaspreviouslydescribed.7

Attheendofthe1hincubationperiod,mediawasremoved andtheepithelialcellswashedthricewith1mL phosphate-buffered saline (PBS;Oxoid).Adherent epithelial cellswere lysedwith1mLof0.1%(v/v)TritonX-100(Sigma)for20min at37◦_{Cand 5%CO}

2.Thenumberofadherentbacteriawas

determinedbyplatingten-foldserialdilutionsofthelysate,in triplicate,onto7H11agarplates.Colonieswerecountedafter 3weeksofincubationat37◦_C.

Invasionassay

Theinvasionassaywasperformedaspreviouslydescribed.7

After2hincubation,monolayersweretreatedwithamikacin (300␮gmL−1_{;Sigma)for1hat37}◦_Cina5%CO

2atmosphere.

Themediawasremovedandplatedonto7H11platesto con-firmthedeathofnon-invadedmicrobes.Theamikacinwas removedbywashingthricewithPBS.Adherentepithelialcells werelysedandthenumberofviableinvadedorganismswas determinedasintheadhesionassay.

Cytokineanalysis

Following4hofinfection,spentmediawasremoved,thewells washedwithPBS,and freshtissueculturemediaadded.At 24,48,and72hpost-infection,cellculturesupernatantswere removed and filteredthrougha0.2␮mfilter(PALLLife Sci-ences).Bovineserumalbumin(BSA;Sigma)wasaddedata finalconcentrationof0.5%(w/v)andthesampleswerestored at −80◦C. The cytokine levels in supernatants were mea-suredusingaBio-PlexProHumanCytokineMulti-PlexPanel (Bio-Rad)inaBio-Plex200System(Bio-Rad),accordingtothe manufacturer’sinstructions.

Statisticalanalysis

Adhesion/invasion assays were performed at least three independenttimesinduplicate.Cytokineexperimentswere performedattwoindependenttimesand assayedin dupli-cate.Thesignificanceofdifferencesbetweenthestrainswas determinedbyone-wayanalysisofvariance(ANOVA).Alldata

150

A

B

Wild-type Mutant Complemented

Strain Strain Wild-type Mean % invasion Mean % adhesion Complemented Mutant 80 60 40 20 0 0

Fig.1–TheroleofM.tuberculosispili(MTP)intheadhesion(A)toandinvasion(B)ofepithelialcells.A549epithelialcells wereinfectedwithawild-type,MTP-deficientmutant,andMTP-overexpressingcomplementedstrainsfor1h(adhesion assay)or2h(invasionassay).Amikacin-treatedand-untreatedinfectedepithelialcellswerewashed,lysed,andplatedonto agarplatestoenumerateviableinvadedandadherentbacilli,respectively.Resultsareexpressedasthemeanpercentage adhesiontoorinvasionofthemutantandcomplementedstrains,relativetothatofthewild-type.Errorbarsare±1×SEM.

wereanalyzedusingSPSS22.0statisticalsoftware.p<0.05was consideredsignificant.

Results

and

discussion

Theentryoftuberclebacilliintothealveolarspaceofthelungs isfollowedbytheinvasionorinternalizationoftheorganism byalveolar macrophages.11 _{This interaction has important}

implicationsforthepathogen’ssurvivalandreplication, eva-sionofthehostimmuneresponse,andinitsdissemination. Althoughlesswidelystudied,M.tuberculosisalsohasthe abil-itytoinvadeandreplicateinepithelialcells.12–14_Mehtaetal.13

showedthatwhilstM.tuberculosisinvadesmacrophagesmore efficiently than epithelial cells,the intracellularreplication withinthelattercelltypewassignificantlyhigher.Epithelial cellsmay,therefore,providealessharshintracellular envi-ronmentforthepathogen thanmacrophages.Furthermore, theyarepresentingreaternumbersthanmacrophageswithin alveoli15_{and,thus,mayrepresentthefirsthostcelltypethat}

thebacilliencounterinthelung.

Using immunofluorescence microscopy with anti-MTP antibodies,Alteri16 _{detected expressionofMTP in a}

previ-ousstudywithA549epithelialcells.Thisfindingsuggested a possible role of MTP inthe M. tuberculosis-epithelial cell interaction.ToascertainwhetherMTPenableM.tuberculosis

entryintoepithelialcells,wecomparedadhesionand inva-sioncapabilitiesofaMTP-deficientandaMTP-overexpressing

M.tuberculosisstraininA549cells.MTP-deficiencysignificantly diminishedadhesiontoandinvasionofA549cellscompared with the parental strain, by 69.39% (p=0.047) and 56.20% (p=0.033), respectively (Fig. 1). Complementation restored adhesion(p=0.027)andinvasion(p=0.027)tothewild-type levels(Fig.1).IncreasedMTPexpressionbythecomplemented straindidnot,however,significantlyalteradhesion(p>0.999) toandinvasion(p>0.999)ofepithelialcellscomparedwiththe wild-type(Fig.1).

Ourfindingsaresimilartothoseofother studies,which haveshownthatcurliatedbacteriaarebetterabletoadhereto andenterepithelialcellsthannon-curliatedbacteria. Curli-ated E. coli strains were reported to be better at adhering touroepithelialcells17_{andhumanlaryngealepithelial}

(Hep-2) cells.18 _{Theyalsodisplayedincreasedinvasionofhuman}

cervicalepithelial(HeLa)cells19_{andHep-2cells.}20_Similarly,

curliatedSalmonellatyphimuriumpromotedattachmentto cul-turedmousesmallintestinalepithelialcells.21

The current results differ from our previous study, which found that the adherence of M. tuberculosisto THP-1macrophageswasdependentonthedensityofpiliation.7

The complemented strain expressed a 51-fold higher mtp

expressionthan the wild-type, drivenbythe constitutively expressed hsp60 promoter of the pMV261 vector used for complementation.6 _{This MTP-overexpression significantly}

increased adhesion to THP-1 macrophages, but not A549 epithelialcells,comparedwiththewild-type.Thisobservation maypossiblyindicatedifferentmechanismsorhostreceptor interaction ofMTPwiththesetwohostcell typesor, alter-natively, varying expressionofthe same hostcell receptor betweenmacrophagesandepithelialcells.

Theonlydefinitiveadhesin/invasin previouslydescribed forM.tuberculosisistheHBHA.Petheetal.1_{reporteda60%and}

80%reductionintheadhesiontoandinvasionofA549 pneu-mocytes, respectively,byaHBHA-deficientstraincompared withthewild-type.LossofHBHAdidnotinfluenceadhesion toandinvasionofmacrophages.1_{Duetothesignificant}

reduc-tioninadherencetoandinvasionofepithelialcells,HBHAwas regardedastheprincipaladhesinofepithelialcells.However, ourstudyconfirmsthatM.tuberculosispossessesmorethana singleadhesin,witharedundantfunction,whichmayaccount forthesuccessofthepathogen.Therapeuticstrategies target-ingmultipleadhesinsmay,therefore,provemostbeneficialto incapacitatingthepathogen.

Inadditiontoservingasanadhesinandinvasinof epithe-lialcells,wepreviouslyreporteda42%and69%decreasein

adhesiontoandinvasionofTHP-1macrophages,respectively, bytheMTP-deficientstrain.7_{Thedifferenceinadhesionand}

invasionratessuggestthatMTPmayplayamoresignificant roleas anadhesininepithelialcells thaninmacrophages, whilst the opposite may be true for its function as an invasin.

Petheetal.1_{suggestedthatfollowingentryandreplication}

within macrophages, M. tuberculosis enters non-phagocytic cells, which facilitate extrapulmonary dissemination. The functionofMTPinbothphagocyticandnon-phagocytichost cellinteractionsimpliesthatMTPisamajoradhesinand par-ticipatesin multiplestages ofM. tuberculosispathogenesis. Duringhostcolonization,bacteriaarealsoknowntodevelop intostructuredmicrobialcommunitiesorbiofilms,whichare associatedwithdrugtolerance.22_{Inadditiontoservingasan}

adhesinofhostcells,MTPmediatetheformationofinvitroM. tuberculosisbiofilms.6

Giventhecollectivedataonbothadhesins,itseemsthat MTPmaybeamarginallybetteradhesinthanHBHAandthat thelattermay,inturn,bemoreeffectiveasaninvasin com-paredwiththeformer.Thiscanonlybeconfirmedbytesting thesedeletionmutantsunderthesameexperimental condi-tions.However,boththeseadhesins/invasinsappeartoplay majorroles in the virulenceof this pathogen and suggest thatincombination,theymayrepresentpowerfultargetsfor adjunctiveimmuno- or chemotherapyor potential vaccine development.

The outcome of M. tuberculosis infection is dependent onboth the virulenceofthe pathogen and the host cellu-lar defence mechanisms. M. tuberculosis-infected epithelial cells are known to secrete cytokines and chemokines, including interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), which contributeto the host inflamma-toryresponse.10_{Pilidonotonlyfunctioninthehost-pathogen}

interaction by enabling adhesion to and invasion of host cells,but the interaction ofpili withhost cell receptors is essentialtotheinductionofinflammationviatheproduction ofinflammatorymarkers,suchascytokines.23_Several

stud-ieshavedemonstratedthatpathogen-inducedinflammation onepithelialcellscanbeattributedtopathogen-associated molecularpatterns,suchaspiliorflagella,whichstimulate pro-inflammatorysignals.24–27_{Inordertodeterminewhether}

MTPareactivatorsoftheepithelialcellcytokineresponse,we comparedtheinvitrocytokineprofilesofA549cellsinfected withthewild-type,mtpmutant,andcomplementedstrains, usingamultiplexassay.

TheA549cellswerefoundtoproduceIL-1␤,IL-4,IL-6,IL-8, granulocyte colony-stimulating factor (G-CSF), interferon-gamma(IFN-␥),MCP-1,andTNF-␣inthisstudy,althoughat lowconcentrationsforsomeanalytes.TitersforIL-1␤,IL-4, IL-6,G-CSF,andIFN-␥werenotsignificantlydifferentforthe epithelialcellsinfectedwiththewild-type,mutant,and com-plementedstrainsatallthreetimepointstested(Fig.2a,b, c,eandf).Similarly,therewerenosignificantdifferencesin IL-8, MCP-1,and TNF-␣levels elicitedbythe MTP-deficient andMTP-proficientstrainsatmostofthetimeintervals,with afewexceptions(Fig.2d,gandh).Theonlyindicationthat MTPinducethecytokineresponsewastheobservationthatat 48hpost-infection,themutantstraindisplayedasignificant

reductioninTNF-␣productioncomparedwiththewild-type (p=0.033)(Fig. 2h). However, at24 and 72h post-infection, themutantstrainshowed,onaverage,higherlevelsofTNF-␣ inductionthanboththewild-typeandcomplementedstrains (Fig. 2h).The mutantstrain alsoinduced the lowest levels ofIL-4,IL-8,andIFN-␥at48hpost-infectioncomparedwith theMTP-proficientstrains, butthisdidnotreachstatistical significance(Fig.2b,dandf).

These findings suggest that, apart from TNF-␣ (and to a lesserextent IL-4, IL-8, and IFN-␥) at48h post-infection, MTP-mediated entry into epithelial cells does not trigger responses of the cytokines tested. These findings contrast other studies that have associatedcurlipilias inducersof the immune system. Tükel et al.28 _{showed that S.}

enter-ica serotype Typhimurium curliare recognized by Toll-like receptor 2, resulting in the activation of IL-8. Bian et al.29

demonstratedthatcurliatedE.coliresultintheactivationof IL-6, IL-8, and TNF-␣.Macrophages and human embryonic kidneycells,andnotepithelialcells,werethehostcell mod-elsinthesestudies.However,othershavedemonstratedan associationbetweenbacterialinvasionandcytokine produc-tion byepithelial cells.30–32 _{Interestingly,the mutantstrain}

induced significantly higher levels of IL-8 than the wild-type at 72h post-infection (p=0.005) (Fig. 2d). In addition, significantly more MCP-1 (p=0.048) and TNF-␣ (p=0.004) were secreted bymutant-infected epithelial cellsat72 and 24hpost-infection,respectively,comparedwiththe comple-mentedstrain(Fig.2gandh).Whilstnotreachingstatistical significance,themutantstrainshowed,onaverage,elevated levelsofallcytokinesdetectedinthisstudy,exceptIL-4and IFN-␥,at24hpost-infectionthanthewild-typeand comple-mentedstrains(Fig.2).Similarly,at72hpost-infection,only theG-CSFconcentrationswerenotthehighestforthemutant strainsamples(Fig.2).Inaddition,at48hpost-infection,the mutantstraininducedthehighestconcentrationsofIL-1␤, G-CSF,and MCP-1(Fig.2a,eand g).Itis, therefore,tempting tospeculate thatMTP-mediated invasionofepithelial cells mayconferanadvantagetothepathogenbydampeningthe epithelialcellcytokineresponse,whichisakeyfactorin infec-tioncontrol.ThiswouldrepresentanadditionalstrategyofM. tuberculosistoensuresurvivalbyescapingthehostimmune system. Alternately, the decreased cytokine levels may be attributed to increased cytotoxicityof wild-type and com-plementedstrain-infectedepithelialcells,duetothehigher bacterialloadsthanforthemutant-infectedepithelialcells. Ithasrecentlybeenreportedthatapositivecorrelationexists betweenM.tuberculosisadhesiontoandinvasionof epithe-lial cellsand itscytotoxic effect.33 _{Another possiblereason}

forthis phenomenoncouldbethatMTP-deficiencytriggers theexpressionofotheradhesins,whichinducecytokine pro-duction. Futurecytotoxicitystudiesand cytokineassayson epithelialcellstreatedwithpurifiedMTPproteinwouldhelp toverifythesepostulates.

To conclude, we have shown that MTP function in the adherence and entryof M.tuberculosis into epithelial cells. However,MTPdidnotlargelyinfluencetheoverallcytokine responses ofM. tuberculosis-infectedepithelial cells. Future

in vivostudies willaddress theimportanceoftheseinvitro

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Time (hours post infection)

IL-1

β

concentration (pg mL

–1)

72 24 48

Time (hours post infection)

72 24 48

Time (hours post infection)

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Key: Strain:

* P<.05 when compared to wild-type

† P<.05 between mutant and complemented strains

48

Time (hours post infection)

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24 48

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Time (hours post infection)

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Time (hours post infection)

72 † † * * 0 1500 1000 1000 500 800 600 400 200 0 IL-8 concentration (pg mL –1) MCP-1 concentration (pg mL –1) TNF-α concentration (pg mL –1) G-CSF concentration (pg mL –1) 0 0.40 0.35 60 40 20 20 15 10 5 0 0 0.20 0.10 IL-4 concentration (pg mL –1) IL-6 concentration (pg mL –1) INF-γ concentration (pg mL –1) 0 1.50 2.50 2.00 1.50 1.00 0.50 0 1.00 0.50 0

Wild-type Mutant Complemented

Fig.2–TheroleofM.tuberculosispili(MTP)inepithelialcellcytokineproduction.A549epithelialcellswereexposedtothe wild-type,MTP-deficientmutant,andMTP-overexpressingcomplementedstrainsfor4h.Extracellularbacteriawere removedbywashing,freshmediaadded,andcellculturesupernatantsat24,48,and72hpost-infectionwereassayedfor levelsofthecytokinesIL-1␤(A),IL-4(B),IL-6(C),IL-8(D),G-CSF(E),IFN-␥(F),MCP-1(G),andTNF-␣(H),usingaBio-Plex assay(Bio-Rad).Errorbarsare±1×SEM.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorswouldliketothanktheNationalResearch Foun-dationofSouthAfricaandtheCanonCollinsEducationaland LegalAssistanceTrustforfinancialsupport.WealsothankDr. LenineLiebenberg(CAPRISA)forassistancewiththecytokine dataanalysis.

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