The past, present, and future of Leishmania genomics and transcriptomics

The

past,

present,

and

future

of

Leishmania

genomics

and

transcriptomics

Cinzia

Cantacessi

,

Filipe

Dantas-Torres

,

Matthew

J.

Nolan

,

and

Domenico

Otranto

1

DepartmentofVeterinaryMedicine,UniversityofCambridge,Cambridge,UK

2_{DepartamentodeImunologia,CentrodePesquisasAggeuMagalha˜es,Fiocruz-PE,Brazil} 3

DipartimentodiMedicinaVeterinaria,Universita`degliStudidiBari,Bari,Italy

4

RoyalVeterinaryCollege,UniversityofLondon,NorthMymms,UK

Ithasbeennearly10yearssincethecompletionofthe firstentire genomesequenceofaLeishmaniaparasite. Genomic and transcriptomic analyses have advanced our understanding of the biology of Leishmania, and shed new light on the complex interactions occurring within the parasite–host–vector triangle. Here, we re-viewtheseadvancesandexaminepotentialavenuesfor translationofthesediscoveriesintotreatmentand con-trolprograms.Inaddition,weargueforastrongneedto explorehowdiseaseindogsrelatestothat inhumans, and how an improved understanding in line with the ‘One Health’ concept may open new avenues for the controlofthesedevastatingdiseases.

Burdenofleishmaniasisandtheneedfora‘OneHealth’ initiative

Leishmaniasesareagroupofdiseasescausedbydigenetic protozoaofthegenusLeishmania,whicharetransmitted by phlebotominae sand flies (Table 1). Based on recent estimates,upto0.4millionand1.2millioncasesofvisceral (VL)andcutaneousleishmaniasis(CL),respectively,occur eachyearin98countriesandthreeterritorieswherethese diseasesareendemic[1].Despitetheirwidespread distri-bution, over 90% of global VL cases occur in only six countries(India,Bangladesh,Sudan,SouthSudan, Ethio-pia,andBrazil),whilemostcases(70–75%)ofCLoccurin ten countries (Afghanistan, Algeria, Colombia, Brazil, Iran,Syria,Ethiopia,NorthSudan,CostaRica,andPeru)

[1]. In mostcases, leishmaniases are zoonoses,affecting thepoorin ruralandnaturalareas, whereaplethora of domestic and wild reservoir hosts and sand fly vectors maintain the infection [2]. For instance, 13 out of the 21human-infective Leishmania have alsobeen reported indomesticdogs,thelatterhavingamajorrolein main-tainingandtransmitting theinfection toother receptive

hostsviathesandflyvectors[3](Table 1). Inaccordance withtheconceptof‘OneHealth’,definedas‘amovementto forge co-equal,allinclusive collaborationsbetween physi-cians, [...], veterinarians and other scientific-health and environmentally related disciplines [...] to improve and defend the health and well-being of all species’ (http:// www.onehealthinitiative.com),successfulcontrolstrategies against human leishmaniases must include preventative measures focussed on the human and animal hosts and arthropod vectors,as wellas ontheenvironmentswhere thelatterperpetuate[3].Toachievethesegoals,athorough understanding ofthe host–pathogen–vectortriangle, and particularlyoftheirintimateinteractionsatthemolecular level,isimperative.Recentadvancesin-omicstechnologies, includinggenomicsandtranscriptomics,togetherwiththe considerabledecreaseinthecostofthesetechniques, pro-videexcitingopportunitiestorevealdetailsoftheintimate relationsbetweenLeishmania parasites,humanand ani-malhosts,andsandflyvectors.Inthisreview,weprovidean overview ofa range of milestone studies that have used genomics and transcriptomicstechniquestoimprove cur-rentunderstandingofthebiologyofLeishmania,aswellas ofthemolecularinteractionsbetweenthisparasiteandits vertebrateandarthropodhosts.Inaddition,giventhe inti-materelationsbetweenhumanandcanineleishmaniasesin endemicareas,andinlinewiththe‘OneHealth’movement, wearguethatcurrentandfutureeffortsshouldbedirected towards integrating -omics technologies (i.e., genomics, transcriptomics, proteomics, metabolomics, and interac-tomics)toachieveabetterunderstandingofthesimilarities anddifferencesbetweenhumanandcanineinfections,with theultimateaimofdevelopingnewdiagnostics,and treat-mentandcontrolstrategiesagainstthisdevastatinggroup ofdiseases.

Thefightagainstleishmaniasis:howcan-omicshelp? Thecontrolofleishmaniasesgenerallyreliesontheearly diagnosis and treatmentof human cases,vector control, and, in some cases,management of reservoir hosts (i.e., treatmentand/orelimination)[3].However,thecontrolof leishmaniases, as with any vector-borne disease, is not trivialduetochallengesrelatingtointerventionprograms,

1471-4922/

ß2015ElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http:// creativecommons.org/licenses/by/4.0/).http://dx.doi.org/10.1016/j.pt.2014.12.012 Correspondingauthor:Cantacessi,C. ([email protected]).

Keywords: leishmaniases; Leishmania infantum; high-throughput sequencing; genome;transcriptome;bioinformatics;sandfly;metazoonosis;host-parasite inter-actions;OneHealth.

mostlyin developingcountries, wheretheburden of dis-easeisheavier(duetoacombinationoffactorsincluding, but not limited to, a lack of political will, of human resources, andofinfrastructure). Inaddition,ourlimited knowledgeofthehost–pathogen–vectortriangle, particu-larlyoftheirintimateinteractionsatthemolecularlevel, impairsthedevelopmentofmoreaffordableandeffective controltools,suchasantivectorvaccinesandmoreeffective chemotherapeutics.

-Omics technologies are increasingly being applied to investigations of determinants of disease phenotype [4], modeofactionofcurrent drugs[5], andparasite biology

[6].Thesestudieshaveimprovedourunderstandingofthe pathogenesis of disease in humans andpossible mecha-nisms ofresistance to antileishmanial drugs. Without a doubt,-omicsapproachesarelikelytorevealdetailsofthe intimaterelationsbetween hosts,parasites, andvectors. Thisrefinedknowledgewillfosterthedevelopmentofnew

Table1.Principalcausativeagentsofhumanleishmaniases

Leishmaniaspecies Principal tropisma

Geographicaldistributionb _{Notesontheinfectionindogs}c

Leishmaniaaethiopica C OldWorld:Ethiopia,Kenya

Leishmaniaamazonensis C NewWorld:Argentina,Bolivia,Brazil,Colombia,Ecuador, FrenchGuiana,Peru,Suriname,Venezuela

VLcasesinBrazil

Leishmaniaarchibaldid _{V OldWorld:Ethiopia,Kenya,Lebanon,Sudan VLcasesinSudan}

Leishmaniabraziliensis C,MC NewWorld:Argentina,Belize,Bolivia,Brazil,Colombia,Costa Rica,Ecuador,Guatemala,FrenchGuiana,Honduras,Mexico, Nicaragua,Panama,Paraguay,Peru,Venezuela

CLcasesinArgentina,Bolivia, BrazilColombia,Peru,and Venezuela

Leishmaniacolombiensis C NewWorld:Colombia,Panama,Venezuela VLinadoginVenezuela

Leishmaniadonovani V OldWorld:Bangladesh,Bhutan,China,Cyprus,Djibouti, Ethiopia,India,Iraq,Israel,Kenya,Nepal,SaudiArabia, Somalia,SriLanka,Sudan,Ukraine,Uganda,Yemen

Dogsarecommonlyinfectedin somecountries(e.g.,Sudan), buttheirroleasreservoirsis unknown

Leishmaniagarnhamid C NewWorld:CostaRica,Venezuela

Leishmaniaguyanensis C NewWorld:Argentina,Bolivia,Brazil,Colombia,Ecuador, FrenchGuiana,Guyana,Peru,Suriname,Venezuela

CLcasesinColombia

Leishmaniainfantum V,C OldWorld:Afghanistan,Albania,Algeria,Armenia,Azerbaijan, BosniaandHerzegovina,Bulgaria,CentralAfricanRepublic, China,Cyprus,Croatia,Egypt,France,Gambia,Georgia,Greece, Iraq,Iran,Israel,Italy,LibyanArabJamahiriya,Jordan, Kazakhstan,Kirgizstan,Lebanon,Macedonia,Malta,Morocco, Mauritania,Monaco,Montenegro,Oman,Pakistan,Palestine, Portugal,Syria,Romania,Senegal,SaudiArabia,Slovenia, Spain,Sudan,Tunisia,Turkmenistan,Turkey,Ukraine, Uzbekistan,Yemen.NEWWORLD:Argentina,Bolivia,Brazil, Colombia,CostaRica,ElSalvador,Guatemala,Honduras, Mexico,Nicaragua,Paraguay,Venezuela

VLcasesusuallyfoundinareas wherehumancasesare reported.Autochthonouscases reportedindogsintheUSA(no humancasesreportedsofar)

Leishmaniakillickid C OldWorld:Algeria,LibyanArabJamahiriya,Tunisia

Leishmanialainsoni C NewWorld:Bolivia,Brazil,FrenchGuiana,Peru,Suriname

Leishmanialindenbergi C NewWorld:Brazil

Leishmaniamajor C OldWorld:Afghanistan,Algeria,Azerbaijan,BurkinaFaso, Cameron,Chad,Egypt,Ethiopia,Georgia,Ghana,Guinea, Guinea-Bissau,India,Iraq,Israel,LibyanArabJamahiriya, Jordan,Kazakhstan,Kenya,Kuwait,Mali,Morocco,Mauritania, Mongolia,Niger,Nigeria,Oman,Pakistan,Palestine,Saudi Arabia,Syria,Iran,Senegal,Sudan,Tunisia,Turkmenistan, Uzbekistan,Yemen

CLinEgyptandSaudiArabia

Leishmaniamexicana C NewWorld:Belize,Colombia,CostaRica,Ecuador,Guatemala, Mexico,UnitedStates

CLinEcuadorandUSA

Leishmanianaiffi C NewWorld:Brazil,FrenchGuiana,

Leishmaniapanamensis C,MC NewWorld:Colombia,CostaRica,Ecuador,Guatemala, Honduras,Nicaragua,Panama

CLinEcuadorandColombia

Leishmaniaperuviana C NewWorld:Peru CLinPeru

Leishmaniapifanoid C NewWorld:Venezuela CLinEcuador

Leishmaniashawi C NewWorld:Brazil

Leishmaniatropica C OldWorld:Afghanistan,Azerbaijan,Egypt,Ethiopia,Greece, India,Iraq,Israel,Iran,Jordan,Kenya,Morocco,Namibia, Pakistan,Palestine,SaudiArabia,Syria,Turkmenistan,Turkey, Uzbekistan,Yemen

CLcasesinIndia,Iran,Israel, Morocco,andSyria

Leishmaniavenezuelensis C NewWorld:Venezuela

a_{Abbreviations:C,dermotropic;MC,mucotropic;V,viscerotropic.}

b_{Based on[63,64].}

c_{Basedon[54,65,66].Inaddition,LeishmaniaarabicahasbeenreportedindogsinSaudiArabia[67].Moreover,otherLeishmaniaspecies(e.g.,Leishmaniaequatorensis} andLeishmaniautingensis)[68,69]havebeendescribedfromwildlifeand/orsandflies,buthavenotyetbeendetectedinhumansordogs.

d_{Speciesstatusisunderdiscussion[63,70].}

controltools(e.g.,antivectorvaccines)thatcouldassistthe fight against leishmaniases. The determination of the wholegenome sequencesof arangeofLeishmania para-sites causing both VL and CL represents the first step towards these goals, providing the scientific community withasolidinfrastructureforpostgenomicinvestigations oftheparasitebiology,pathogenicity,andmastery mech-anismsofmanipulationofbothinsectandvertebratehosts.

TheLeishmaniagenomes:a‘toolbox’tounderstand host–parasiteinteractions

Efforts todetermine the whole genome sequence ofkey

Leishmaniaspeciesinfectinghumanswereconsolidatedin 1994inRiodeJaneiro(Brazil),withtheestablishmentof the Leishmania Genome Network (LGN) initiative. Not onlydidthisnetworkrepresenttheresearchers’firstmove toexpandexistingknowledgeofthefundamental molecu-larbiologyofthisparasite,withaviewtowardspromoting thediscoveryofnoveltreatmentandcontrolstrategies,but it also saw the support of the FIOCRUZ and UNICEF/ UNDP/WorldBank/WHOSpecialProgrammeforResearch andTraininginTropicalDiseases[7].In2005,theseefforts provedsuccessful,withthepublicationofthefirstcomplete genome sequenceof Leishmania major (causing CL) [8], soon followedby those ofLeishmania infantum (causing VL)andLeishmaniabraziliensis(causingmucocutaneous leishmaniasis; MCL) [9]. In recent years, the advent of high-throughput sequencing technologies (Box 1) has assisted relentless progress in the genomics of human leishmaniases,with the completionofthe wholegenome sequencesofLeishmaniamexicana(CL;[10]),Leishmania donovani (VL; [11]) and Leishmania amazonensis (CL;

[12])(Box1).Theavailabilityofthesegenomesequences hasprovidedunprecedentedopportunitiestoperform de-tailedcomparativeanalysesofLeishmaniaspecies associ-atedwith differenthuman diseasesat ascalepreviously unimaginable[9,12].

Thegenomes ofLeishmaniavaryfrom29Mb(L. ama-zonensis;[12])to33Mbinsize(L.major,L.infantumand

L. braziliensis; [9]) and are organised into a variable number of chromosomes(i.e., 34 in L. amazonensis and

L. mexicana,35inL.brasiliensis,and36inL.major,L. donovani, and L. infantum) [12]. Despite the striking variabilityinpathogenicityandtissuetropismofdifferent

Leishmania species,theirgenomes areremarkably simi-lar,displayingahighdegreeofconservationingene con-tent and architecture (synteny) [9,12]. The genomes of

Leishmaniaspp.arecharacterisedbyahighgenedensity, thepresenceoflongarraysofpolycistronicgeneclusters, andthealmostcompleteabsenceofintrons[7].However, carefulexaminationofprotein-codinggenesinLeishmania

allowedtheidentificationofarelativelysmallnumberof species-specific genes,the majority ofwhich encode pre-dicted proteins ofunknown function [10]. Only a few of thesegenescouldbeassociatedtospecifictissuetropism. Forinstance,LinJ.28.0340,agenespecifictoL.infantum

andoccurringasapseudogeneinL.major,L.braziliensis, andL.mexicana[10],hasbeenimplicatedintheabilityof thelattertospreadandsurviveinvisceralorgansofthe vertebrate hosts [13]. Indeed, when a L. donovani gene orthologueofLinJ.28.0340wasintroducedintotransgenic

L. major, the latter displayed a significantly increased capacity to survive in visceral organs of BALB/C mice

[13]. Conversely, the spleens andlivers of mice infected withtheLinJ.28.0340/L.donovaninullmutantwere char-acterised by significantly reduced parasite burdens com-paredwith thoseinfectedwiththewildtypeL.donovani

counterpart,thusprovidingsolidevidenceforaroleofthis gene in the visceralisation of the infection [13]. Among other genes thought to have key roles in the ability of specieswithintheL.donovanicomplextocolonizevisceral organs, those belonging to the A2 gene family are also presentaspseudogenesinL.major[14].Thesegeneswere firstidentifiedinL.donovaniandshowntobeexclusively expressed by the amastigote stage (cf. [14]) (Figure 1); subsequently,thesegenesweredemonstratedtobe essen-tialforthesurvivalofL.donovaniinvisceralorgans,while transgenic L. major expressing A2 genes displayed in-creased survivalinthe spleensofinfectedmice[14]. De-spitetheevidenceforaroleofA2genesinthepathogenesis of VL, molecules encoding A2 proteins have also been identifiedinLeishmaniaspeciesresponsibleforCL,such

Box1.DNAsequencingtechnologiesandLeishmaniagenomics:adecadeofprogress

In2005,theLeishmaniamajorgenomewaspublishedinthejournal

Science [8]. Thesuccessful completionof thefirst whole genome sequence of a Leishmania species resulted from a collaboration between the Wellcome Trust Sanger Institute (UK), the WTSI-coordinated EULEISH consortium, and the Seattle Biomedical Re-search Institute (USA) [8]. The sequencing strategy utilised a combinationofhigh-andlow-coveragelargeinsertclonesequencing andawholechromosomeshotgunapproach,precededby purifica-tion of singleor co-migrating chromosomes using pulse-fieldgel electrophoresis(PFGE)[8].In2007,thegenomesoftwoadditional

Leishmaniaspecies, thoseofLeishmaniainfantumandLeishmania braziliensis, were completed by a multi-institutional network of scientists led bytheWTSI[9]; togenerateanapproximate sixfold coverageofthecompletegenomesequencesofthesetwospecies, researchers utilisedwhole-genomeshotgunsequencingofplasmid clonescontaininggenomefragmentsofvariablelength(upto4kb). Four years later, a similar approach was used to sequence the genomeofLeishmaniamexicana[10].Inthesameyear,thereference genomeofLeishmaniadonovaniwasthefirsttobecompletedusing a next-generation sequencing strategy [11]; in particular, using a

pyrosequencing approach (454 Roche), Downing and colleagues generatedatotalof1.29millionsingle-endand3.58million paired-endreads [withanaveragelengthof167basepairs(bp)],96% of which wereassembled intoan initial setofreferencecontigsand scaffolds [11]; subsequently, 17 clinical isolates of L. donovani

obtainedfromNepalese andIndianpatients withVL(including the isolatefromwhichthereferencegenomesequencewas obtained), weresequencedusinghigh-throughputIlluminasequencing, gener-atingatotalof41Gbofsequencedata[11].Then,the76-bp paired-endreadsfromeachisolateweremappedtothereferencegenome sequence for SNP analysis, which resulted in new insights into

Leishmaniapopulationgeneticsandmechanismsofemergingdrug resistance[11].ThemostrecentLeishmaniagenomesequence(i.e., that of Leishmania amazonensis [12]) was generated using a combination of 454 and Illumina sequencing. The approximately 179000454 reads corresponded to a twofold coverage of the L. amazonensisgenomeandwereassembledinto27856contigsthat, together with the 4411 scaffolds derived from the assembly of approximately37 million76-bppaired-endIllumina reads,resulted inthefinalassemblyof2627scaffolds[12].

as L. amazonensis and L. mexicana [10,12]. While the presence of theseproteins in Leishmania parasites with skintropismhasbeenattributedtofunctionaldivergence betweenOldWorldandNewWorldspecies[14],theirrole in thepathogenesisofCLisyettobeascertained. Inter-estingly, a recent comparative analysis of the genomes and transcriptomes of two phenotypically distinct sub-strainsofL.donovani(i.e.,onecausingVLandone respon-sible for a large number of cases of CL in Sri Lanka) revealed an increased copy number of A2 genes in L. donovani causing VL, which was also associated with significant upregulation in A2 mRNA transcription and proteinexpressioninstrainscausingVL[15].Inthesame study,Zhangandcolleagues[15]identifiedthepresenceof severalnonsynonymousSNPs ingenesfromtheL. dono-vaniCLstrain. Amongthesewas amolecule encodinga ras-like smallGTPase-RagCprotein;insertionofthe cor-respondingorthologousgenefromtheL.donovaniVL iso-late into the CL counterpart resulted in a significant increaseinparasiteburdensinthespleenofinfectedmice

[15].ThesedataprovidedevidenceoftheimpactofSNPs on gene function and phenotype, thus refining current understandingof their potentialimpact onthe pathoge-nicityofdifferentstrainsofLeishmania.

While comparative analyses of the whole genome sequencesofLeishmaniaspeciescausingCLandVL rep-resentasolidbasisforin-depthinvestigationsofthe inti-matemechanismsofhost–parasiteinteractionsthatresult indifferentcoursesofinfection,studiesoftheregulationof parasitegene expressionthroughoutitslifecycle inboth thevertebratehostsandthesandflyvectorsarelikelyto contributetoabetterunderstandingofthepathogenesisof disease. Clearly,theavailabilityof anarrayofgenomes, together with an explosion in microarray and high-throughputtranscriptomicsequencing technologies,have facilitatedsuchstudies(e.g.,[16–19]).However,thesame organisation into polycistronic transcription units that makes the genomes of CL- and VL-causing Leishmania

sostrikinglysimilar [7]hasbeendeemed responsiblefor the lack of extensive gene expression regulation at the

Amasgotes of viscerotropic Leishmania express A2 genes, implicated in visceralisaon of infecon [15]

Simultaneously, Cox2 genes overexpressed by infected macrophages promote parasite survival [25]

Peritrophins and chins expressed by the sand fly midgut serve as a barrier to the migraon of Leishmania to the thoracic midgut, unl their degradaon by proteolyc enzymes [36]

Salivary components, such as maxadilan and hyaluronidases, exacerbate parasite infecvity and promote the infecon process [71]

Sand fly 7

8 9

6

5 1

2

Mammalian host 3

4

TRENDS in Parasitology

Figure1.LifecycleofLeishmaniaspp.andexamplesofmoleculesputativelyinvolvedinparasiteinfectivityandvisceralisationofinfection.Phlebotominaesandflies release Leishmaniainfective stages (i.e., metacyclic promastigotes) to the mammalian hosts during blood feeding (1); the parasites invade macrophages and granulocytes (2 and 3) and develop to amastigotes inside the phagolysosome (4); the amastigote stages replicate within the phagolysosome by simple division (5); then, amastigote-containingmacrophagesareingestedbysusceptiblesandfliesduringthebloodmeal(6);theparasitesarereleasedfromtheinfectedmacrophageswithinthesandfly midgut (7), where they transform into procyclic promastigotes and divide. Then, the parasites migrate towards the stomodeal valve (anterior midgut) and transform into different promastigote subtypes that ultimately form metacyclic promastigotes (8). These infective stages are then released into a new mammalian host during a subsequentbloodmeal(9)[15,25,36,71].Abbreviation:Cox2,prostaglandin-endoperoxidesynthase2.

transcriptionallevel[20].Indeed,mostLeishmaniagenes havebeenshowntobeconstitutivelyexpressedthroughout thetransitionfrompromastigotetoamastigotestage[21], with post-transcriptional events, including mechanisms that control the abundanceofmRNAs, translation rates and post-translational protein stability, hypothesised to have key roles in the regulation of protein abundance

[21].However, themarkedvariation in chromosomeand genecopynumbersamongstrainsofL.infantum,L. mex-icana,L.braziliensis,andL.majorunveiled,forthefirst time, a degree of aneuploidy in the genomes of these parasites[10].Accordingly,unstableploidyamongstrains ofL.infantum,aswellasvariablechromosomalcontents among cells, revealed that the Leishmania genome is characterised by ‘mosaic aneuploidy’ [11,22]. Therefore, ‘genomeplasticity’and‘genedosage’,ratherthan differen-tial expression of single genes and gene products, are increasingly being considered as two of the keys to the differenttissuetropismofLeishmaniaspp.[22].

TranscriptomicsunveilsLeishmania-mediated regulationofhostgeneexpression

Several transcriptomic studies have investigated Leish-mania-inducedregulation ofgene expression in infected tissues with the aim to link such responses to disease outcome.Asanexample,forVL-causingLeishmania, Beat-tieandcolleagues[23]usedwhole-genomearray technolo-gies to compare the gene expression profiles of liver-resident macrophages (Kupffer cells) from mice infected byL.donovanitothoseofuninfectedmacrophagesexposed toinflammatorystimuli. The authorsshowed significant upregulation of genes within the retinoid X receptor a

pathway(i.e.,Rxra), linkedtolipidmetabolism,in unin-fected macrophages exposed to inflammation compared with the infected counterpart [23]; pharmacological per-turbation oftheactivity ofthispathway inKupffer cells resulted in an increased resistance of these cells to

Leishmaniainfection,whichledtospeculationthateither thispathwayhasaroleintheusageoflipidsand choles-terol by theparasite, or that Leishmanialipids regulate theactivationofinnateimmuneresponsesthatfollowthe infection [23].ForCL-causing Leishmania,Maretti-Mira and colleagues [24] utilised high-throughput RNA-Seq technologies to characterise and compare the transcrip-tomesoftissuefragments obtainedfrom humansubjects withCLandMCLcausedbyL.braziliensis[24].The out-comesfromthisstudyhighlightedsignificantupregulation of genes involved in biological pathways linked to the recruitment and activation of immune cells (including lymphocytes,granulocytes, naturalkillercells, and anti-gen-presenting cells) and to regulation of inflammatory responsesintissuesfromsubjectswithCL[24].This sug-gested that the inability of the host to mount effective immune responses against the parasite at the site of cutaneousinfectionislinkedtotheprogressionofdisease

[24].Inanefforttocharacterisedifferencesinmacrophage gene expression that might contribute to the ability of different Leishmaniaspp.tocause localised (CL)or sys-temicinfections(VL),Gregoryandcolleagues[25]used a DNAmicroarrayapproachtoperformcomparative analy-sesofthetranscriptomesofmurinemacrophagesinfected

byL.majorandL.donovani.Interestingly,bothparasites induced a similar differential regulation of relatively small numbers ofmacrophagegenes, with mostofthese genes unsurprisingly linked to the development of immuneresponses[25].Theonlynoticeabledifferencein geneexpressionprofilingbetweenL.major-andL. dono-vani-infectedmacrophageswasaremarkable increasein levelsoftranscriptionofmRNAsencoding prostaglandin-endoperoxidesynthase (Cox2) inthe latter, whichledto speculationthatthispathwayisinvolvedinthe pathogen-esisofVL[25](Figure 1).

Clearly,the availability ofhigh-throughput transcrip-tomictechnologieshasresulted inrapidexpansionofthe alreadysubstantialplethoraofknowledgeofthemolecular interactions occurring between Leishmania and the hu-man host; nevertheless, significant variation in host responsestoinfectionhasbeendescribedinseveralstudies (cf.[26]),althoughareviewofthisvariationisbeyondthe scope ofthepresent article.However, thesetechnologies havealsoenabledprogresstowardstheexplorationofthe molecularrelationshipsbetweentheparasiteandthesand flyvectorandthepatternsofLeishmaniadevelopmentinto itsinfective,nondividing metacyclicform[27].

TranscriptomicsinLeishmania–sandflyinteractions TransmissionofLeishmaniafromaninfectedtoa suscep-tible host requires development of the parasites in the midgut of a competent sand fly vector. Macrophages containingLeishmaniaamastigotesare ingestedby sand flyvectorsviaabloodmealand,oncereleasedintheinsect midgut, develop through several developmental stages into infective, metacyclic promastigotes [26] (Figure 1). ThereproductivemodeofLeishmaniaparasiteshas tradi-tionally been considered clonal, based onstrong linkage disequilibrium (cf. [28]); however, several studies have provided solid evidence of the occurrence of genetic ex-changebetweenspeciesand/orstrainsofLeishmania(i.e.,

L.majorandL.infantum)duringgrowthanddevelopment in the sand fly vector, with successful transmission of the hybrid progeny to a susceptible vertebrate host

[28–32]. The range of vertebrate and invertebrate host speciesthatLeishmaniacaninfect,aswellasthemultiple formsofdiseasethatitcauses,havebeenpartlyattributed totheabilityofthisparasitetoundergogeneticexchange in the sand fly vector (cf. [29]). Clearly, the molecular interactionsthat occurat theparasite–sand flyinterface are keyprocessesthat determinethesuccessful develop-ment and transmission of Leishmania; therefore, a de-tailed understandingofthesemechanismshas becomea priority. PreviousstudieshadusedSangersequencingof cDNA libraries from the midgut of sand fly vectors of both CL- and VL-causing Leishmania (i.e., Phlebotomus papatasi, vector of L. major and Lutzomyia longipalpis, vectorofL.infantum;[33,34])toidentifymolecules puta-tively involved in the development of the parasites in theirinsectvectors.WhilesandflyinfectionsbyL.major

andL.infantumwereconsistentlyassociatedwith down-regulation of molecules encoding microvilli-like proteins and chymotrypsin and upregulation of trypsin-encoding transcripts, the transcription profiles of peritrophin-like molecules were inconsistent between P. papatasi

andLu. longipalpis[33,34].Peritrophins arethe protein componentoftheperitrophicmatrix(PM),anextracellular chitin-containing structure that encapsulates the blood meal following its ingestion by the sand fly [35]. The formation of the PM (immediately following the blood meal) has longbeen considered advantageous for Leish-mania,becausetheparasitesarethoughttobeprotected fromtheactionofthesandflyproteolyticenzymesduring the vulnerable time of development to promastigotes

[35]. Several key investigations have contributed to furtherunderstandingoftherelationsbetween Leishman-iapromastigotesandthesandflyPM(e.g.,[36]).In partic-ular, while previous studies hypothesised a role of

Leishmania chitinases in the disintegration of the sand fly PM (cf. [36]), current evidence supports the theory that the breakdown of the PM is independent from the activityofLeishmaniaenzymesandthatparasite promas-tigotes escape the PM by migrating through a posterior opening that formsirrespective ofthe infection statusof the sand fly [36]. In the same study, Sadlova and Volf

[36]showedthat theanteriorplugofthePMservesasa ‘barrier’ for parasite migration to the thoracic midgut, until its degradation from sand fly proteolytic enzymes is complete [36]. The elucidation of patterns of sand fly gene expression during the disintegration of the PM in the presence (or not) of Leishmania parasites, and during migrationofthelatterfromtheabdominaltothe thoracicmidgut,mayhelptoeitherconfirmorconfutethis point.

Togetherwithstudiesofthemidgutofsandflies,other investigations used transcriptomic technologies to shed lightonthemolecularmechanismsthatgovernthe devel-opmentofLeishmaniaparasitesintotheirinfective meta-cyclic stage [37]. While little information isavailable on sandflymolecularpathwaysactingastriggerof Leishman-iametacyclogenesis,recentstudieshighlightedtheroleof key genes and gene products in the differentiation of promastigote stagesintometacyclicformsinthesandfly vector. Among these molecules, a hydrophilic acylated surface protein (HASPB) and a small hydrophilic endo-plasmic reticulum (ER)-associated protein (SHERP) showed increased expression in the metacyclic stages

[38]; in addition, creation of HASPB and SHERP null mutants in L. major resulted in the accumulation of non-infective parasite stages in the midgut of the sand fly vector, thusproviding evidence forthe essentialityof thesemoleculesforparasitedevelopment[38]. Investiga-tionsofpatternsofgenetranscriptionduringLeishmania

metacyclogenesisinvitrohaveledtotheidentificationof genes and gene products potentially related to parasite infectivity(e.g.[37,39–41]).Forinstance,recentfunctional studiesofessentialmoleculesinL.majormetacyclic pro-mastigotes highlighted major roles of mitogen-activated proteinkinases(i.e.,MAPK4)andmetallopeptidasesofthe M24Afamilyintheestablishmentofintracellular macro-phage infections [40] and proliferation in infected cells

[41].Thesedataprovidedasolidbasisfortheexploration oftheroleofthesemoleculesasnoveltargetsfor interven-tionstrategies.

In recent years, the search fornew andeffective pre-ventativemeasuresagainstLeishmaniatransmissionhas

alsoinvolvedthecharacterisationofkeycomponentsofthe salivaofthesandflyvectors(e.g.,[27,42–46]).Theinterest of the scientific community in salivary gland transcrip-tomes (‘sialotranscriptomes’) is mainly derived from knowledgethatselectedsalivaproteinshavecrucialroles infacilitatingthesuccessfulestablishmentofLeishmania

parasitesinvertebratehosts, includingtheregulation of theimmuneresponseatthesiteofbite[27,46,47]. There-fore, sialotranscriptomes of several competent sand fly vector species are now available (e.g., [43–45,48–51]), which,insomecases,haveledtotheselectionofkeysand flymoleculesthatareinvolvedintheblood-feedingprocess and that may assist the immunoevasive strategies of

Leishmania[47,52](Box2).Forinstance,apotent vasodi-lator(maxadilan)abundantlydetectedinthesalivaofLu. longipalpis[53]hasnotbeenidentifiedintranscriptomic data sets from the salivary glands of Lutzomyia ayacu-chensis[44].Similarly,amaxadilanhomologueidentified in Lutzomyia intermedia showed only 34% identity to maxadilan from Lu. longipalpis [50]. It is worth noting thatbothLu.intermediaandLu.ayacuchensisarevectors ofdermotropicLeishmaniaspecies,whereasLu. longipal-pis,whosesalivacontainslargeamountsofmaxadilan,is themainvectoroftheviscerotropicL.infantumintheNew World[54](Figure1).Whiletheseobservationssuggested aroleofmaxadilaninvisceralisationofL.infantum infec-tion[55],the absenceofmaxadilanhomologuesfromthe salivaof sand fly vectors of VL in the Old World raises questionsabouttherole/sofothersalivarycomponentsin diseaseprogression.Indeed,otherenzymes,suchas hya-luronidasesandapyrases,havebeenidentifiedusing tran-scriptomic andproteomic technologies fromseveralsand fly vectors of VL in both the Old and New Worlds

[45]. Theseenzymes have beenshown topositively con-tributetothespreadofLeishmaniaparasitesbypromoting

Box2.Sand-flysalivaryglandsecretedproteinsand

Leishmania:examplesfromasuccessfulpartnership

The salivary glands ofsand fly vectors secrete several proteins known to facilitate the process of host invasion by Leishmania

[27].Amongtheseproteins,maxadilan,aknownvasodilator,was firstdescribedinLutzomyialongipalpisandshowntoexacerbate theinfectivityofLeishmaniamajorviatheinhibitionoflymphocyte proliferation[47].Sincethisfirstreport,arangeofothermolecules hasbeendescribedfromthesalivaryglandsofanarrayofsandfly speciesthatcontributetothesuccessfulestablishmentof Leishma-nia infections. Among these molecules, protein homologues of salivary hyaluronidases secreted by Phlebotomus papatasi were demonstratedtohavearoleintheseverityofskinlesionscausedby

L.major[71].Whilethemechanismsthatresultintothisoutcome areyettobeelucidated,Volfovaandcolleagues[71]hypothesised thattheneutrophilsrecruitedthroughtheenzymaticactivityofsand flyhyaluronidases(viathepresenceofhyaluronanfragments)may be exploited by Leishmania as ‘Trojan horses’, thus facilitating macrophageinfection[71].Interestingly, asecretedendonuclease identifiedfromthesialotranscriptomeofLu.longipalpishasbeen recentlyimplicatedintheabilityofLeishmaniatoevadekillingby neutrophils [47]. This protein was shown to interfere with the microbicidalactivityofneutrophilsrecruitedatthesiteofasandfly bitebydisintegratingtheneutrophilextracellulartraps(NETs)that ensnareLeishmaniaparasites,thussignificantlycontributingtothe immunoevasive strategies of the parasite and enhancing its infectivity[47].

theenlargementofthefeedinglesionandthediffusionof othersalivaryactivecompounds(hyaluronidases)and pre-ventinghaemostasis(apyrases)[45].

Besides containingcomponents essentialto the infec-tionprocess,thesalivaofsandfliescontainsmoleculesthat canelicitspecificimmuneresponsesthatareindicativeof hostexposuretosandflybites(e.g.,[56,57]).Inparticular, three proteins from thesaliva ofP. perniciosus(i.e.,two yellowproteinsandanapyrase),expressedinrecombinant form,wereshowntobeusefulindeterminingtheintensity ofexposuretosandflybitesinexperimentallybittenmice anddogs[57].Whilecross-reactivity betweenanti-P. per-niciosusantibodiesandthosefromcloselyrelatedsandfly species was not assessed [57], the authors hypothesised that this may occur. Both yellowproteins and apyrases have been detected in the saliva of a range of sand fly species.However, subtledifferences insequencemay re-sultinvaryingimmunogenicproperties;future investiga-tionsusingtranscriptomicandproteomictechnologiesmay assistelucidatingthispointvia,forinstance,the genera-tionofwholetranscriptand/orproteindatasetsfromsand fly vector species, with the ultimate aim of identifying suitabletargets forthedevelopment ofcommercial diag-nostictoolstoassesstheriskofhumanandcanine trans-missionin bothendemicandnonendemic areas,andthe evaluation of the effectiveness of antivector campaigns

[56];thisimprovedknowledgecouldalsoaidcurrentefforts aimedatdevelopingrecombinantvaccinescontaining im-munogenic components from both the parasite and the sandflyvectors.Inaddition,thusfar,nodataareavailable ontheeffectsofLeishmaniainfectionsontheglobal tran-scriptional profiles of sand fly vectors. Future studies could,forinstance,utiliseRNA-Seqtechnologiesto inves-tigatedifferencesingeneexpressionprofilingof Leishman-ia-infected and uninfected sand flies. Exploring and identifying molecular pathways involved in the para-site–vector–hostinteractionsmayleadtotheidentification of new molecular pathways implicated in the infection process,whichwouldbeinstrumentalforrefiningcurrent control strategies against sand flies. Undoubtedly, some challengesexist inperforming large-scaletranscriptomic studiesofspeciesforwhichareferencegenomeis unavail-able;amongthesechallenges,thedenovoassemblyof full-lengthtranscriptsinabsenceofreferencesequencesisone ofthemostsignificant[58].Nevertheless,otherresources, suchasthegenomesandtranscriptomesofselected mos-quitospecies[59]thatarephylogeneticallyrelatedtosand flyvectors ofLeishmania [60], could beexploited forthe accurate reconstruction of(at least) a proportion of full-lengthsandfly transcripts,thus reducingoverallproject costsandlimitingpotentialbiasesintroducedby denovo

assembly.

Concludingremarksandresearchneeds

Overthepastdecade,advancesingenomicsand transcrip-tomics technologieshave contributed toconsiderably en-hanceourknowledge ofthe setofmolecularinteractions thatoccurwithinthehost–parasite–vectortriangle. How-ever, some gaps still exist in our understanding of the similaritiesand/ordifferencesbetweenhuman leishmani-asesandthediseaseinanimalreservoirhosts.Dogs,for

instance,representthemostimportanthostreservoirfor

L.infantum(causingVL)[3,61].Therefore,differencesand similaritiesbetweenhumanandcanineinfectionsshould be comprehensively analysed. However, most studies of

Leishmania immunobiology and genetics, as well as of host–parasiteinteractions,utilisemurinemodelsof infec-tionas‘mirrors’ofhumandisease[61].Giventhat trans-mission of key Leishmania species(e.g., L. infantum)to humans strictly relieson the circulation of the parasite amongcaninepopulations,elucidatingwhetherdog leish-maniasis servesas a model forhuman infections should becomeapriority. Thiscouldprovideavenuesforstudies aimed, for instance,at evaluating the ‘translatability’ of novel treatment and vaccine strategies from humans to dogs and vice versa. The availability of in vivo canine models of leishmaniasis [62], together with advances in genomicsand/ortranscriptomics,proteomics,and metabo-lomics technologies, may assist this quest. Forinstance, RNA-Seqandhigh-throughputproteomicsplatforms pro-videagoldenopportunitytomonitorchangesinhostgene transcription and protein expression throughout the course of canine and human infections, thus enabling onetodrawparallelsbetweenthem.Similarly,large-scale analyses of metabolites produced during the course of infection, both by the parasite and the vertebrate host, mayrepresent agoldmineforthe identificationofnovel diagnostic biomarkers,as wellasofpotentialnew Leish-mania‘Achilles’heels’thatcouldassistcurrentprograms aimed at breaking thetransmission cycle ofhuman and canineleishmaniases.

Acknowledgements

Part of this article was conceived within the framework of of the EurNegVecCOSTActionTD1303.FundingfromtheIsaacNewtonTrust/ WellcomeTrust ISSF/Universityof CambridgeJoint Research Grants SchemetoC.C.isgratefullyacknowledged.

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