Influence of iron deprivation on virulence traits of mycobacteria

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Influence

of

iron

deprivation

on

virulence

traits

of

mycobacteria

Rahul

Pal

_,

_Saif

_Hameed

_,

_Sharda

_Sharma,

_Zeeshan

_Fatima

AmityUniversityHaryana,AmityInstituteofBiotechnology,Gurgaon,India

a

r

t

i

c

l

e

i

n

f

o

Received18June2016 Accepted30August2016 Availableonline17October2016

Keywords: Iron Morphology Biofilm Anti-TBdrugs

a

b

s

t

r

a

c

t

NovelstrategiestocombattheeverincreasingburdenofdrugresistanceinMycobacterium tuberculosis(MTB)causingtuberculosis(TB)remainsaglobalconcern.TheabilityofMTB tosenseandadapttorestrictedironconditionsinthehostileenvironmentisessentialfor theirsurvivalandconfersthebasisoftheirsuccessasdreadfulpathogen.Thestrikingand clinicallyrelevantvirulencetraitofMTBisitsabilitytoformbiofilmsandadheretothe hostcells.Thepresentstudyelucidatedtheeffectofirondeprivationonbiofilmformation andcelladherenceofMycobacteriumsmegmatis,anon-pathogenicsurrogateofMTB.Firstly, weshowedthatirondeprivationleadstoenhancedcellsedimentationrateandaltered colonymorphologydepictingalterationsincellsurfaceenvelopeproperties.Weexplored thatbiofilmformationandcelladherencetopolystyrenesurfaceaswellashumanoral epithelialcellswereconsiderablyreducedunderirondeprivationbothinpresenceof2,2 BP(ironchelator)andsiderophoremutant011-14strain.Wefurtherinvestigatedthatthe potencyofthreefirstlineanti-TBdrugs(Isoniazid,Ethambutol,Rifampicin)toinhibitboth biofilmformationandcelladhesionwereenhancedunderirondeprivationincontrasttothe drugswhentestedalone.Takentogether,byvirtueoftheindispensabilityofironfor func-tionalvirulencetraitsinmycobacteria,irondeprivationstrategiescouldbefurtherexploited againstthisnotorioushumanpathogentoexplorenoveldrugtargets.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Tuberculosis(TB)isamongthemajorcausesofmortalityand morbidityinhumansaffectingnearlyonethirdoftheglobal populationannually.1_{OneofthehallmarksofMycobacterium}

speciesistheexistenceofcomplicatedbutuniquecellenvelop whichmakesthemlesspermeableandrenderingthemdrug resistanttherebydifferentiatingfromotherclassofbacteria.2

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](Z.Fatima).

1 _{Theseauthorscontributesequallytothisstudy.}

Therefore,tofindnovelstrategies,addresstheissueofdrug resistance,andsurmounttheMycobacteriumtuberculosis(MTB) infectionstillremainsatpriority.

Targeting iron homeostasis of the invading pathogens including MTB to impede the fast growing resistance has emerged asoneofthe strategiesthathavebeen efficiently adoptedandwidelygainedprominence.3_{Thestruggleforthe}

limited amount ofiron in the hostile environment is cru-cial for establishment of infection in MTB. Moreover, iron

http://dx.doi.org/10.1016/j.bjid.2016.08.010

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

deprivationisalreadyknowntoenhancethedrug suscepti-bilitiesofknownanti-TBdrugsinmycobacteria.4_Thepresent

studyaimedtoaddtotheexistingknowledgeabouttherole ofironinsurvivalandvirulenceofMycobacteriumsmegmatis,a surrogateforMTBbecauseofitsclosehomology,fastgrowth andnon-pathogenicnature.Weshowedthatirondeprivation leadstoalteredcellsurfaceproperties,biofilmformationand celladherencetopolystyreneaswellashumanoral epithe-lial cells. All these unreported phenotypes and disrupted mechanismsarenecessaryforMTBtosustainvirulenceand representsalternativeparadigmforantimycobacterial inter-vention.

Materials

and

methods

Materials

All media chemicals Middlebrook 7H9 broth, Middlebrook 7H10 agar, albumin/dextrose/catalase (ADC), and oleic acid/albumin/dextrose/catalase (OADC) supplements were purchasedfromBDBiosciences(USA).Tween-80,ethambutol (EMB), and isoniazid (INH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rifampicins, crystal violet, Tris–HCl, ferrozine, were purchased from Himedia (Mum-bai, India). Dimethyl sulfoxide (DMSO), potassium chloride (KCl),sodiumchloride(NaCl),disodiumhydrogen orthophos-phate (Na2HPO4), potassium dihydrogen orthophosphate

(KH2PO4),glycerol, Ferroussulphate (FeSO4), were obtained

fromFischerScientific;methanol(CH3OH),TLCsilicagel60

F254(20×20)waspurchasedfromMerck.Chloroform(CHCl3)

waspurchasedfromCDH,sodiumacetate,iodineballswere purchasedfromQualigens.

Bacterialstrainsandcultureconditions

Mycobacteriumsmegmatis,mc2155,wasusedasaparent wild-typestrain andmutant 011-014 asexochelinsiderophore mutant (mimicking iron deprivation) for all the experi-ments.Bacteriaweremaintainedon7H10agarsupplemented with10% (v/v) oleic acid/albumin/dextrose/catalase (OADC; BD Difco) and grown in Middlebrook 7H9(BD Biosciences) brothsupplementedwith0.05%Tween-80(Sigma),10% albu-min/dextrose/catalase (ADC; BD Difco), and 0.2% glycerol (FischerScientific)in100mlflasks(SchottDuran)and incu-batedat37◦CtilltheOD600isequalto1.0.Stockculturesof

logphasecellsweremaintainedin30%glycerolandstoredat −80◦_C.

Ferroxidaseassay

Wholecellproteinwasextractedandproteinconcentration was determined by Lowry method to proceed for ferrox-idase assay by using a method as previously described.5

Bacterialcellswere growninmiddlebrook7H9brothinthe absence (control) and presenceof 2,2BP (irondeprivation) at concentration (35␮g/ml). The assay was performed by usingferroussulfateastheelectrondonorand 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine) as a chelatortospecificallydetectthe ferrousironremainingat

theendofthereaction.Eachassaymixtureof0.2mlcontained (50mMNa-acetatebuffer,pH5,and0.1–0.3␮g)proteinextract, andthereactionwasstartedbyadditionof0.2mMFeSO4.

Sam-pleswerequenchedbyaddingferrozineat3.75mM,andthe Fe(II)oxidationwasdeterminedbymeasuringtheabsorbance ofresidualFe(II)-ferrozineat570nmbyspectrophotometer.

Cellsedimentation

Cell sedimentation assay was performed as described elsewhere.6 _{Cultures atOD}

600∼1.0–1.4 of the control, iron

deprivedandmutantstrainsin7H9+ADC+glycerol+tween, wereadjustedintriplicatetoOD590∼1.0andkeptunshaken

at37◦C.At3and22h,theupper1mlwasremovedforOD590

measurements.

Colonymorphology

ColonymorphologywasobservedbyadjustingtheOD600∼0.1

of the M. smegmatis strains (control, iron deprived and mutant)in1×PBS salineandspotting5␮lonagarPlate7H 10+ADC+glycerolfollowedbyincubationoftheplatesat37◦C for 48h. Theimages were observed bymicroscope at10× magnification.6

Lipidextractionandthinlayerchromatography(TLC)

CellsofM.smegmatiscontroland2,2BPtreatedat exponen-tial phasewere usedforlipid extractionbymodified Folch method.7,8_{Briefly,thecellswereharvestedat10,000rpmfor}

10min.Cellswerehomogenizedinaqueoussolutionfor3min andsuspendedinCHCl3andCH3OHinratioof(1:2).Cellswere

shakenwelland centrifugedat2000rpmat4◦Cfor15min. Supernatantwastransferredtoanotherglassvial andthen remainingCHCl3 wasaddedand filteredthroughWhatman

No.1filterpaper.Theextractwasthenwashedwith0.88% KCltoremovethenon-lipidcontamination.Thelowerdense layerofchloroformcontaininglipidwastakenbyglass Pas-teurpipettein5mlglassvialwithTefloncapping.Thevials arestoredat20◦Cuntilfurtheranalysis.Extractedlipidswere thenresolvedbyTLCusingaluminum-backedsilicagelplates (silica gel 60 F254; Merck).The lipid extractobtained from control and 2,2 BP was loaded on TLC plate at adistance of2cmupfromtheplateend.Chloroform–methanol–water (65:25:4; v/v/v) was used for developing the plates. Devel-oped chromatogram was dried at room temperature for 2min and then exposed to iodine fumes generated by iodinecrystalballsplacedinglasschambertovisualizethe lipids.

Biofilmformationandcelladhesiononpolystyrene

M. smegmatisbiofilmformationwasestimatedasdescribed previously6,9,10 _{on 96-well polystyrene plates. Briefly, cells}

were grown in 7H9 middlebrook broth till the OD600∼1.0.

100␮lofmediawasdispensedtoeachwellofthe96wellplates withorwithoutadditionofdrug.Culturewerethenadjusted to0.1ODanddilutedintheratioof1:100inthemiddlebrook mediaand100␮lofdilutedculturewaspipettedineachwellof 96-wellflatbottommicrotitreplateandincubatedat37◦Cfor

48h.ThewellswererinsedtwicewithdistilledH2Oand125␮l

of0.1%solutionofthecrystalvioletwasadded.Plateswere incubatedfor10minfollowedbywashingwithdistilledH2O

2–3times,driedandthenobservedunderthelightmicroscope at100×.Forquantitativeassayofbiofilm,200␮lof95%ethanol wasaddedtoeachcrystalvioletstainedwellandplateswere incubatedfor10minatroomtemperature.Contentsofeach wellweremixedbypipetting,andthen125␮lofthecrystal violet/ethanolsolutionwastransferredfrom eachwell toa separatewellofanopticallyclearflatbottom96-wellplateand opticaldensities(OD)measuredat600nmusing spectropho-tometer. Inhibitionofbiofilmwas calculatedas percentage inhibition/reductioningrowth.Forcelladhesionassaysame procedurewasfollowedexceptthatprimarilytreatedand non-treatedcellsweregrowntillOD6001.0andafterwashingthe

non-adheredcells,theyweredirectlyquantifiedthrough crys-talvioletwithoutformingbiofilm.

AdherenceofMycobacteriumtohumanbuccalepithelial cells

Celladherenceassayweredevelopedonmycobacteriausing aprotocoldescribedelsewherewithmodifications.9,11

Epithe-lialcellswereobtainedfrommouthcavityoftheauthorwho voluntarilyagreedtodonate.Thecellswerewashed2–3times inPBSandthepelletswerethenresuspendedinPBStogive approximately(0.5OD600)byusingspectrophotometer.

Bac-terialcellsweregrowninmiddlebrook7H9brothunderiron deprivation(35␮g/ml)andincubatedovernightat37◦C.The culturewasadjustedtogiveanapproximatelyOD650of0.5,

washedtwiceinPBS,centrifugedfor10minat10,000rpmand resuspendedinPBS.Thetestwasperformedbytakingequal volumesofbuccalepithelialcells(OD600of0.5)andbacterial

suspensionsthatwere mixedandincubatedundershaking (120rpm)at37◦Cfor2–3h.Afterincubation,2–3␮lofcarbol fuchsindyetostainMycobacteriumcellsandcrystalvioletto stainepithelialcellwereaddedtoeachtubeandthemixture wasgentlyshaken.10␮lofthestainedsuspensionwere trans-ferredtoaglassslide,coveredwithcover-slipandexamined underlightmicroscope at40×.Atotalof50epithelialcells werecountedandthemeanpercentageofadherencewas cal-culatedusingthe numberofbacteriaaddedper 50cells as denominator.12_{Cellswerescoredasadherentwhenattached}

toatleast40%oftheepithelialcells,whilenon-adherentwhen attachedtolessthan10%oftheexaminedepithelialcells.13

Statisticalanalysis

All experiments were performed in triplicates (n=3). The resultsare reportedasmean±standard deviation(SD)and analyzedusingStudentt-testwhereinonlyp<0.05was con-sideredasstatisticallysignificant

Ethicsapproval

The study was approved by the Research Ethics Com-mittee of the Amity University Haryana (reference no. AUH/EC/RP/2016/05).

Results

and

discussion

Irondeprivationleadstoenhancedcellsedimentationrate andalteredcolonymorphology

Toensuretheirondeprivedcondition,firstlywedid ferrox-idaseenzymaticassaytoestimatethenon-oxidizedferrous ironpresentinthereactionmixture.Asexpected,wefound significantlyreducedferroxidaseactivity ofM.smegmatisin thepresenceofanironchelator2,2BPconfirmingthe exist-ingirondeprivedcondition(Fig.1).Irondeprivationisknown todisruptthemembraneintegrityofmycobacteria.4

There-fore,wespeculatedthatirondeprivationcouldfurtherleadto alterationsincellsurfaceproperties.Itisalreadyknownthat cell sedimentationrate isinverselycorrelatedwiththe cell surface hydrophobicity(CSH),6,14 _{hence,measuringthe cell}

sedimentationratecouldgiveanestimateofanychangein CSHleadingtoalterationsincellenvelop.Ourobservations showedthatcellsedimentationrateisconsiderablyenhanced underirondeprivation(Fig.2a).Tofurtherconfirm,weusedan exochelinsiderophoremutant(011-14) whichmimicsiron deficientcondition.Toouranticipation,thecell sedimenta-tionratewasalsoenhancedinthesiderophoremutant(Fig.2a) strengtheningthenotionthatCSHofenvelopisalsoregulated byiron.TheseresultsconfirmthatCSHisdiminishedunder irondeprivationcorroboratingwiththeearlierreportof dis-ruptedmembraneintegrity.6_{Thiswasalsoapparentfromthe}

factthatthecolonymorphologyofcontrolcellwasdistinct fromthatoftheirondeprivedandmutant(011-14)cells.The controlcellcomposedofsmoothcolonywithdefinedborder ascomparedtoirondeprivedcellandmutant(011-14)which showedroughcolonyandundefinedborders(Fig.2b). Further-more,theseobservationscouldalsobeduetothefactthat mycobacteriahaveuniquecellenvelopeknownas mycomem-branecomprisingmycolicacidswhichareknowntogovern pathogenicityofmycobacteriaandthatirondeprivationmay possiblyaffect mycolicacid synthesisas well.Toascertain whether the observed alteration in morphology could be attributedtochangesinlipidprofile,weperformedaTLCof

∗ 0 2 4 6 8 10 12 2.2, BP Control (Ferroxidas e assa y ) O.D 57 0

Fig.1–Effectofirondeprivationonferroxidaseactivity. Enzymeferroxidaseactivityquantifiedbyusingferroxidase assaydepictedasbargraphasdescribedinmaterialand methodsinpresenceof2,2BP(35␮g/ml)inM.smegmatis.

MeanofOD570 nm±SDofthreeindependentsetsof

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 2,2,BP Control Δ011-14 Sedimentation rate/hour 0 0.2 0.4 0.6 0.8 1 1.2 0 3 22 O. D59 0 Time(hrs) Control 2,2,BP 011-14

a

b

c

2,2, BP Control

Solvent front

Fig.2–Effectofirondeprivationoncellsurfaceproperties.(a)RelativesedimentationofM.smegmatiscellsunderiron deprivation.LeftpanelshowsOD590ofuntreated(control),2,2BP(35␮g/ml)andsiderophoremutant011-14cellsdepicted

onY-axiswithrespecttotimenotedat3and22honX-axis.RightpaneldepictssedimentationratesperhouronY-axisof

2,2BP(35␮g/ml)andmutant011-14cellswithrespecttocontrolonX-axis,calculatedbyestimatingthedifferencein OD590from0till22hperunittimeinterval.Meanofthreeindependentsetsofexperiments±SD(shownbyerrorbars)are

depicted.(b)Alteredcolonymorphologyunderirondeprivation.Colonymorphologyofcontrol,irondeprived(2,2BP)and mutant011-14cellswereobservedon7H10-basedmedium.Correspondingareaofcolonyborderswerefocused (10×-magnification)andoneofthecolonyborderdepictedbyanarrowisenlargedandshownintheinset.(c)Thinlayer chromatogramshowingalteredtotallipidprofileunderirondeprivation.

totalcellularlipidsunderirondeprivation.Thechromatogram (Fig.2c)showedthattotalcellularlipidsincontroldisplayed variouslipidsclassesattheirrespectivepositionscontraryto theirondeprivedlipidswhichwere notdetectedatsimilar positions.However,furtherworkisstillneededtovalidatethe disruptionofmycolicacidsynthesisunderirondeprivation.

Irondeprivationaffectsbiofilmformation

Biofilmsare surfacelinkedmicrobialcolonyenclosedinan extracellularpolymericmatrix.Thisisoneofthemajorkey factorsforMTBhavingimplicationsonvirulence, pathogeni-cityandtherebyfacilitatesenvironmentalsurvival.13_Biofilms

renderMTBhighlyresistanttoanti-TBdrugshence search-ing for mechanisms counteracting biofilm formation is an attractive area of research. Moreover, reduced CSH under irondeprivationobservedfromthisstudyandreducedCSH already knownto negatively inhibitbiofilm,6 _{compelled us}

tostudy theeffectofirondeprivationonbiofilm formation of M. smegmatis. For this, we performed biofilm forma-tion assay as described in material and methods by both qualitativeandquantitativemethods.Thecrystalviolet stain-ing result showed that biofilm formation was significantly

inhibited in irondeprived condition in contrast to control (Fig.3a)therebysupportingtheearlierobservationof indis-pensabilityofexochelinsiderophoreinbiofilmformation.15

We also tested whether iron deprivation could enhance the antibiofilmeffectofknownanti-TBdrugsviz.isoniazid (INH),ethambutol(EMB)andrifampicin(RIF).Interestingly,we observedthatirondeprivation(both2,2BPand011-14)leads toenhancedbiofilm inhibitionincontrast totheinhibition causedbythedrugsalone(Fig.3a).Theinhibitorybiofilmeffect under irondeprivationwasfurthervalidated quantitatively whichshowedgoodcorrelationwithourcrystalvioletstaining result(Fig.3b).Thisisalsocommensuratewithourabove find-ingofdecreasedCSHandalteredcolonymorphologywhich mayhavemadetheMycobacteriummoresusceptibletoknown antibiotics.Thusirondeprivationleadstodiminishedsurvival afterexposuretoknownanti-TBdrugs.

Irondeprivationaffectsmycobacterialcelladherenceto polystyrenesurfaceandhumanoralepithelialcells

The compromised biofilm formation gives us clue to sub-sequently examine the effect of iron deprivation on cell adherence to an abiotic surface, being the primary step

Control INH +Δ011-14 EMB + 2.2, BP EMB +Δ011-14 EMB INH + 2.2, BP INH RIF 50µm 50μm 50μm 50μm 50μm 50μm 50μm Δ011-14 2.2, BP RIF+ 2.2, BP RIF+Δ011-14 50μm 50μm 50μm 50μm 0 20 40 60 80 100 120 % Gr owth Control 2.2, BP Δ 01 1-14 INH INH + 2.2, BP INH + Δ 01 1-1 4 EMB EMB + 2.2, BP EMB + Δ 011-1 4 RIF RIF + 2.2, BP R IF + Δ 01 1-1 4 ∗ ∗ ∗ ∗ _∗ ∗ 50μm 50μm

a

b

Fig.3–Effectofirondeprivationonmycobacteriumbiofilm.Irondeprivation(2,2BP(35␮g/ml)andsiderophoremutant 011-14)showinginhibitedbiofilmformationofMycobacteriumsmegmatisinpresenceofthreeknownanti-TBdrugs,i.e. INH,EMBandRIF(1␮g/ml,0.0625␮g/ml,0.0625␮g/ml).(a)Crystalvioletstainingshowingtheinhibitedbiofilmformation underirondeprivation(2,2BP(35␮g/ml)andsiderophoremutant011-14).(b)Biofilminhibitionunderirondeprivation(2,2 BP(35␮g/ml)andsiderophoremutant(011-14)inpresenceofthreeknownanti-TBdrugs,i.e.INH,EMBandRIF(1␮g/ml, 0.0625␮g/ml,0.0625␮g/ml)depictedasreductioningrowthpercentquantifiedbyusingcrystalvioletdyeasbargraph. MeanofOD600±SDofthreeindependentsetsofexperimentsaredepictedonY-axisand*depictspvalue<0.05.

0 20 40 60 80 100 120 % Growth Control _Δ01 1-14 2.2, BP INH INH + Δ 01 1-14 INH + 2.2, BP EMB EMB + Δ 011-1 4 EMB + 2.2, BP RIF R IF + Δ 01 1-1 4 RIF + 2.2, BP ∗ ∗ ∗ ∗ ∗ ∗

Fig.4–EffectofirondeprivationonMycobacteriumcelladhesion.Irondeprivation(2,2BP(35␮g/ml)andsiderophoremutant 011-14)inhibitsthecelladhesionofMycobacteriumsmegmatistomicrotiterpolystyrenesurfaceinpresenceofthreeknown anti-TBdrugs,i.e.INH,EMBandRIF(1␮g/ml,0.0625␮g/ml,0.0625␮g/ml).Reductioningrowthpercentisquantifiedby usingcrystalvioletdyeanddepictedasbargraph.MeanofOD600±SDofthreeindependentsetsofexperimentsare

depictedonY-axisand*depictspvalue<0.05.

Control _Δ011-14 2.2, BP

a

b

Fig.5–EffectofirondeprivationonMycobacteriumcelladherencetoepithelialcells.(a)Control(untreated)cellsappeared adheredtohumanbuccalepithelialcellswhileirondeprivedcells(2,2BP(35␮g/ml)andsiderophoremutant011-14)are notadheredtotheepithelialcells(magnification40×).(b)Meanpercentageadherenceofmycobacterialcellsdepictedasbar graphsshowingOD600±SDofthreeindependentsetsofexperimentsonY-axisand*depictspvalue<0.05.

for biofilm formation. Cell adhesion is a gradual pro-cess that starts with adhesion, surface attachment, and maturation.16–19_{Toourexpectation,wefoundthatiron}

depri-vationcausesdiminishedcelladherence ofM.smegmatisto polystyrenesurface(Fig.4).Thiswasrevalidatedwhenknown anti-TBdrugsviz.INH,EMBandRIFweretestedfortheir effi-cacytodisruptcelladhesion.Interestingly,weobservedthat irondeprivationleadstoenhancedinhibitionofcelladhesion topolystyrenesurfaceincontrasttotheinhibitioncausedby thedrugswhentestedalone (Fig.4)implyingimpairedcell adhesionunderirondeprivation.Thisobservationbecomes noteworthy from the fact that there is also a correlation betweencelladhesionandmycolicacidwhichiswellknown tohaveimplicationsonvirulenceandpathogenicityofMTB.20

Inhibited cell adherence under iron deprivation on polystyrene surface and in order to verify our above find-ing,wecheckedthe adherenceofMycobacteriumonhuman buccal epithelial cells which isa critical step before inva-sionandpathogenesis.Theabilityofmostinvadingbacterial pathogensincludingMTBtoadheretheepithelialandmucosal surfacesiscrucialforpathogenicity.Interestingly,weobserved thatirondeprivation(2,2BPandsiderophoremutant)showed inhibited adherence ofMycobacterium on epithelial cells as comparedtountreatedcells(Fig.5a).Toourexpectation,the controlcellsshowedmeanpercentadherenceof52.6%in com-parison tothe iron deprived and siderophore mutantcells whichonlyshowedmeanpercentadherenceof9.3%and8.6%, respectively(Fig.5b)Together,thisstudyreinforcethatiron deprivationinhibitspotentialvirulencetraitsofM.smegmatis

resultingininhibitionofbiofilmformationandcelladherence.

Conclusion

Thedatagenerated from this study clearlyestablishes the indispensabilityofironforvirulencetraitsofMycobacterium.

ThustargetingthepathwaysthatrequireironinMycobacterium

couldbeanovelandpromisingantitubercularstrategy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Funding

FinancialassistanceintheformofYoungScientistawardto Z.F.fromBoardofResearchinNuclearSciences(BRNS), Mum-bai(2013/37B/45/BRNS/1903)isdeeplyacknowledged.

Acknowledgments

WearegratefultoProf.SarmanSinghandProf.GrahamF. Hat-fullforprovidingM.smegmatismc2_{155referencestrainand}

siderophoremutantasgenerousgiftsrespectively.

r

e

f

e

r

e

n

c

e

s

1. MaJ,YangB,YuS,etal.Tuberculosisantigen-induced expressionofIFN-␣intuberculosispatientsinhibits productionofIL-1␤.FASEBJ.2014;28:3238–48.

2.HettEC,RubinEJ.Bacterialgrowthandcelldivision:a mycobacterialperspective.MicrobiolMolBiolRev. 2008;72:126–56.

3.HameedS,PalR,FatimaZ.Ironacquisitionmechanisms: promisingtargetagainstMycobacteriumtuberculosis.Open MicrobiolJ.2015;31:91–7.

4.PalR,HameedS,FatimaZ.Irondeprivationaffectsdrug susceptibilitiesofmycobacteriatargetingmembrane integrity.JPathog.2015;2015:1–10.

5.KimC,LorenzWW,HoopesJT,DeanJF.Oxidationof phenolatesiderophoresbythemulticopperoxidaseencoded bytheEscherichiacoliyacKgene.JBacteriol.2001;183:4866–75.

6.JametS,SlamaN,DominguesJ,etal.Thenon-essential mycolicacidbiosynthesisgeneshadaandhadccontributeto thephysiologyandfitnessofMycobacteriumsmegmatis.PLoS One.2015;10:2015.

7.FolchJ,LeesM,StanleyS.Asimplemethodfortheisolation andpurificationoftotallipidesfromanimaltissue.JBiol Chem.1957;226:497–509.

8.PalR,HameedS,KumarP,SinghS,FatimaF.Comparative lipidomeprofileofsensitiveandresistantMycobacterium tuberculosisstrain.IntJCurrMicrobiolApplSci.2015;(Special Issue1):189–97.

9.SharmaS,PalR,HameedS,FatimaZ.Antimycobacterial mechanismofvanillininvolvesdisruptionofcellsurface intensity,virulenceattributeandironhomeostasis.IntJ Mycobacteriol.2016,

http://dx.doi.org/10.1016/j.ijmyco.2016.06.010.

10.CraviotoA,GrossRJ,ScotlandSM,etal.Anadhesivefactor foundinstrainsofEscherichiacolibelongingtothetraditional infantileenteropathogenicserotypes.CurrMicrobiol. 1979;3:95.

11.RamsugitS,GumaS,PillayB,etal.Pilicontributetobiofilm formationinvitroinMycobacteriumtuberculosis.AntonieVan Leeuwenhoe.2013;104:725–35.

12.AshiruOT,PillayM,SturmAW.Adhesiontoandinvasionof pulmonaryepithelialcellsbytheF15/LAM4/KZNandBeijing strainsofMycobacteriumtuberculosis.JMedMicrobiol. 2010;59:528–33.

13.AbdullaAA,AbedTA,SaeedAM.Adhesion,autoaggregation andhydrophobicityofsixLactobacillusstrains.BrMicrobiol ResJ.2014;4:381–91.

14.GuptaKR,KasettyS,ChatterjiD.Novelfunctionsof(p)ppGpp andcyclicdi-GMPinmycobacterialphysiologyrevealedby phenotypemicroarrayanalysisofwild-typeandisogenic strainsofMycobacteriumsmegmatis.ApplEnvironMicrobiol. 2015;81:2571–8.

15.OjhaA,HatfullGF.TheroleofironinMycobacteriumsmegmatis

biofilmformation:theexochelinsiderophoreisessentialin limitingironconditionsforbiofilmformationbutnotfor planktonicgrowth.MolMicrobiol.2007;66:468–83.

16.BusscherH,WeerkampA.Specificandnon-specific interactionsinbacterialadhesiontosolidsubstrata.FEMS MicrobiolRev.1987;46:165–73.

17.MohamedN,TeetersM,PattiJ,HookM,RossJ.Inhibitionof

Staphylococcusaureusadherencetocollagenunderdynamic conditions.InfectImmun.1999;67:589–94.

18.DunneWJr.Bacterialadhesion:seenanygoodbiofilms lately?ClinMicrobiolRev.2003;15:155–66.

19.AbidiSH,AhmedK,SherwaniSK,BibiN,KazmiSU.Detection ofMycobacteriumsmegmatisbiofilmanditscontrolbynatural agents.IntJCurrMicrobiolApplSci.2014;3:801–12.

20.BendingerB,RijnaartsHHM,AltendorfK,ZehnderAJB. Physiochemicalcellsurfaceandadhesivepropertiesof coryneformsbacteriarelatedtothepresenceandchain lengthofmycolicacids.ApplEnvironMicrobiol. 1993;59:3973–7.