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Mitochondria : A target for bacteria
Lobet, Elodie; Letesson, Jean-Jacques; Arnould, Thierry
Published in:
Biochemical Pharmacology
DOI:
10.1016/j.bcp.2015.02.007
Publication date:
2015
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Early version, also known as pre-print
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Citation for pulished version (HARVARD):
Lobet, E, Letesson, J-J & Arnould, T 2015, 'Mitochondria : A target for bacteria', Biochemical Pharmacology, vol.
94, no. 3, pp. 173-185. https://doi.org/10.1016/j.bcp.2015.02.007
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Commentary
Mitochondria:
A
target
for
bacteria
Elodie
Lobet
a,
Jean-Jacques
Letesson
b,
Thierry
Arnould
a,*
aLaboratoryofBiochemistryandCellularBiology(URBC),NAmurResearchInstituteforLIfeScience(NARILIS),UniversityofNamur,61RuedeBruxelles,
5000Namur,Belgium
bResearchUnitinMicroorganismsBiology,UniversityofNamur,61RuedeBruxelles,5000Namur,Belgium
ARTICLE INFO Articlehistory:
Received1December2014
Receivedinrevisedform12February2015 Accepted12February2015
Availableonline20February2015 Keywords: Mitochondria Bacteria Metabolism Calcium mtROS Immunity ABSTRACT
Eukaryoticcellsdevelopedstrategiestodetectanderadicateinfections.Theinnateimmunesystem, whichisthefirstlineofdefenceagainstinvadingpathogens,reliesontherecognitionofmolecular patterns conserved among pathogens. Pathogen associated molecular pattern binding to pattern recognitionreceptortriggerstheactivationofseveralsignallingpathwaysleadingtotheestablishment ofapro-inflammatorystaterequiredtocontroltheinfection.
Inaddition, pathogens evolved to subvertthose responses (withpassive andactive strategies) allowingtheirentry andpersistenceinthehostcellsandtissues.Indeed,severalbacteriaactively manipulateimmunesystemorinterferewiththecellfatefortheirownbenefit.Onecanimaginethat bacterialeffectorscanpotentiallymanipulateeverysingleorganelleinthecell.However,themultiple functionsfulfilledbymitochondriaespeciallytheirinvolvementintheregulationofinnateimmune response,makemitochondriaatargetofchoiceforbacterialpathogens astheyarenotonlyakey componentofthecentralmetabolismthroughATPproductionandsynthesisofvariousbiomoleculesbut theyalsotakeparttocellsignallingthroughROSproductionandcontrolofcalciumhomeostasisaswell asthecontrolofcellsurvival/programmed celldeath.Furthermore,considering thatmitochondria derivedfromanancestralbacterialendosymbiosis,itisnotsurprisingthataspecialconnectiondoes existbetweenthis organelle and bacteria. In this review,we willdiscuss differentmitochondrial functionsthatareaffectedduringbacterialinfectionaswellasdifferentstrategiesdevelopedbybacterial pathogens to subvert functions related to calcium homeostasis,maintenance of redoxstatus and mitochondrialmorphology.
ß2015ElsevierInc.Allrightsreserved.
Abbreviations:A/A, ammonia/ammonium; AIM2, absent in melanoma 2; AMPK, AMP-activated protein kinase; APC, antigen presenting cell; CpG-ODN, CpG-oligodeoxynucleotide;DAMP,damages-associatedmolecularpattern;DC,dendriticcells;DRP1,dynamin-relatedprotein1;ECSIT,evolutionarilyconservedsignalling intermediateinTollpathways;Eis,enhancedintracellularsurvival;ER,endoplasmicreticulum;ERRa,estrogen-relatedreceptoralpha;ETC,electrontransportchain;GAPDH, glyceraldehyde3-phosphatedehydrogenase;GPX,glutathioneperoxidase;HIF-1a,hypoxia-induciblefactor-1alpha;IFNb/IFNg,interferon-beta/interferon-gamma;IL-4, interleukin-4;IRF,interferonregulatoryfactor;JNK,JunN-terminalkinase;LLO,listeriolysin;LPS,lipopolysaccharide;MAPK,mitogen-activatedproteinkinase;MAVS, mitochondrialantiviralsignallingprotein;MCU,mitochondrialcalciumuniporter;MDA-5,melanomadifferentiation-associatedgene-5;mtDAMP,mitochondrialDAMP; mtDNA,mitochondrialDNA;mtROS,mitochondrialROS;MyD88,myeloiddifferentiationprimaryresponsegene88;NFAT5,nuclearfactorofactivatedT-cells5;NFkB, nuclearfactor-kappaB;NRL,NOD-likereceptor;NOX,NADPHoxidase;OMM,outermitochondrialmembrane;OPA1,opticatrophy1;OXPHOS,oxidativephosphorylation; PAMP,pathogen-associatedmolecularpattern;PGC-1b,PPARgammacoactivator-1beta;PI3K,phosphoinositide3-kinase;PMN,polymorphonuclearneutrophil;PPA, propionicacid;PPAR,peroxisomeproliferator-activatedreceptor;PPP,pentosephosphatepathway;PRR,patternrecognitionreceptor;PRX,peroxyredoxins;RLR,Rig-Ilike receptor;ROS,reactiveoxygenspecies;SAM,sortingandassemblymachinery;SERCA,sarco/endoplasmicreticulumcalciumATPase;SLO,streptolysin;SOD,superoxide dismutase;STAT6,signaltransducerandactivatoroftranscription6;STING,stimulatorofinterferongenes;TXSS,typeXsecretionsystem;TACE,TNFa-convertingenzyme; TCA,tricarboxylicacid;TcdB,ClostridiumdifficiletoxinB;TCR,T-cellreceptor;TFAM,mitochondrialtranscriptionfactorA;TIM,translocaseoftheinnermembrane;TLR, Toll-likereceptor;TNFa,tumour-necrosisfactoralpha;TOM,translocaseoftheoutermembrane;TRAF6,TNFareceptor-associatedfactor6;UCP-2,uncouplingprotein-2;VacA, vacuolatingcytotoxinA;VDAC,voltage-dependentanionchannel.
* Correspondingauthor.Tel.:+3281724125;fax:+3281725306.
E-mailaddresses:[email protected](E.Lobet),[email protected](J.-J.Letesson),[email protected](T.Arnould).
ContentslistsavailableatScienceDirect
Biochemical
Pharmacology
j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm
http://dx.doi.org/10.1016/j.bcp.2015.02.007
1. Introduction
Mitochondria are dynamic organelles with a morphology
controlled by fusion and fission events [1] that evolved from endosymbiotic
a
-proteobacteria belonging to Rickettsia gender[2].Theystillpresentmanysimilaritieswithprokaryoticcellssuch
as a double membrane, the ability to produce ATP through
thegenerationof a protongradient generatedacross theinner
membrane or the fact that they have their own genome and
bacterialtyperibosomes[2].Thebacterialoriginofmitochondriais furthersupportedbythefactthatvariousantibiotics,especially bactericidal ones such as quinolones, aminoglycosides and
b
-lactams are alsoable to inducemitochondrial dysfunction and reactiveoxygenspecies(ROS)production[3].The mitochondrial DNA (mtDNA) encodes two ribosomal,
22 transfer RNA and only 13 peptides of the mitochondrial proteins involved in the oxidative phosphorylation (OXPHOS) system.Mostofthemitochondrialproteomeisthusencodedby thenucleargenome[4].
Mitochondrial double membrane resultsin theformation of foursub-compartments. Firstly, theouter mitochondrial mem-brane(OMM) contains numerousporins that make it passively permeable to small molecules (<5kDa). Secondly, the inter-membranespace(IMS)sequestersnumerousofproteinsactingas damages-associatedmolecular patterns(DAMPs), such as cyto-chrome c,endonuclease G,apoptosis-inducing factor (AIF), and several pro-caspases, which are also recognised by pattern recognitionreceptors(PRR)[5].Indeed,theirreleaseinthecytosol will induce inflammation and/or cell death. As subversion of mitochondrial death pathways has already been extensively reviewed [6], mechanisms related to apoptotic cell death will notbedevelopedinthisreview.Thirdly,theinnermitochondrial
membrane (IMM) contains the different complexes of the
respiratoryelectrontransportchain(complexesI–IV)as wellas theFo-F1ATPsynthase(complexV),whichareresponsibleforATP productionbytheOXPHOS.Thismembraneishowevermuchmore impermeablethantheOMM.Furthermore,cardiolipin,a phospho-lipid found exclusively in inner mitochondrial membrane and bacterialplasmamembrane,maketheIMMlessfluid[7]. Conse-quently,metaboliteshavetouseavarietyofselectivetransporters tocrosstheinnermembrane.Thesurfaceofthismembraneforms cristaetoincrease theabilityto produceATP in thematrix. In addition,many mitochondrial proteins encoded by thenucleus willneedtheimportandsortingmachinerypresentinbothOMM andIMMtoreachthedifferentsub-compartments[8].According tothecompartmenttheyreach,proteinswillusedifferenttransport complexes:translocaseoftheoutermembrane(TOM)/translocase of the inner membrane 23 (TIM23)/presequence
translocase-associated motor (PAM) for the matrix, TOM/TIM23 or TOM/
TIM22forIMM,TOM/mitochondrialinter-membranespace assem-bly(MIA)forIMSandTOM/sortingandassemblymachinery(SAM) for
b
-barrelproteinslocatedintheOMM[8].Finally,thematrix containsmultiplecopiesofmtDNAorganisedintonucleoidsaswell as the machinery that is necessary to transcribe and translate mtDNA-encodedgenes. Reducingagents(NADHand FADH2)are alsogeneratedinthematrixbythetricarboxylicacid(TCA)cycleand thefattyacidb
-oxidation(FAO)[9].Evenifmitochondriastillsharesomefeatureswithitsbacterial ancestor,theorganellealsoacquirednewcharacteristicssuchasa dynamicmorphologyof themitochondrial networkthat affects bothmitochondrialactivityandfunction.Accordingtocelltypes andfunctionalstatus,mitochondriacanthusshiftfromseparated rounded/fragmentedmitochondriaintointerconnectedand elon-gated tubular network [1]. This very dynamic organelle thus continuouslyadaptsthemorphologyandmovetospecificcellular sub-compartmentsusingdifferentcomponentsofthecytoskeleton
[1].Themitochondrialmorphologyisdeterminedbythebalance betweentwoopposingprocessesthatoccurcontinuallyinthecell: themitochondrialfissionandfusion thataremediatedbylarge GTPases related to the dynamin superfamily [10]. The fusion occurs in two steps: first the fusion of OMM mediated by the homo-/heterodimerisation of mitofusin1/2 (MFN1/2) and then opticatrophy1(OPA1)that formshomodimersleadingtoIMM fusion. Fission process requires the recruitment of dynamin-relatedprotein1(DRP1)totheoutermitochondrial membrane, whereitwillassembletoformaconstrictionringleadingtothe fission. Four different receptors for DRP1 located in the outer membrane have beenidentified so far:mitochondrial fission 1 (FIS1), mitochondrial fission factor (MFF) and mitochondrial dynamicsproteinof49and51kDa(MID49andMID51)[1].Itis important to note that mitochondrial morphology influences themitochondrial (dys)function while mitochondrialfunctional status also controls the dynamics and shape of the organelle
[11].Indeed,extremelydepolarisedandfragmentedmitochondria aredegradedbymitophagy,aspecificformofautophagy[1].The best-characterisedmitophagypathwayinvolves therecruitment ofParkin(anubiquitinligase)fromthecytosoltotheOMM by PTEN-induced putative kinase 1 (PINK1). This relocation also allowsParkintopoly-ubiquitinateproteinslocatedintheOMM, leadingtotheirdegradationbythe26Sproteasome[12]. 2. Mitochondrialtargetingbybacteria
Whiletheimpactofthemitochondriafunctionalstatusonthe efficiency and persistence of infection and/or trafficking (for intracellular bacteria) is still poorly understood, the effects of bacteria infection on several parameters of the mitochondrial populationstarttobebetterdelineated.
First,toimpactmitochondria,bacterialeffectorsneedtocross severalbarriers.Theyhavefirsttobesecretedoutofthedifferent layers of the bacterial envelope through dedicated secretion systems,thentoreach (andpassthrough)thehostplasmaand organelle membranes. Several bacterial effectors, collectively calledABtoxin,once secretedin theextracellularmedium, are abletotranslocateinsidetheeukaryoticcellcytoplasm[13].TheB domainisresponsibleforthecellulartropismandofteninduces thereceptormediated-endocytosis[14]oftheholotoxinfollowed bythetranslocation oftheAdomainintothecytoplasm ofthe targeted cell. The A domain has an activity responsible for the‘‘toxiceffect’’suchastheglycosyltransferaseactivityofthe ClostridiumdifficiletoxinB(TcdB)[14]ortheporeformingactivity of the Helicobacter pylori VacA toxin [15]. Moreover, in Gram-negative bacteria, somecomplex secretion systems are able to delivereffectorsdirectlyfromthebacterialcytoplasmintohost cytoplasm [16] as observed for EspF of the enteropathogenic Escherichia coli [6]. It is important to note that, to impact mitochondria, effectors do not necessarily have to enter host cells. Indeed, pore-forming toxins can induce mitochondrial dysfunction and organelle fragmentation just by inducing ion fluxesthroughtheplasma membrane(e.g.listeriolysin (LLO)of Listeriamonocytogenes[17]).Onceinhostcells,toxinsthatdirectly target mitochondria have to enter and reach the appropriate mitochondrial sub-compartment. They usually interact with mitochondrial translocase complex to be imported (e.g. OMM: NeisseriaPorB[18],IMM:H.pyloriVacA(vacuolatingcytotoxinA)
[15] and mitochondrial matrix: enteropathogenic E. coli MAP effector[19]).
Asmitochondriaevolved fromanancestralbacterium, some similaritiesareobservedbetweenproteinscontaining mitochon-drial targeting sequence (MTS) and sequences targeting to
bacterial cytoplasmic membrane such as the presence of a
acidsequencesthatarecomposedofpositivelychargedresidues locatedattheN-terminaldomainoftheprotein[20,21]. Further-more,NeisseriaPorBuses anevolutionaryconserved ‘‘bacterial-like’’machinerytoreachtheOMM ofinfected cells.Indeed,the SAM complex, the functional equivalent of
b
-barrel assembly machinerylocatedatoutermembraneofgram-negativebacteria, iscomposedofseveralsubunits[18].Evenifthedifferentsubunits areessentialfortheinsertionofeukaryoticb
-barrelproteininto theOMM,PorBinsertionisexclusivelydependentontheSam50 subunit,theonlysubunitforwhichabacterialhomologdoesexist[18].
3. Energymetabolism
In mammalian cells, ATP is mainly produced from the
catabolism of glucose and fatty acids by co-regulated and interconnectedpathwaysthatinvolvesglycolysisand
b
-oxidation, respectively,followedbyTCAcycleandOXPHOS.Afteritsentryin the cell, glucose is phosphorylated by hexokinases forming glucose-6-phosphate(G6P),whichpreventsthereleasefromthe cell and allows its glycolytic degradation [22]. Glycolysis is a cytosolic10-reactionsmetabolicpathwayleadingtothe conver-sionofG6Pintotwomoleculesofpyruvateaccompaniedbytheproduction of two ATP and two NADH. The pyruvate is then
importedandconvertedinacetyl-CoAinthemitochondrialmatrix, whereit willbefully oxidisedinto CO2 by TCAcycle,allowing the formation of more reducing molecules such as NADH and FADH2.Reducing agents, includingthose generated byFAO are thenoxidised,viatheOXPHOStogenerateaprotongradientcross theIMM.Theproton-motiveforceofthisgradientwillnextbeused bytheFo-F1ATPsynthase(complexV)toproduceATP[22].Besides theproductionofATP,mitochondriaalsocontribute,atleastpartly, tothebiosynthesisoflipids[23],steroids,aminoacids,nucleotides andintheassemblyofiron-sulphurclusters[24].
3.1. Mitochondrialmetabolismandimmuneresponse
UnliketheparagraphsaboutmitochondrialROS,calciumand morphologydescribedbelowinwhichwewilldevelopexamples of direct manipulation of mitochondrial functions by bacterial effectors,thepresentsectionaboutmetabolismwillonlypresent generalandglobalimpactofbacterialinfectiononcellmetabolism, thataffectmitochondria.Someoftheexamplesdescribedmight thusrefertoindirecteffectsofbacterialinfectiononmitochondria, taking part in a broader metabolic reprogramming during cell activationthatmightsubsequentlyaffecttheorganelle.
Whilethecommonpathwaytoproduceenergyfromglucosein mammaliancellsincludestheOXPHOS[22],insomeconditions, cellsusepreferentiallyandalmostexclusivelyactivatedglycolysis toproduceATP,eveninthepresenceofoxygen,aconditionthat coincides with a repression of the TCAcycle and theOXPHOS
[9].Thisphenomenon,calledaerobicglycolysisorWarburgeffect, wasfirstdescribedintumourcells inwhichitallowstosupply the anabolic demand associated withthe high proliferation of these cells [25]. Indeed, the partialglucose oxidation provides precursorsforthebiosynthesisofcarbohydrates,fattyacidsand amino acidsas wellasnucleotides and NADPHbythepentose phosphate pathway (PPP), a pathway that also oxidises G6P
[26]. As the biosynthesis of new molecules consumes both glycolysisandTCAcycleintermediates,anapleroticreactionsare requiredtoreplenishthosepathways. Oneofthose reactionsis theglutaminolysisthatconsistsinthedegradationofglutamine into
a
-ketoglutarate,anintermediateoftheTCAcycle[9].More recently,several studies have shown that theaerobic glycolysiscancontributetodifferentphysiologicalprocessessuch asthematuration/differentiationand theactivation ofimmune
cells duringinfection,including a severe metabolic reprogram-ming [22]. In fact, activated innate immune cells, including neutrophilsanddendriticcells(DCs),mostlyrelyonglycolysisand glutaminolysistoproducetheirenergy[22].In neutrophils,this metabolicshiftfromoxidativemetabolismtoglycolysisisunder thecontrolofToll-likereceptor(TLR)inducedhypoxia-inducible factor1alpha(HIF-1
a
)[22].ItincreasestheproductionofNADPH bythePPP,whichisrequiredtofueltheoxidativeburstbyNADPH oxidase(NOX)systemactivatedinresponsetomicrobialexposure[22].However,evenifthemitochondrialrespirationisnotrequired for cell ATP supply, the mitochondrial membrane potential is involved in the temporal regulation of apoptosis induction following neutrophil activation as the elimination of activated neutrophils by this cell death pathway is crucial to prevent unnecessaryextendedinflammation[27].InDCs,the reprogram-mingduringactivationisdependentonTLR-inducedHIF-1
a
and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt/PKB) pathwayand seemstoberequiredfortheantigenpresentation andcytokineproduction[22].Thismetabolicshiftcouldalsobe supported by a stronger recruitment of the glycolytic enzyme hexokinaseII(HKII)tothemitochondria[28].Besideimmunecellactivation,metabolismisalsoinvolvedin celldifferentiationandpolarisation.Indeed,macrophages differ-entiate from circulatingmonocytes once theyinfiltrate tissues.
Circulating monocytes, according to the environment they
encounter,areimmunologicallyimprintedtotolerant (immuno-suppression)ortrained(innateimmunememorywhichisantigen unspecific)[29].Thisimprintingwilldeterminefunctionalfateof monocytes and macrophages, which is caused by a glycolytic switch under the control of the activation of Akt/mammalian targetofrapamycin(mTOR)/HIF-1
a
pathway[30].Macrophages candisplaydifferentphenotypesaccordingtheirlocationandthetype of stimuli they respond to. The two best phenotypes
characterisedareM1andM2macrophages[22].Thepolarisation M1(classicalactivation)isinducedinresponsetodifferentpathogen associatedmolecularpattern(PAMPs)suchaslipopolysaccharide (LPS) and interferon-gamma (IFN
g
). On the other hand, the M2 polarisation (alternativeactivation) is developed in response to interleukins-4 and -13 (IL-4 and IL-13), two cytokines. M1 macrophages take part in the acute phaseof inflammation and aremoreefficienttoeliminatebacteriawhileM2macrophagesare mainlyinvolvedintheresolutionphaseofinflammationandhealing process [31].Classical macrophageactivation inresponse toLPS exposureislinkedtotheactivationofglycolysis[32].Inaddition,in LPS-stimulatedmacrophages,glutaminolysis-deriveda
-ketogluta-rateleadstotheaccumulationofsuccinate,which,onceexported from the mitochondria, is able to stabilise HIF-1a
, the master regulatorofmetabolicswitchassociatedwithmacrophageclassical activation[32].However,mitochondrialmetabolismisalsoinvolved inM1macrophagefunctionsasmtROStakepartintheirbactericidal activityandpro-inflammatorycytokinesecretion(seeSection4.1.1). While M1macrophages mainlyrely on aerobicglycolysisto acquire their bactericidal activity,M2macrophageactivation is supportedbyFAOandmitochondrialoxidativemetabolism.This metabolic programme is regulated by signal transducer and activator of transcription 6 (STAT6), a masterregulator of Th2 polarisation in the immune response. This transcription factor controls the expression of peroxisome proliferator-activated receptors (PPARs) and PPAR gamma coactivator-1 beta (PGC-1b
),well-knownregulatorsthatpromotemitochondrial biogene-sistosustainthisactivation[33].Adaptive immune cells, undergo several phases after an activating/challengestimulus.Indeed,Tlymphocytes,aftertheir activationbytheDC-presentedantigenrecognition,will prolifer-ateintoclonalpopulationsandthenestablishanantigenspecific memory[22].Atthebasalstate,CD4+T-cellsproduceenergyusing
theoxidationofglucoseandfattyacidsandtheOXPHOSpathways
[34].Whenactivated,theyalsoswitchfromoxidativemetabolism to glycolysis and glutaminolysis. The glycolytic switch is also inducedina PI3K/Akt-dependentpathway,inresponsetoCD28 binding[35].Additionally,T-cellreceptor(TCR)binding,activates extracellular signal regulated kinase (ERK)/mitogen-activated proteinkinase(MAPK)pathway,thatinturn,inducesglutamine uptake and promote the expression of catabolic enzymes of theglutaminolysis[36].Metabolismmightalsoplayanimportant roleintheactivationof CD4+ T-lymphocytesby controllingthe expressionofkey signallingmolecules,suchIL-2and IFN
g
.The mechanismwouldinvolveglyceraldehyde3-phosphate dehydro-genase(GAPDH),oneoftheenzymesoftheglycolysis,thatbinds the30-untranslatedregion(UTR)ofIL-2andIFNg
mRNAsleading totheinhibitionoftheirtranslation.Whenglycolysisisactivatedin T cells, GAPDH is required to promote the glycolytic flux and, consequently,isnotabletobindthesemRNAsanymore,allowing thesynthesisofIL-2andIFNg
byactivatedCD4+Tcells[37].MemoryTcellsarealsomodulatedbymetabolism.Forinstance, CD8+Tcellsareabletoenterinquiescentstatetoparticipatein thememoryresponse,allowingfasterandstrongeractivationin response tosubsequent pathogen exposure. Unlike activatedT cells,memoryTcellsproducetheirenergybythemitochondrial oxidativemetabolismandespeciallybytheFAO[38].Forexample, TNF
a
receptor-associated factor 6 (TRAF6) induces FAO and mitochondrial biogenesis required for the establishment of a memory response against L. monocytogenes and this process is dependent on AMP-activated protein kinase (AMPK) signalling[38].The importanceofmetabolism forT-cellsduringbacterial infectionhasrecentlyandextensivelybeenaddressed[39].
This shift fromoxidative metabolism to glycolysisobserved duringimmunecellactivationseemstobeageneralresponseto microbialexposureasitisalsoobservedinvarioustissuessuchas livercellsfrominfectedmiceexposedtothepathogenicbacteria Salmonella enterica serovar typhimurium (S. typhimurium), lung cells from Mycobacterium tuberculosis-infected mice, intestinal epithelia fromL. monocytogenes-infectedmice and HepG2 cells infectedbyChlamydiapneumoniae.Inallthesemodels,itwasalso shownthatthemetabolicreprogrammingwasunderthecontrolof HIF-1
a
[9].3.2. Manipulationofmitochondrialmetabolismbybacterial pathogens
Legionella pneumophila is known tosecrete a mitochondrial carrierproteinduringinfection.Thiseffector,targetedtotheinner mitochondrialmembrane,isabletotransportATPfromthematrix to the IMS. However, the mechanism by which this effector supports L. pneumophilainfection still needs to be determined
[40].AsEisenreichandcollaboratorshaverecentlyandextensively reviewed the multiple facets by which metabolism could be affected/manipulatedbybacterialpathogens[9],wewillfocusin therestofthereviewonmitochondria-associatedcellsignalling andmitochondrialmessengerproductionaffectedbybacteria. 4. Mitochondria-associatedcellsignalling
4.1. mtROSandmitochondria
Mitochondrialrespirationisresponsiblefortheproductionof mtROSas metabolic by-products.Indeed,mtROS are generated throughthemonovalentreductionofamoleculeofO2byelectrons leakingfromthemitochondrialelectrontransportchain(ETC)[41]
(Fig.1A).mtROSabundanceishowevertightlyregulatedattwo differentlevels:theirproductionandtheirdegradation.First,the
quantity of mtROS produced depends on the mitochondrial
respiration rate. Indeed, an increase in mtROS production is observedwhentheproton-motiveforceisenhanced [41]. Addi-tionally,ithasbeensuggestedthatuncouplingprotein2(UCP2),a ubiquitously expressed uncoupling protein, is involved in the regulation of mtROS production [42,43] as, when activated, it reduces theprotongradientacrosstheIMM.However,it isnot
clear whether UCP2 down-regulates mtROS production only
throughitsputativeuncouplingactivityorbytheother mecha-nismsuchastheregulationoftheglutathionesystem[44].
mtROSdegradationisalsotightlyregulated.Indeed,O2()are converted inH2O2 by superoxide dismutases(SODs). Thethree membersofthisenzymefamilydisplaydifferentlocationsinthe cell:SOD1locatedinthemitochondrialIMSandcytosol,SOD2in themitochondrialmatrixandSOD3isanextracellularform.H2O2 isthenabletodiffusefreelyacrossthemitochondrialmembraneto reachtocytosol.InadditiontoSODs,thereareotherenzymesthat are able to detoxify H2O2: peroxyredoxins (PRXs), glutathione peroxidases (GPXs) and catalase. On the one hand, GPXs are thoughttoscavengemtROSwhenfoundathighlevelstoprevent damagesintheorganelleandkeeptheconcentrationofmtROSin therangeofconcentrationsthatiscompatiblewithROSsignalling function.Ontheotherhand,PRXs,which haveahighsubstrate affinityandarehighlyexpressed,arethoughttobeinvolvedinthe terminationofmtROSsignallingthatoccursatlowconcentrations
[41].Animportantpointthatneedstobetakenintoaccountinthe modulation of mtROS signalling is the mitochondrial location itself.Indeed,mtROSareshort-livedmolecules,sotheirefficiency dependsontheproximitybetweenthesiteofproductionandthe siteoftargets[41].
Inallofthestudiesmentionedinthisreview,itisassumedthat mtROS are only generated in response to bacteria exposure. However,itisimportanttonotethatmostofthestudiesreferenced below detected and/or quantified (mitochondrial) ROS content using older technologies based on fluorescent probes such as dihydroethidium (DHE and MitoSox, its mitochondrial targeted form)ordichlorodihydrofluoroscein(DCF)probes.Theseprobes, evenifcommonlyusedandaccepted,presentsomedisadvantages
(when compared to more recent methods) such as a lack of
specificityorevenanartificialinductionofROSproductionwhen toohighconcentrationsareusedastheycanreactwithoxygenand thusartificiallyelevateROSabundance.Inthefuture,studiesto analyse ROS in cells exposed to bacteria should opt for new detectionmethodssuchasfluorescentprotein-basedredoxprobes. For additional information, Dikalov and Harrison recently reviewed the advantages and limits of various old and recent methodsforROSdetection[45].
4.1.1. mtROSandimmunity
4.1.1.1. mtROSandPRR. mtROShavebeenreportedtobeinvolved in the clearance of differentintracellular pathogens such as L. monocytogenes, S. typhimurium or Toxoplasma gondii [43,46– 48].During infection,mtROSproductionis inducedin response toPRRactivation.PRR-inducedsignallingleadstotheactivationof nuclear factor-kappaB (NF
k
B) and interferon regulatoryfactor(IRF) pathways, that both promote the expression of
pro-inflammatory cytokines and chemokines as well as type I
interferon[49].Thosemediatorsarepartofanarsenalrequired forthedevelopmentofanadequateimmuneresponseleadingto the eradication of pathogens [49]. PRR family contains three different classes of receptors sensing different PAMPs/DAMPs leadingtothedevelopmentofaneffectiveimmuneresponse:TLR, Rig-Ilikereceptor(RLR)andNOD-likereceptor(NRL)[49].
TLRarereceptorslocatedattheplasmamembraneoratthe endosome/endoplasmicreticulum(ER)membranesthatcansense PAMPs associated with bacteria, viruses, fungi and parasites
[49].InLPS-stimulated macrophages,UCP2expressionis down-regulated through cell signalling dependent on Jun N-terminal kinase (JNK)/p38 pathways, promoting mtROS production
fol-lowed by a subsequent MAPK activation and the onset of
respiratory burst [43]. More recently, it was shown that, in RAW264.7 macrophages, TLR signalling is able to trigger the mtROSproductionand therecruitmentofmitochondriaaround the phagosome [47]. Indeed, in response to TLR1/2/4 binding, TRAF6istranslocatedatthemitochondriawhereitinteractswith evolutionarilyconservedsignallingintermediateinTollpathways (ECSIT),aproteinthatcontributestotheETCcomplexassembly. TRAF6interactionwithECSITthenpromotesitsaccumulationat the mitochondrial surface, triggering an increase in mtROS production[47](Fig.1A).
ItisimportanttonotethatwhileTLRsinducepro-inflammatory responsesinordertoeradicateinvadingpathogens,theyalsotake partinresolutionofthisinflammation/oxidativestressasinorder tomaintainmitochondriaintegrityandtopreventcelldeath,host cells are able to respond by the activationof a transcriptional
programmeleadingtotheexpressionofgenesinvolvedin anti-inflammatoryandanti-oxidantresponsessuchasIL-10andheme oxygenase-1(HO-1),respectively.Thisprocessisaccompaniedby astimulationofmitochondriabiogenesis[50].Forexample,a LPS-mediatedTLR4challengemightalsoleadtothedevelopmentofa cellresponseaimingtorecoverfromenergyimpairment[51].Itis knownthat,inmacrophages,theearlyeffectofaLPSstimulationof TLR4 is mitochondrial dysfunction, which, if prolonged,can be detrimentalforthehostcell.TLR4,byitsinteractionwithmyeloid differentiation primary responses gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-
b
(TRIF), is able to activate the pro-survival PI3K/Akt pathway leading to the expressionofthemitochondrialtranscriptionfactorA(TFAM).This signallingpathwayleads,infine,toanincreaseintheabundanceand activityoftwodifferentsubunitsoftherespiratorychaincomplex IV,cytochromeoxidase-1and-4(COX1andCOX4),encodedbythe mitochondrialandnucleargenomes,respectively[51].Alternatively, mtROS can also regulate TLR signalling. For example, nuclear factor of activated T-cells 5 (NFAT5) is a
Fig.1.Mitochondria-associatedredoxsignallinginhost–pathogenrelationship.(A)mtROSandimmuneresponse.mtROS(yellowstars)areproducedasby-productsof mitochondrialrespiration.TheyareinvolvedinPRRregulation.(1)ActivationofTLR1/2/4leadstomtROSproductioninaTRAF6/ECSIT-dependentmanner.ThesemtROS participatetobacterialkillingsuchasL.monocytogenes[46,47].(2)mtROSpromoteRLRsignallingbyactivatingMAVSdependentpathway.MAVSsignallingisprimarily involvedinantiviralresponse(fullarrow)butitcanalsotakepartinbacterial-inducedinterferon-bproduction(e.g.duringL.pneumophilainfection)(dottedarrow)[53,56]. (3)NLRX1isabletopromotemtROSproduction[120]whilemtROScaninduceNRLP3[58].mtROScanalsomodulatecytokinesignalling(4)byinducingtheirtranscription
[69,70],(5)bymodulatingtheirreceptoravailability[71]or(6)bytakingparttothecytokine-inducedeffect[46].Greenarrows:positiveeffect;redarrows:detrimental effect.(B)ExamplesofbacterialpathogensmanipulatingmtROSproduction:M.tuberculosis[73],C.trachomatis[76],E.chaffeensis[84],P.aeruginosa[83],H.pylori[79],heat killedE.coli[80]andASD-associatedmicrobiome[82].Thetablesummarisesthebacterialspecie,theeffectorinvolved,thetargetcell/tissue,theeffectorimpactonhostcell andthefinalphenotypeobserved.Abbreviations:ECSIT(evolutionarilyconservedsignallingintermediateinTollpathways),FAO(fattyacidb-oxidation),IFNg(interferongamma), IL-1b/2/6(interleukin-1b/2/6),MAVS(mitochondrialantiviralsignallingprotein),MCP1(monocytechemoattractantprotein-1),mtROS(mitochondriareactiveoxygenspecies),NLR (NOD-likereceptor),OXPHOS(oxidativephosphorylation),PGC-1b(peroxisomeproliferator-activatedreceptorgammacoactivator1-beta),STING(stimulatorofinterferongenes), TACE(TNFaconvertingenzyme),TCA(tricarboxylicacid),TLR(Toll-likereceptor),TNFR(TNFareceptor)andTRAF6(TNFreceptor-associatedfactor6).
transcriptionfactoractivatedinresponsetodifferentstimulisuch as LPS-inducedTLR4 signalling or NaCl induced-osmotic shock
[52]. A recent study showedthat these stimulilead to a ROS-dependentexpressioninhibitionofasubsetofNFAT5targetgenes
[52].Inaddition,theseauthorshaveshownthatROSoriginseems tobecrucialtoqualitativelyselectgenesregulatedbythisfactor ascytosolicROS(generatedbythexanthineoxidase)willpromote a pro-inflammatory response (IL-6 transcription) while mtROS inducehypertonicresponse[52].
RLRarecytoplasmicreceptorsbindingmostlyvirus-associated PAMPssuchasdoublestrandedRNA[49].Mitochondriaarealso known to provide a platform for signalling induced by two different RLR: RIG-I and melanoma differentiation-associated gene-5(MDA-5).RIG-IorMDA5hasbeenshowntosignalthrough mitochondrial antiviral signalling protein (MAVS), a nuclear encoded protein located in OMM [53]. When activated, MAVS interactswithstimulatorof interferongenes(STING)locatedin
the ER membrane and TRAF3/6 leading to IRF3/7 and NF
k
Bactivation[49].
TheexpressionofRLRmightalsoberegulatedandcontrolled bymtROS;cellsdeficientforautophagyprotein-5(ATG5/cells), thatcontainhighmtROSconcentrations,up-regulateRLR expres-sionaswellasactivesignallingandarealsomoreresistanttoviral infection [54] (Fig. 1A). However, mtROS are not the only
mitochondrial feature to regulate MAVS pathways. Indeed,
mitochondrial dynamics and membrane potential are also
requiredforefficientMAVSsignalling[53].Furthermore,different mitochondrial proteins have beenreported toact as activators suchasTOM70orinhibitorssuchasmitochondrialE3ubiquitin proteinligase1(MUL1)ofMAVSsignalling[53].
Interestingly, the mitochondrial protein ECSIT is not only involvedintheregulationofTLRsignallingasitalsoparticipatesin RLRinducedsignalling.Infact,ithasbeenshownthattripartite motif59(TRIM59)is abletoinhibitRLR-inducedIRFand NF
k
B activationbyitsinteractionwithECSIT[55].WhiletypeIinterferon istypicallyconsidered asanantiviralcytokine[56],ithasbeen shownthat the gene encoding this cytokine is alsoinduced in responsetobacterialinfection[56].Forexample,ithasbeenshown thatRIG-I/MDA-5/MAVSpathwayisinvolvedintheinductionof IFNb
in response to L. pneumophila, a facultative intracellular bacterium(Fig. 1A).Furthermore, authors showedthat SdhA, a bacterial effector translocated in host macrophages during L. pneumophila infection, is able to inhibit the IFNb
production probablybyinhibitingtheMAVSprotein[56].NLRisthethirdtypeofPRRs.Inresponsetothedetectionof cytosolicPAMPs/DAMPs,NLRscaninduceacaspase1-dependent maturationofpro-inflammatorycytokinessuchasIL-1
b
andIL-18[49]. The most-described family member is NLRP3 and recent studies showed that mitochondria play a crucial role in the activation of NLRP3 inflammasome by both the production of mtROS (Fig. 1A) and the mtDNA release out of the organelle
[57,58]. NLPR3 activation requires the combination of two different signals. The priming signal, coming from TLR4 and cytokinesreceptors,thateitherinducesNLRP3transcriptional up-regulation [59] and/or NLRP3 deubiquitination, in a mtROS-dependent manner [60]. The second signal, which leads the oligomerisation,canbetriggeredbydifferentPAMPsandDAMPs, includingextracellular ATP, lysosomal destabilisation, (mt)ROS, oxidisedmtDNAandporeformingtoxins-inducedionsfluxessuch as K+ efflux, a mechanism used by several bacteria such as L. monocytogenesorStaphylococcusaureustoactivateNLRP3 inflam-masome[61].Themolecularmechanismsbywhichthosedifferent signalsactivateNLRP3arestillpoorlyunderstood[61].However, NLRP3 activation seems to depend on a NLRP3 translocation
from ER to mitochondria, in a MAVS-dependent manner [62]
[58].Inmacrophages,mitochondrialdysfunctioncausedbyNLRP3
inducers (such as nigericin and monosodium urate) leads toa decreaseinNAD+concentration,whichinturnleadstoadecrease insirtuin2(SIRT2),aNAD+-dependentdeacetylase,activityleading to theaccumulation of acetylated
a
-tubulin that takes partindynein-dependent mitochondrial transport by microtubules
[63].Theseauthorssuggestthatcytoskeleton-dependenttransport of mitochondria would promote NLRP3 activation by apposing mitochondrialapoptosis-associatedspeck-likeproteincontaining a CARD(ASC)and NLRP3(on theER) [63].No matterwhatthe molecularmechanismofactivationis,itseemsthatmitochondrial dysfunction-mediatedNLRP3activationisalsorequiredtocontrol theinfectionofseveralfacultativeorobligateintracellularbacteria suchasBrucellaabortus[64]orC.pneumoniae[65].Interestingly, somebacterialpathogensarealsoabletomodulateNRLactivation asitisknownthatNRLP3canbeinhibitedbytoxinssecretedby several bacteriasuchasYersiniaenterocolitica (YopEandYopT), Yersinia pseudotuberculosis (YopK), M. tuberculosis (Zmp1) and Streptococcuspneumoniae(pneumolysin).Thesetoxinscaneither
block inflammasome oligomerisation, prevent ligand to be
recognised or modulateNF
k
B/MAPKsignalling toimpair IL-1b
expressionandactivation[66].
Finally,bacteriaactivateotherinflammasomesuchasabsentin
melanoma-2 (AIM2) inflammasome, which recognise cytosolic
double stranded DNA. It hasrecently been shown that mtROS might potentiate indirectactivation of AIM2 inflammasome in responsetoFrancisellainfection[67].Theseauthorsshowedthat mtROSmightbeinvolvedintheregulationofAIM2activationby promotingbacterial DNA release.Indeed, Francisella novicida (a non-virulentstrain)issusceptibletomtROSinduced-membrane damages,whileFrancisellatularensis(apathogenicstrain)isnot, leading to a differential DNA release in the cytosol and a differentialAIM2activation[67].
4.1.1.2. mtROSinductionduringinfection. AsmtROSproductionin response to infections is dependent on respiration, different strategiescanbedevelopedtoincreasetherespiratoryflow:(1)
promoting the mitochondrial biogenesis and (2) enhancing
OXPHOSactivitythroughFAOfuelling.Indeed,thosehypotheses were verified in infection models. First, in IFN
g
-stimulated macrophages,theactivationofoestrogen-relatedreceptoralpha (ERRa
)andPGC-1b
,twokeyregulatorsofmitochondria biogene-sis[50],leadstoanincreaseintheexpressionofseveralOXPHOS components [46]. Second, a recent study on S. typhimurium infectionsuggeststhatmtROSproductioninresponsetobacterial infection depends on FAO [68]. Indeed, the activation of glucocorticoid receptor and the stimulation of Janus kinase (JAK)/STATsignallingpathwaypromoteaCCAATenhancerbinding protein (CEB/P)and STAT3-dependent transcription of immuno responsive gene-1 (Irg1) encoding a mitochondrial proteinthat regulatesthemitochondrialuptakeandFAO,andisrequired(but notsufficient)formtROSproductionbymurinemacrophagesin responsetoS.typhimuriuminfection[68].4.1.1.3. mtROS and cytokine signalling. mtROS are known as modulators of cytokine signalling involved in immunity, as demonstratedinvariousmodels[41].Thefirstlevelofregulation is themodulation ofcytokine synthesis. Indeed, anincrease in mtROSabundanceisobservedinthespleenofL. monocytogenes-infectedmiceinvalidatedforUCP-2(Ucp-2/mice),a phenome-nonthatisaccompaniedwithbothanincreaseinexpressionof pro-inflammatory cytokines such as IFN
g
, IL-6, IL-1b
and monocytechemotacticprotein-1(MCP1)andadelayofinfected animal death [69] (Fig. 1A). Additionally, in T lymphocytes activation, mtROS generated in response toTCR-mediated Ca2+ influx,allowtheactivationofthetranscription factorNFATand subsequentIL-2 production [70].Secondly, mtROS arenot onlyable to regulate cytokine expression but also control their signallingefficiencybyindirectlylimitingtheavailabilityoftheir receptor.Forexample,TNFreceptor-1(TNFR1)bindingleadstoa Ca2+-dependentmtROSproduction[71].Subsequently,mtROSare able toactivate theprotease TNF-
a
converting enzyme (TACE) leadingtothereceptorsheddingwhichlimitsfurtherorprevent uncontrolledinflammation[71](Fig.1A).Finally,mtROScanalso takepartinthecytokine-induceddownstreamsignallingasithas been shown, in L. monocytogenes infected-macrophages, that bactericidaleffectofIFNg
isdependentonPGC-1b
/ERRa
induced mtROS[46](Fig.1A).TheimportanceofmtROSintheregulationof aproperimmuneresponsesignallingwasrecentlysupportedby theobservationthatsplenocytes,isolatedfromUcp-2/mice,that containhighmtROSconcentrations,failed toproduce immuno-globulinG(IgG)inresponsetoLPSstimulationbothinvitroand invivo[72].Insummary,itseemsthatROScanparticipatetotheimmune response in different ways: ROS can induce bacterial damages leadingtoantimicrobialactivityorareabletomodulateinfected eukaryotecellsignalling.
4.1.2. Modulationofredoxsignallingbybacteria
Considering the deleterious effects of ROS for bacteria in infectedcells,itnot surprisingthatseveralbacteriadeveloped strategiestolimittheirproduction.Forexample,M.tuberculosisis knownto decreaseROS production by theN-acetyltransferase activity of the effector enhanced intracellular survival (Eis). Indeed,whencellsareinfectedwithmutantMycobacteriathatdo not expressEis,anincrease in JNK dependent-ROSproduction accompaniedbythestimulationofautophagyandan inflamma-toryresponseisobserved[73].ItseemsthatEisdown-regulates
the LPS-mediated phosphorylation of JNK by acetylating
and activating MAPK phosphatase-7 (MKP7), a JNK-specific
phosphatase[74](Fig.1B).
IfROSareprimarilyahostdefencemechanism,theycanalsobe detrimentalforthehostcells.TheROSdualeffectininfectedcellsis wellillustratedin M.tuberculosis-infectedmacrophages.Indeed, tumour necrosis factor alpha (TNF
a
) binding to its receptor triggersmtROSproduction,whichinitiallytakepartinbactericidal activity[75].However,inthismodel,mtROSwillrapidlyinduce necrosis, which results in bacterial release in the permissive extracellular environment[75].Some bacteria even manipulatehost cell to promote mtROS production. Indeed, Chlamydia
trachomatis, an intracellularbacterium responsiblefor a severe sexuallytransmissibledisease,inducesROSproductiontopromote itsgrowth[76].Indeed,onceinsidetheepithelialcell,thebacteria secreteeffectorsintothecytoplasmthroughthetype3secretion system(T3SS),whichleadstoapotassiumexitfromthecytoplasm. ThispotassiumeffluxleadstoROSproductionbytheNOXsystem, whichinturns,diffusetothemitochondrialmatrixandactivate NLRX1toproducemoreROSintheformofmtROS[76](Fig.1B). ROS inductionin C. trachomatis infected cells takes partin the activationof caspase-1 bythe NLRP3 inflammasome,a process requiredforitsintracellulargrowth[76,77].
mtROScanalsodirectlycausethesymptomsassociatedwith the disease. For example, during H. pylori infection, redox signallingtakespartintheinflammationandoncogenesisofthe gastricmucosaeobservedinpatients[78],aphenotypethatcould beexplainedbythemtROSproductiontriggeredbythepathogen anditsabilitytoinhibitDNArepairmachinerycausingnuclearand mitochondrialgenomicinstability[78](Fig.1B).Indeed,mtDNA, eveninthenucleoidorganisation,isparticularlysensitivetothis genomicinstabilityconsideringitsproximitytothesiteofmtROS production and the absence of histone complexes [79]. TLR4 dependentmtROSaccumulationalsomediatesmtDNAdamagesas observedinheatkilledE.coliinjectedmice[80](Fig.1B).
Onecanalsomentionthatpathogenicbacteriaarenottheonly onestoinducemtROSproduction.Indeed,fermentationproducts ofthecommensalgutmicrobiome,suchasshortchainfattyacids (SCFA), are alsoknown toinduce mtROS production [81]. It is interesting to notethat propionic acid(PPA) overproduction, a metabolic product of bacterial fermentation from commensal bacteriaofpatientswithASD(AutismSpectrumDisorders)suchas Clostridia,BacteriodetesandDesulfovibriomightalsobeinvolvedin theinductionofmitochondrialdysfunctionobservedinpatientgut andbrain[82].PPAisalowmolecularweightorganicacidthatcan accumulatein thecytosolcausingintracellularacidification and subsequentcarnitine dependent-mitochondrialdysfunction that impairsfattyacidmetabolism[82](Fig.1B).
Asmentionedpreviously,regulationofROScontentoccursat two different levels: their production and their scavenging/ detoxificationrate.Hereafteraretwoexamplesofbacteriawhose
virulence is dependent on the modulation ROS content by
interfering with their detoxification. Firstly, most of bacteria possesstheir ownROSdetoxificationenzymes.However, itwas unclearifthoseenzymestakepartinthebacterialvirulenceduring infection.ArecentstudyanalysedthepotentialimplicationLsfA,a 1-cysPRXoftheopportunisticpathogenPseudomonasaeruginosa duringinfection.ItwasshownthatLsfAisinvolvedinthebacterial resistance to NOX burst occurring in infected macrophages
[83]. Even if no data are available about the impact of those enzymesonmitochondrialROS,itisreasonabletothinkthatthey mightbeinvolvedintheirdetoxificationaswellasitappearsthat themtROSparticipatetobacterialkillingininfectedmacrophages
[84](Fig.1B).Itwouldthusbeinterestingtostudytheimpactof those bacterialenzymesonthemitochondrial ROScytotoxicity. Secondly,itiscrucialforsomebacteriatopreventprogrammecell deathoftheirhostcell.Forexample,Ehrlichiachaffeensissecretesa mitochondrial-targeted effector during monocyte/macrophage infection.Thisbacterialtoxinseemstoup-regulatethe mitochon-drialdetoxificationenzymeMnSOD/SOD2,whichresultsinalower ROScontentandaninhibitionofapoptosis[84](Fig.1B).However, amoresystematicanalysisandscreenofthedifferentantioxidant enzymeswould benecessarytoaddresstheoriginand mecha-nisms leading to the altered ROS production in response to bacterialpathogens.
4.2. Calciumsignallingandmitochondria
Free calcium bivalentcation is a key signallingmolecule in eukaryotic cells. In basal state, cytosolic and mitochondrial calciumconcentrations([Ca2+]c;[Ca2+]mt,respectively)arevery
low (ranging between 100 and 200nM) when compared with
millimolar concentration found in the extracellular fluids [85]
(Fig.2A).Thisgradientconcentrationismaintainedbytheactionof severalligandand/orvoltage-dependentchannels(uniportersand antiporters)andATP-consumingpumpsforcalciumlocatedinthe plasmamembraneandmembranesoforganellessuchasmainly the ER and mitochondria [86]. In response to various stimuli, channelslocatedintheplasmamembraneortheERmembrane do open, allowingcalcium (in)fluxes orrelease, that leadstoa sudden (and often transient) 10-fold increase in the cytosolic calciumconcentration(>1
m
M)(Fig.2A).Thesechangesincalcium concentrationcanbetranslatedintodifferentcellresponsessuch asmusclecontractionorvesiclesecretiondependingoncelltype andconditions[85].Evenifthemajorintracellularcalciumstoreis theER(acalciumreservoirinwhichcalciumconcentrationreaches 0.5mM),mitochondriaalsocycleand/oraccumulate calciumin the matrix [86] (Fig.2A).In conclusion, mitochondria regulate calciumsignallingbytakingandreleasingCa2+locally,allowing changes in concentration in microdomains [86]. Indeed, when calcium concentration increases in the cytosol, calcium alsoaccumulatesin mitochondriathat act asa ‘‘calciumbuffer’’. To reachthemitochondrialmatrix,cytosoliccalciumhastocrossboth mitochondrialmembranes.TheionscrosstheOMMbytheprotein voltage-dependentanionchannel-1 (VDAC1)also knownas the mitochondrialporinthatfunctionsasagatekeeperfortheentry and exit of mitochondrial metabolites) and the IMM by the recently identified mitochondrial calcium uniporter (MCU), a highly specific transporter [87] (Fig. 2A). In some conditions, calciumexportmightbemediatedbythePTP,evenifthisisstill controversial[88]butcalciumeffluxcouldalsobemediatedbythe exchangewithsodium,which,inturn,ispumpedoutsideofthe matrixbyasodium/protonexchange[89](Fig.2A).
Calcium homeostasis isnot only regulatedby mitochondrial bioenergeticsand redoxstatusbutalsocontributes tomaintain several functions in mitochondria (Fig. 2A). Calcium, ATP and ROS concentrations are thus tightly interconnected and their regulationinvolvesseveralcrosstalks[89].Firstly,calciumentryin the mitochondrial matrix is dependent on the mitochondrial
membranepotential.Secondly,calcium entryand accumulation intothematrixcanbemodifiedbyROS[89]whileitspositiveeffect
on TCA and respiratory chain can modulate ROS formation
[89].Mitochondrialactivityisdependentoncalciumconcentration asmitochondrialcalciumstimulatestheTCAcycleandOXPHOS through positive allosteric regulation of 3 enzymes (pyruvate dehydrogenase, isocitrate dehydrogenase and
a
-ketoglutarate dehydrogenase)[89].Calciumalsostimulatesenzymesbelonging to (Cytochrome c oxidase, Fo-F1 ATP synthase) or regulates (glycerophosphatedehydrogenaseandadeninenucleotide trans-locase)theOXPHOS[89].Moderateincreasein[Ca2+]mttherefore resultsin a higherATP production [89]. However,calcium also regulatesmtROSproduction.Indeed,increasedcalciumnotonly boosts the respiratory chain and thus the associated electron leakage[90],butalsomightdissociatecytochromecfromtheIMM cardiolipinleadingtoanincreaseinelectronleakage[89].Calcium alsoregulatesother mitochondrialfunctionsthan bioenergetics suchasureacycle[89]andtheglutaminolysis[90].Fig.2.Mitochondria-associatedcalciumsignallinginhost–pathogenrelationship.(A)Mitochondrialcalciumregulation:Theverylowbasalcytosoliccalciumconcentrationis maintainedbytheactionofseveralchannelsandATPasespumpslocatedeitherattheplasma,ERandmitochondrialmembranes.Calciumreleasefromthemitochondrial matrixismediatedbysodiumexchangefromtheIMMtothematrix,whichinturnsisexportedfromthematrixbythesodium/protonexchange(calciumfluxesareshownin blue).Changesincytosoliccalciumconcentrationcanleadtotheactivationofsignallingpathways.Atmoderateconcentrations,calciumpromotesseveralmitochondrial functions(greenarrows)suchasTCAcycle,OXPHOS(andsubsequentmtROSproduction(yellowstars)),FAO,glutaminolysis,andureacycle[89]. However,athigh concentrations,calciuminducesmitochondrialdysfunction(redarrows),PTPopeningandcelldeath[89].(B)Examplesofimmunefunctionsmodulatedbycalcium signalling:macrophagephagocytosis[92],neutrophilschemotaxis[93],lymphocytesactivation[95]andantigenprocessingandpresentationbyAPCs[94].(C)Examplesof bacterialpathogensmodulatingcalciumsignalling:GroupAStreptococcus[98],C.difficile[101],H.pylori[99,102],C.septicum[100],L.monocytogenes[102]andShigella
[103].Thetablesummarisesthebacterialspecie,theeffectorinvolved,thetargetcell/tissue,theeffectorimpactonhostcellandthefinalphenotypeobserved.Abbreviations: A/A(ammonia/ammonium),APC(antigenpresentingcell),FAO(fattyacidb-oxidation),IP3R(inositol-3-phosphatereceptor),MCU(mitochondrialcalciumuniporter),OXPHOS (oxidativephosphorylation),PTP(permeabilitytransitionpore),SERCA(sarco/endoplasmicreticulumcalciumATPase),SLO (streptolysinO),TCA(tricarboxylic acid),TcdB (ClostridiumdifficileToxinB),VacA(vacuolatingcytotoxinA)andVDAC(voltage-dependentanionchannel).
Ifphysiologicalrangeofcalciumconcentrationshasapositive impact on different mitochondrial functions, sustained high concentration of this ion is detrimental for mitochondria and caninducemitochondrialdysfunction[89].Itiswelldescribedthat calciumoverloadleadsto anincrease intheIMM permeability thatcanresulttocelldeath[91].
4.2.1. Calciumsignallingandimmunity
Mitochondriaassociatedcalciumsignallingisinvolvedinthe regulationoftheimmuneresponse.Calciumsignallingpromotes Mycobacteriumbovisphagocytosisandprocessinginmacrophages
[92](Fig.2B).Secondly,mitochondrialUCP2down-regulatesthe polymorphonuclearneutrophils(PMN)chemotaxisbyincreasing the[Ca2+]c alsoresponsiblefortheincreasedexpressionof
b
2-integrinsallowingtheinitialfirmadhesionofPMNtoendothelial cells [93] (Fig. 2B). Finally, calcium also regulates adaptiveimmunity by promoting T lymphocytes activation by two
mechanisms[94].Ontheonehand,calciumcontrolstheantigen processingandpresentationinantigenpresentingcells(APCs),a step that is required to get T lymphocytes activated (Fig. 2B). Indeed,ithasbeenshownthattheinhibitionofFo-F1ATPsynthase andMCU,bothinhibittheantigenprocessing[94].Ontheother hand,calciumcanregulateT-cellactivationitself.Whenexposed to an appropriate stimulus, mitochondria relocate near the immunologicalsynapse toregulatetheCa2+fluxes and provide localenergyrequiredforT-cellactivationandcytokinesecretion
[95,96](Fig.2B).
The importance of calcium signalling during intracellular bacterial infection is now emerging. Interestingly, a recent screening(usingalibraryof640drugstargetingvarioushostcell functions (host-directed antimicrobial drugs)) devoted to test drugsabletomakeTHP-1cellsresistanttofourdifferentbacteria (B.abortus,Coxiellaburnetii,L.pneumophilaandRickettsiaconorii) revealedthatmostofthesedrugstargeteitherGprotein-coupled receptors, membrane cholesterol distribution and intracellular calciumsignalling[97].Thefactthatnumerousdrugsmodulating calciumsignallingininfectedcellswereabletoprotectTHP-1cells against four different bacteria species that display different lifestylesemphasisesthecentralroleofcalciumsignallingduring bacterial infections. In thefollowing paragraph, recent mecha-nismsbywhichbacteriaexertanimpactoncalciumhomeostasis andcalciumsignallingwillbediscussed.
4.2.2. Subversionofcalciumhomeostasisbybacteria
Bacterial pathogens can manipulate calcium signalling in eukaryotic cells in order to modulate mitochondria-dependent apoptosis. Group A Streptococcus (GAP), extracellular bacteria foundon oropharynx and damaged skin, haveto reach deeper tissuestocauseinfection[98].Themechanism usedby GAPto achievethisgoalistosecretestreptolysinO(SLO),apore-forming moleculethatinducesprematuredifferentiationandapoptosisof keratinocytes.Thoseporesincreasethepermeabilityoftheplasma membraneandallowcalciuminfluxinthecytosol,leadingtoER stressandmitochondrialdysfunctionthatsubsequentlytriggers apoptosis[98](Fig.2C).
H. pyloriinfection leads togastric cell death caused by the ammonia/ammonium(A/A)producedbythebacteria[99].These authorsshowedthatA/A-associatedcytotoxicitywasmediatedby theactivationofaN-methyl-D-aspartatereceptor,whichleadsto calcium entry. Cytosolic calcium is thus first taken by the ER, mediated by the sarco/endoplasmic reticulum calcium ATPase (SERCA)pumps, and thentransferred tomitochondria.Calcium alsoinducesthetranscriptionofcelldeatheffectorssuchasBAK andBAXandactivatescalpainandcathepsinB,whichbothtake partinA/Acytotoxicity.Theconsequencesofthesechangesresult inOMMdamage,ATPdepletionandfinallycelldeath[99](Fig.2C).
Clostridium septicum isa commensal gutbacteriumthat can sporadically cause necrosisof thehumanskeletal musclecells. Thesebacteria secrete
a
-toxinintheextracellular environment that inserts into myoblast plasma membrane, forms a Ca2+ permeablechannel,andtriggersacalciuminflux[100].Subsequent calpainactivationandreleaseofcathepsinsfromlysosomesthen modifycytoskeletonorganisation.Intheseconditions,theincrease in cytosolic calcium concentration also leads to mitochondrial dysfunction,adecreaseinATPproductionaswellasnuclearDNA damages,andotherhallmarksofnecrosis[100](Fig.2C).C.difficileisanextracellularbacteriumresponsiblefor pseudo-membranous colitis.C. difficile secretestoxins (TcdAand TcdB) thatwilldisrupttheepithelialbarrier.Arecentstudyshowedthat TcdBseemstoinduceapoptosisinepithelialthroughtheinhibition of the mitochondrial ATP sensitive potassium channel, which accompaniedwith anincreasein [Ca2+]c, leadstomitochondrial membranehyper-polarisationandapoptosis[101](Fig.2C).
Whilesevereandsustainedchangesincalciumconcentration in responsetoinfectionmightlead,infine,tocelldeath,some pathogensdosubvertcalciumsignallinginacell death-indepen-dent manner. Forexample, in eosinophilsexposed to purified VacA,aporeformingtoxinsecretedbyH.pylori,thetoxinisableto induce Ca2+ release from intracellular stores. This increase in cytosoliccalciumconcentration leadstoan increasein mtROS production,subsequentNF
k
Bactivationandchemokine produc-tionresponsiblefortherecruitmentofinflammatorycellsinthe gastric mucosae observed in infected patients [102] (Fig. 2C). AnotherillustrationisgivenforLLO,abacterialtoxinsecretedby L.monocytogenesduringinfection,isabletooligomerisetoform poresintheplasmamembraneofinfectedHeLacells.Thecalcium influxandthemetaboliteleakagemediatedbytheseporesinduce atransientmembranepotentialdisruption,mitochondrial frag-mentation,andadecreaseintheATPproduction.The mitochon-drial fragmentation might thus induce a ‘‘transient metabolic slow down’’ required for the establishment of the bacterial replicative niche [17] (Fig. 2C). Eventually, Shigella is an intracellular bacterium knownto induce actin re-organisation duringinfection.Arecentstudyshowedthatsuchcytoskeleton alterationslimitthediffusionofsmallsolutesandthereforelead to confinement of calcium signalling at bacterial entry sites[103].Thismicroenvironmentpromotesa long-lastingcalcium signallingtakingpartincalciumdependent-proteaseactivation, known to be involved in Shigella infection. This calcium rich environmentalsoallowstheinductionATPproductionrequired fortheactinre-organisationbymitochondriathataretrappedin the polymerised actin, while it prevents the spreading of apoptotic signalling associated with sustained and largely diffused high calcium concentrations at early stages of the infection[103](Fig.2C).
4.3. mtDAMPs
As mentioned in Section 1, in addition to PAMPs, PRR also recognise DAMPs, molecular patterns that are sequestered in healthytissuesbutexposed/releasedwhencellsaredamaged,in responsetotraumaornecrosisforinstance[49].Asmitochondria stilldisplaysomeprokaryote-relatedfeatures,several mitochon-drial componentscouldberecognisedas mitochondrial DAMPs (mtDAMPs).
Firstly,mtDNA,whichlikethebacterialgenomeisenrichedin unmethylated CpGs [104] is able to activate TLR9 [105]. For example, injection of endogenously oxidised mtDNA (but not nuclearDNA)inmiceleadstothedevelopmentofarthritisinmice, a phenotype that couldbe supported by thefact that oxidised mtDNAinducesNF
k
B-dependentTNFa
productionbymonocytes/ macrophages [106]. In addition, mtDNA are able to stimulateneutrophilsinresponsetoTLR9bindingandactivation[105]. How-ever,it seems that, according to celltype, unmethylated CpG-oligodeoxynucleotide(CpG-ODN)isabletopromoteapro-or anti-inflammatory response [107]. In immune cells such as macro-phages,achaperoneUnc93b1isabletoshuttleTLR9fromtheERto thelysosomes,wherereceptor processingand bindingby CpG-ODNleadstotheclassicalMyD88-dependentpro-inflammatory response[107].However,innon-immunecells,mitochondriatake partinthedevelopmentofananti-inflammatoryresponse.While in neuronsand cardiomyocytes, Unc93b1 is less abundant and TLR9ismainlyfoundintheER[107].Consequently,TLR9binding by CpG-ODN triggers a different signalling in those cell types. Indeed,intheER,TLR9interactswithSERCA2(acalciumATPase pump),decreasesitsactivitylimitingthecalcium(re)entryinthe ERlumenandsubsequentlyreduces thecalciumtransfertothe mitochondria. A higher AMP/ATP ratio associated with lower mitochondrialmatrixcalciumconcentrationalsoleadstoAMPK activation, a condition known to promote autophagy and cell survival[107,108].
Secondly,similartoprokaryotes,mitochondrialtranslationis initiated bythe additionof N-formylmethionine [109]. Conse-quently,mitochondrialN-formylpeptidescantrigger inflamma-tion.Theyarerecognisedbyformylpeptidereceptor-1(FPR-1)in PMN,whichleadstotheiractivationinaMAPK-dependentmanner
[110].Thesamegroupalsorecentlyshowedthat mtDAMPsare able to promote PMN adherence to endothelial cells (EC) and enhancesystemic endotheliumpermeability[111].Additionally, othermitochondrialproteins,suchasthemitochondrial transcrip-tionfactorTFAM arealsoabletoactasmtDAMPs [112].TFAM releasebynecroticcellpotentiatescellstoproduceCXCchemokine ligand-8 (CXCL-8) in response to N-formyl-peptide exposure
[112]. In addition, some mitochondrial proteins, such as heat shockproteinsof60and70kDa(HSP60andHSP70)arestillableto triggerinflammationviaTLR4activationinmacrophages,leading totheproductionofIL-6andTNF
a
,whentheyarereleasedinthe bloodstreamforexampleaftersurgery[113,114].Furthermore, mitochondria extracts from H2O2-stressed THP-1 cells induce strongerTLR4-dependentinflammatoryresponsewhencompared withmitochondrialextractsofcontrolcells[115].Eventually,very recently,astudyshowedthatinresponsetotraumainduced-cell injury,mtDAMPsreleasedintheextracellularfluidscouldactivategd
-Tlymphocytes(assessedbyanincreaseinTLRexpressionand cytokinesproduction),whichareknowntobeinvolvedinhealing processes[116].As mentioned in Section 1, mitochondrial morphology and functionsaretightlyrelated.Itisthusnotsurprisingthatseveral bacterialpathogenscan altermitochondrial morphologyduring infectiontomanipulatemitochondrialfunctions.
5. Mitochondrialdynamics
Inrecentyears,severalbacterialeffectorshavebeenreportedto induceorinhibitmitochondrialfragmentationtocontroldifferent cellresponses. While notexhaustive,here are fewexamples of recentlycharacterisedeffectorsknowntomodulatemitochondrial morphology during bacterial infection. First, as described in Section 4.2.2, subversion of calcium homeostasis by bacteria duringinfection,L.monocytogenessecretesapore-formingtoxin calledLLO.Poresformedattheplasmamembranebythisbacterial
effector allow calcium influx in the cytoplasm leading to
mitochondrial fragmentation [17]. These authors show that alterationsof mitochondrialmorphology occurrapidlyafter(or evenbefore)bacterialentry,aretransientandaccompaniedwith a decrease in ATP production. They also hypothesised that mitochondrial fragmentation observed during L. monocytogenes infectionmight mediate a ‘‘metabolicslow-down’’ required for
theestablishmentofthebacterialreplicationniche[17]butthis
remains to be experimentally demonstrated. The mechanism
involved in the LLO-induced fragmentation is not clearas LLO couldinduceanatypicalfissionmediatedbyaDRP1-and OPA1-independent mechanism [117]. Secondly, VacA is also a pore-formingtoxinsecretedduringH.pyloriinfectionthatlocalises,at leastpartially,atthemitochondria.VacAwouldinduceactivation andrecruitmentofDRP1resultinginmitochondrialfragmentation ininfectedcells. DRP1-inducedfragmentationalsoleadstoBAX activation, cytochrome c release and finally programmed cell death[118].Themolecular mechanismsbywhichVacAinduces DRP1-mediatedmitochondrialfragmentationandtheconnection betweenmitochondrialfragmentationandBAXactivationremain tobedetermined[118].Finally,Vibriocholeraesecretes VopE,a T3SS-secreted effector, that inhibits mitochondrial network reorganisation[119].ItwasshownthatincellsinfectedwithV. choleraedepletedforthegeneencodingthiseffector,some T3SS-associated componentsinduce mitochondrial peri-nuclear clus-tering that leads to MAVS aggregation and induction of NF
k
B signalling. The mitochondrial clustering could be mediated by Miro1and Miro2(two RhoGTPases) that sensecalcium fluxes changes in response to V. cholerae infection. However, in cells infected with the WT bacteria, VopE, which acts as a specific GTPaseactivatingprotein(GAP),isabletophysicallyanddirectly interactwithMiro1andMiro2keepingtheminaGDP-boundform allowing the inhibition of mitochondrial clustering and subse-quentpro-inflammatorysignalling.Thus,changesin mitochondri-al morphology during V. cholerae infectionseem to inhibit the developmentofanefficientimmuneresponse[119].6. Perspectives
Iftheimpactandeffectofinfectiouspathogensonmitochondria arerelativelywellstudied,itisnowimperativetoassesstheeffectof mitochondrial activity and metabolic status during infection. Systematicstudies to analysethe effect ofboosted or impaired mitochondrialfunctionson infectionefficiency,intracellular traf-ficking(forfacultativeorobligatebacteria),bacterialresistanceor sensitivitytointra-andextracellulardefencemechanismsshouldbe developedin thefuturetobetterunderstandthe numerousand tightly regulated cross talks between a host and its bacterial pathogens. In addition, mechanisms by which bacteria affects, directlyorindirectly,mitochondriastillneedtobedeterminedas many bacterial effectors are still most likely to be identified. Eventually,theoriginofincreasedmtROSproductiondetectedin responsetobacterialinfectionstillneedtobeaddressedbymodern androbustapproaches todetect ROS,especiallythat sofar, few
elements allow to determine whether the changes in mtROS
abundanceinresponsetobacterialpathogensresultfromatrue increaseintheirproductionorareductionofantioxidantcapacity. 7. Conclusion
Aswehaveseen,mitochondriaarevery importantorganelle allowing many functions in the immune systems, especially
through metabolic control, calcium homeostasis and mtROS
production and degradation [89].Many of thesemitochondrial controlshelpthehosttodefendagainstthepathogens.However, many bacteria (both intracellular and extracellular) have also evolvedanddevelopedseveraltypesofeffectorsandmechanisms that target and alter themitochondria to circumvent or avoid mitochondria-dependentmechanisms,suchasmtROSproduction, thatcouldbedeleteriousorharmfulforthepathogenicbacteria. Finally,theexpectedoutcomeoffuturestudiesshouldbringa
better knowledge of effectors and mechanisms that affect
thisorganelleininfection,datathatcouldalsoprobablyhelpto identifynewhostmitochondrialtargetsandthedevelopmentof innovativemedicationstofightbacterialpathogens.
Acknowledgements
ElodieLobetisadoctoralResearchFellowatFRS-FNRS(Fonds delaRechercheScientifique,Belgium).TheauthorsthankMichel Savelsforhisgreatcontributiontothefigurelayout.Thisworkwas supportedbytheActionsdeRecherchesConcerte´es-Communaute´ Franc¸aisedeBelgique(GrantnumberConventionN808/13-015). References
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