Study of the secretome of Leishmania involved in the infection

! ! ! ! ! Nuno!Pedro!Moreira!Santarém! ! Study!of!the!secretome!of!Leishmania! involved!in!the!infection! ! ! !Tese!do!3º!Ciclo!de!Estudos!Conducente!ao!! Grau!de!Doutoramento!em!Ciências!Farmacêuticas!! 8!Especialidade!de!Bioquimica! ! ! ! (Dissertation! thesis! for! the! degree! of! Doctor! of! Philosophy! in! Biochemistry! by! the! Faculty! of! Pharmacy! in! Porto! University! and! Molecular! and! Cellular! Biology! by! the! Faculty! of! Medicine! in! University!Laval)! ! ! ! ! With!the!supervision!of:! ! Professor' Doutora' Anabela' Cordeiro' da' Silva' (Associate' professor' with' Aggregation'in'the'Faculdade'de'Farmácia'da'Universidade'do'Porto)' ' Professor'Marc'Ouellette'(Scientific'Director'Institute'of'Infection'and'Immunity' CIHR.' ' Burroughs' Wellcome' Fund' Scholar' in' Molecular' parasitology' Canada' Research'Chair'in'Antimicrobial'resistance'Centre'de'Recherche'en'Infectiologie' CHUQ'et'Université'Laval)'! December,!2011 ii" " ' ' ' ' ' ' ' ' ' ' ! Declaração!de!reprodução! ' ' É'AUTORIZADA'A'REPRODUÇÃO'INTEGRAL'DESTA'TESE!APENAS'PARA'EFEITOS' DE.'INVESTIGAÇÃO,'MEDIANTE'DECLARAÇÃO'ESCRITA'DO'INTERESSADO'QUE'A' TAL'SE'COMPROMETE.' ! ! ! iii" " ! Faculty!of!Farmacy!of!Porto!University! &! Faculty!Medicine!University!Laval! ! ! ! ! ! ! ! ! ! Study!of!the!secretome!of!Leishmania! involved!in!the!infection!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Nuno'Pedro'Moreira'Santarém' ! iv" " ! ! ' ' ' ' ' ' The'candidate'performed'the'experimental'work'with'a'doctoral'fellowship'(SFRH'/'BD'/' 37352' /' 2007)' supported) by) the) “Fundação) para) a) Ciência) e'a) Tecnologia”) (FCT;) Portugal)'and'CIHR'operating'grants'from'the'Centre'de'Recherche'en'Infectiologie'du' Centre'de'Recherche'du'CHUL'(Quebec,'Canada).'FCT'also''also'participated'with'grants' to' attend' international' meetings' and' for' the' graphical' execution' of' this' thesis.' The' Faculty' of' Pharmacy' of' the' University' of' Porto' (FFUP;' Portugal),' the' Institute' for' Molecular'and'Cell'Biology'(IBMC;'Portugal),'and'Centre'de'Recherche'en'Infectiologie' du'Centre'de'Recherche'du'CHUL'provided'the'facilities'and'logistic'support.' ' ' ' ' ' ' ' ' v" " AUTHOR’S)DECLARATION! ' Under'the'terms'of'the'Decree_Law'nº'216/92,'of'October'13th,'is'hereby'declared'that' the'following'original'articles'were'prepared'in'the'scope'of'this'dissertation.' ' ' ! PUBLICATIONS! Articles'in'international'peer_reviewed'journals' ' ' In'the'scope'of'this'dissertation' ' I.'Impact'of'continuous'axenic'cultivation'in'Leishmania!infantum'virulence'(Acepted! for!publication!in!Plos!Neglected!Tropical!Diseases)' II.'The'development'of'a'semi_defined'media'for'growth'of'Leishmania!infantum'(To!be! Submited)' III.'Exoproteome'dynamics'in'L.!infantum!(To!be!submitted!to!Journal!of!Proteomic! Research)' IV.'Activation'bone'marrow'derived'dendritic'cells'by'the'exoproteome'from'Leishmania! infantum'(To!be!Submited)' ' ' Participation'in'other'publications'in'related'fields' ' I.'Neves,'B.M.,'Silvestre,'R.,'Resende,'M.,'Ouaissi,'A.,'Cunha,'J.,'Tavares,'J.,'Loureiro,'I.,' Santarem,! N.,' Silva,' A.M.,' Lopes,' M.C.,' Cruz,' M.T.,' Cordeiro' da' Silva,' A.,' 2010,' Activation' of' phosphatidylinositol' 3_kinase/Akt' and' impairment' of' nuclear' factor_ kappaB:' molecular' mechanisms' behind' the' arrested' maturation/activation' state' of' Leishmania'infantum_infected'dendritic'cells.'Am'J'Pathol!177,'2898_2911.' ' II.'Santarem,! N.*,' Silvestre,' R.*,' Cardoso,' L.,' Schallig,' H.,' Reed,' S.G.,' Cordeiro_da_ Silva,'A.,'2010,'Application'of'an'improved'enzyme_linked'immunosorbent'assay'method' for'serological'diagnosis'of'canine'leishmaniasis.'J'Clin'Microbiol!48,'1866_1874.' ' *'The'authors'have'equally'contributed'to'the'work' ' vi" " III.' Silvestre,' R.*,' Santarem,! N.*,' Teixeira,' L.,' Cunha,' J.,' Schallig,' H.,' Cordeiro_da_ Silva,'A.,'2009,'Evaluation'of'Leishmania'species'reactivity'in'human'serologic'diagnosis' of'leishmaniasis.'Am'J'Trop'Med'Hyg!81,'202_208.' ' *'The'authors'have'equally'contributed'to'the'work' ' IV.' Carvalho,' S.,' Cruz,' T.,' Santarem,! N.,'Castro,' H.,' Costa,' V.,' Tomas,' A.M.,' 2008,' Heme'as'a'source'of'iron'to'Leishmania'infantum'amastigotes.'Acta'Trop!109,'131_135.' ' V.'Silvestre,'R*.,'Santarem,!N*.,'Cunha,'J.,'Cardoso,'L.,'Nieto,'J.,'Carrillo,'E.,'Moreno,' J.,'Cordeiro_da_Silva,'A.,'2008,'Serological'evaluation'of'experimentally'infected'dogs'by' LicTXNPx_ELISA'and'amastigote_flow'cytometry.'Vet'Parasitol!158,'23_30.' ' *'The'authors'have'equally'contributed'to'the'work' ' VI.' Tavares,' J.,' Ouaissi,' A.,' Santarem,! N.,' Sereno,' D.,' Vergnes,' B.,' Sampaio,' P.,' Cordeiro_da_Silva,'A.,'2008,'The'Leishmania'infantum'cytosolic'SIR2_related'protein'1' (LiSIR2RP1)'is'an'NAD+'_dependent'deacetylase'and'ADP_ribosyltransferase.'Biochem'J! 415,'377_386.'' ' VII.' Borges,' O.,' Cordeiro_da_Silva,' A.,' Tavares,' J.,' Santarem,! N.,' de' Sousa,' A.,' Borchard,'G.,'Junginger,'H.E.,'2008,'Immune'response'by'nasal'delivery'of'hepatitis'B' surface'antigen'and'codelivery'of'a'CpG'ODN'in'alginate'coated'chitosan'nanoparticles.' Eur'J'Pharm'Biopharm!69,'405_416.' ' ' ' ' ' ' ' ' ' Under'the'terms'of'the'referred'Decree_Law,'the'author'declares'that'he'afforded'a'major' contribution'to'the'conceptual'design'and'technical'execution'of'the'work,'interpretation' of' the' results' and' manuscript' preparation' of' the' published' articles' included' in' this' dissertation.' vii" " ' ' ' ' ' ! ! ! ! ! ! ! ! “Far%better%is%it%to%dare%mighty%things,%to%win%glorious%triumphs,%even%though%checked% by! failure...than! to! rank! with! those! poor! spirits! who! neither! enjoy! much! nor! suffer! much,%because%they%live%in%a%gray%twilight%that%knows%not%victory%nor%defeat.”! _'Theodore'Roosevelt' ' ' viii" " ' ' ' ' ix" " ACKNOWLEDGMENTS!! It'is'difficult'to'find'words'to'translate'my'gratitude'to'all'that'contributed'to'make'this' thesis'possible.' First,'I'must'acknowledge'my'supervisors,'the'Professors'Anabela'Cordeiro_da_Silva'and' Marc'Ouellette.'To'both'I'thank'for'the'advice,'guidance,'and'support.'It'was'truly'an' honor'and'a'privilege'to'have'both'of'you'as'my'supervisors.'It'would'be'ungrateful'of'me' to'let'pass'the'opportunity'to'publically'thank'to'Professora'Anabela'for'always'believing' and'supporting'me'throughout'my'academic'trajectory.'I'will'never'forget.'To'Professor' Marc'Ouellette'I'must'confess'that'when'I'arrived'in'your'laboratory'I'was'feeling'a'bit' scared'because'I'was'afraid'that'I'might'be'outside'of'my'depth,'far'from'the'comfort' zone' of' my' laboratory' in' Portugal.' It' was'truly' important' for' my' confidence' and' self' believe)the)time)I)spent)in)“Mou)team”.' I'will'also'like'to'thank'to'Professor'Claudio'Sunkel,'Director'of'the'IBMC,'and'Michel'G.' Bergeron'MD,'the'director'of'the'CRI'for'opportunity'of'developing'my'research'project' at'their'institutes,'in'which'all'the'necessary'conditions'for'the'success'of'my'work'were' available.' I'also'extend'my'gratitude'to'the'directors'of'the'doctoral'programs,'Professor'José'Costa' Lima'in'the'University'of'Pharmacy'and'Jacques'Landry'in'Laval'University'for'the'help' in'solving'all'the'burocratic'problems'during'these'four'years.' To' all' the' co_authors' of' the' work' developed' during' this' thesis,' thank' you' for' your' contribution,'it'would'not'have'been'possible'to'have'this'thesis'without'your'help.' To' the' Parasite' Disease' group' I' must' thank' you' all' for' the' continuous' support' and' friendship.''Ricardo'Silvestre'(A'veteran'from'many'scientific'battles,'always'there'for'a' discussion,' a' joke,' a' word' of' encouragement,' never' without' an' advice),' Mariana' and' Diana' (What' can' I' say!' There' would' not' be' a' PD' as' we' know' it' without' you' both!' Mariana'is)the)“P”)–'Public'Relations'_'and)Diana)is)the)“D”_'Dénia!'In'fact'not'only'PD' would' be'different' but'also' the' IBMC,' at'least' there'would' no' need' of' canteen'in' the' IBMC' without' Diana!!!' To' you' both' I' thank' the' friendship' and' cooperation' and' the' research' –' MEGASOMAAAAA!!!).' To' Sofia' Lima,' Inês,' Suzana,' Vasco' (Vascão!!!!!),' Joana'Tavares,'Susana,'Marta'and'all''trainees'that'have'passed'in'the'laboratory'during' these'years)(Daniela,)Simão,)Ana)Luisa,)“Seu”)Jorge…)just)to'name'a'few,'you'know'who' you'are!!!),'a'big'thank'you'for'enduring'four'years'of'bad)jokes)and)“excellent”'music' and' all' of' the' constant' vesicle' theories!!!' And' of' course,' Dona' Casimira' from' the' Biochemistry'department'in'the'FFUP!!!!'I'did'not'forget!!!' xvi" " 2.2.1.3'–'Molecular'diagnosis'...................................................................................'27' 2.2.2'–'Treatment'of'the'disease'..................................................................................'29' 2.2.3'–'Control'of'vectors'and'reservoirs'.....................................................................'35' 3'–'Immunopathogenesis'of'Leishmania'spp.'infection'.....................................................'36' 3.1'_'Establishment'of'Leishmania!spp.'on'the'host'........................................................'36' 3.2'–'The'importance'of'Toll_like'receptors'in'Leishmania'spp.'infection'.....................'42' 3.2.1'–'Toll'like'receptors'and'Leishmania'..................................................................'45' 3.3'–'APC'deactivation'induced'by'Leishmania!spp.'......................................................'47' 4'–'Exoproteome..................................................................................................................'49' 4.1'–'Definition'.................................................................................................................'49' 4.2'_'Mechanisms'of'protein'release'in'the'Leishmania!spp.!exoproteome.'...................'49' 4.2.1'_'The'surface'of'the'promastigote'........................................................................'50' 4.2.2'_'The'classical'secretory'pathway'........................................................................'52' 4.2.3'_'Unconventional'secretory'pathway'...................................................................'55' 4.2.3.1'–'Vesicle'release'............................................................................................'56' 4.3'–'Exoproteome'in'Leishmania!spp.!immunopathology'............................................'61' 4.4'–'Approaches'for'the'study'of'the'exoproteome'........................................................'62' ! Chapter!II!P!Objectives!and!results! 1.'Objectives'.........................................................................................................................'65' 2.'Article'name:'Impact'of'continuous'axenic'cultivation'in'Leishmania!infantum' virulence'...............................................................................................................................'69' 3.'Article'name:'The'development'of'a'semi_defined'media'for'growth'of'Leishmania! infantum'............................................................................................................................'109' 4.'Article'name:'Exoproteome'dynamics'in'L.!infantum'..................................................'139' 5.'Article'name:'Exoproteome'from'!Leishmania!infantum'activates'bone'marrow' derived'dendritic'cells'........................................................................................................'181' ' Chapter!III!P!Discussion!and!perspectives! 1.'Discussion.......................................................................................................................'207' 2.'Perspectives'...................................................................................................................'213' 3.'Bibliography'...................................................................................................................'214' 4.'Anexed'files'....................................................................................................................'247' ' ' xvii" " INDEX!OF!FIGURES! Figure'1.'Taxonomy'of'pathogenic'Leishmania'spp.."................................................................."3" Figure'2.'Schematic'representation'of'Leishmania'spp.'developmental'forms."...................."5" Figure'3.'General'life'cycle'of'the'parasites'from'the'genus'Leishmania."..............................."6" Figure'4.'Global'geographic'distribution'of'leishmaniasis"......................................................"19" Figure'5.'Disease'presentations'and'distinctive'clinical'signs'of'Leishmaniasis."................."20" Figure'6.'Cells'involved'in'the'first'steps'of'infection'by'Leishmania!spp.'.........................'37' Figure'7.'Immune'response'associated'with'Leishmania'spp.'infection"..............................."39" Figure'8.'Recognition'of'PAMPs'from'different'classes'of'microbial'pathogens.."..............."43" Figure'9.'TLRs'associated'immune'response."..........................................................................."45" Figure'10'–'Schematic'diagram'summarizing'the'different'secretion'mechanisms'and' their'putative'regulation'in'Leishmania!spp.."..........................................................................."50" Figure'11.'Surface'coats'of'Leishmania'ssp."..............................................................................."51" Figure'12.'Release'of'extracellular'microvesicles'into'the'extracellular'space."....................."57" Figure'13'Involvement'of'secreted'vesicles'in'interactions'of'immune'cells."........................"59" ' " " xviii" " ' INDEX!OF!TABLES! Table'1.'Major'epidemiological'characteristics'of'pathogenic'Leishmania'spp.'.................'17' Table'2.'Standard'treatment'regimens'for'visceral'leishmaniasis.'.....................................'29' ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! xix" " ABBREVIATIONS!LIST! (ABC)'_'ATP_binding'cassette' (APCs')'_'Antigen'presenting'cells'' '(BmDCs)'_'Bone'marrow'derived'dendritic'cells' (CR)'_'Complement'receptor' (CL)'_'Cutaneous'leishmaniasis'' (DNA)'_'Deoxyribonucleic'acid' (DDT)'_'Dichloro_Diphenyl_Trichloroethane'' (ELISA)'_'Enzyme'linked'immunosorbent'assay'' (ER)'_'Endoplasmic'reticulum'' (ESCRT)'_'Endosomal'sorting'complexes'required'for'transport' '(DCL)'_'Diffuse'cutaneous'leishmaniasis'' (DAT)'_'Direct'agglutination'tests'' (fPPG)'_'Filamentous'proteophosphoglycan'' (FCS)'_'Fetal'Calf'Serum'' (GFP)'–'Green'fluorescent'protein' (GPI)'_'Glycosylphosphatidylinositol'' (HIV)'_'Human'immunodeficiency'virus;' (HASPB)'_'Hydrophilic'acylated'surface'protein'B'' (IFNγ)'_'Interferon_γ'' (IL)'_'Interleukine' (LPG)'_'Lipophosphoglycan'' (MS)'_'Mass'spectrometry'' (MHC)'_'MHajor'histocompatibility'complex' (Mb)'_'Mega'Base'' (mRNA)'_'Messanger'RNA'' (moDCs)'_'Monocytes'derived'dendritic'cells'' (MyD88)'_'myeloid'differentiation'gene'88'' (Mya)'_'Million'years'ago'' xx" " (MVs)'_'Microvesicles'' (MCL)'_'Mucocutaneous'leishmaniasis'' (MVBs)'_'Multivesicular'bodies'(MVBs)' '(NNN)'_'Novy_MacNeal_Nicolle'' '(PRRs)'_'Pattern'recognition'receptors'' (PAMPs)'_'Pathogen_associated'molecular'patterns'' (PBMCs)'_'Peripheral'blood'mononuclear'cells'' (PCR)'_'Polymerase'chain'reaction'' (PKDL)_'Post'kala_azar'dermal'leishmaniasis'' (PSG)'_'Promastigote'secretory'gel' (TLRs)''_'Toll_like'receptors'' (RFLP)'_'Restriction'fragment'length'polymorphism'' (RNA)'_'Ribonucleic'acid' (rRNA)'_'Ribosomal'RNA'' (SL)'_'Spliced'leader'' (SHERP)'_'Small'hydrophilic'endoplasmic'reticulum_associated'protein'' '(TH1)'_'T'helper'1' (Tregs)'_'T'regulatory'cells'' (TGF-β) - Transforming growth factor beta' (tER)'_'transitional'endoplasmic'reticulum' (TNF_α)'_'tumor'necrosis'factor'alpha'' (VDE)'_'Vesicle'depleted'exoproteome'' (VL)'_'Visceral'leishmaniasis'' ' (WHO)'_'World'Health'Organization' CHAPTER''......................................................'I' 1" " Chapter!I! Introduction'' ' CHAPTER''.....................................................'I' 2" " 1.!!Leishmania!spp.! ! 1.1!–!Discovery!and!the!first!years! ! In'1900'in'British'colonial'India,'at'the'Army'Medical'School'in'Netley,'a'patient' suffering'from'pyrexia,'anemia'and'enlargement'of'the'spleen,'typical'symptoms'of'kala_ azar,'was'admitted'for'treatment'and'investigation.'William'Boog'Leishman,'at'the'time' Assistant'Professor'of'Pathology'at'Netley,'discovered'in'this'patient'a'great'number'of' heavily_stained'oval'bodies'in'spleenic'cells'and'blood'preparations.'Later,'while'studying' rats'afflicted'with'trypanosomiasis,'he'registered'similar'round'bodies'in'the'spleen,'blood' and' liver' of' the' infected' rodents''(Leishman,' 1903).' Soon' after,' Charles' Donovan' described' similar' bodies' in' cells' from' patients' in' Madras,' in' the' south' of' India,' and' proceeded'with'the'exclusion'of'the'possibility'of'malaria,''a'disease'that'could'produce' similar' symptoms' to' kala_azar' (Donovan,' 1903).' Finally' Ronald' Ross,' a' prominent' authority' in' parasitology' at' the' beginning' of' the' 20th' century,' attributed' to' both' the' discovery'of'the'causative'agent'of'kalaQazar'in'India,'naming'it'Leishmania'donovani' and'creating'the'gender'Leishmania!(Ross,'1903).'' In'less'than'one'decade'the'etiological'agents'of'kalaQazar!(visceral'leishmaniasis)' were' isolated' all' over' the' Mediterranean' basin,' Sudan,' China' and' Russia' (Sen' Gupta,' 1962).'Soon'after,'several'reports'associated'the'gender'Leishmania!with'other'diseases'of' unknown'origin,' like' the' “delhi'sore”' in'India'(Wright,'1903)' or'the' “baurú' ulcer”' and' “espundia”' in' South' America' (Lindenberg,' 1909;' Splendore,' 1911).' Quickly' after' the' discovery' of' the' causative' agent' of' leishmaniasis' it' became' clear' that' these' microorganisms'had'an'extracellular'form'that'was'distinct'from'the'one'found'inside'the' diseased'patients.'In'1904'the'extracellular'form'of'the'protozoan'was'isolated'and'grown' in'laboratory'(Rogers,'1904).'The'transmission'of'the'parasite'became'quickly'matter'of' debate'with'an'extensive'search'for'the'elusive'agent'responsible'for'the'transmission'of' the' disease.' The' correlation' between' endemic' areas' and' the' distribution' of' certain' sandflies'species'eventually'contributed'to'the'discovery'of'these'insects'as'vectors'for'the' dissemination'of'the'disease'(Knowles'et'al.,'1924).'' These'events'were'at'the'genesis'for'the'current'definition'of'Leishmania!spp.!as' obligate' protozoan' parasites' that' are' the' etiological' agent' of' a' group' of' diseases' collectively'known'as'leishmaniasis.' ' CHAPTER''......................................................'I' 3" " 1.2!P!Taxonomy!and!evolution! ' ' The' classification' of' Leishmania! spp.!was' initially' based' on' subjective' extrinsic' characteristics:' clinical' manifestations,' geographic' distribution,' susceptible' vectors' and' morphological' data' (Banuls' et' al.,' 2007).' In' the' last' quarter' of' the' 20th' century' these' criteria'were'substituted'by'more'accurate'intrinsic'criteria'like'patterns'of'polymorphism' exhibited'by'kinetoplast'deoxyribonucleic acid (DNA)'(Rodriguez_Gonzalez'et'al.,'2007;' Wirth'and'Pratt,'1982)'or'antigenic'characteristics'(Anthony'et'al.,'1985;'de'Ibarra'et'al.,' 1982).'These'tools'eventually'lead'to'the'identification'of'31'Leishmania!species'of'which' around'20'are'thought'to'be'pathogenic'to'humans'(Figure'1).' ' ! Figure!1.!Taxonomy'of'pathogenic'Leishmania'spp.'Adapted'from'(Banuls'et'al.,'2007).! ! Phylogenetically'the'Leishmania!spp.'protozoa'are'placed'in'the'genus'Leishmania! belonging'to'the'class'Kinetoplastida!and'the'order!Trypanosomatidae'(Figure'1).''Some' authors' using' molecular' characterization' that' included:' multilocus' enzyme' electrophoresis,' analysis' of' the' ribosomal' ribonucleic' acids' (rRNA)' gene' cluster' by' restriction'fragment'length'polymorphism'(RFLP)'and'sequencing'of'the'small'subunit' rRNA' gene,' proposed' the' separation' of' the' genus' Leishmania! into' two' divisions,! Euleishmania'and'Paraleishmania.!Euleishmania!would!contain'the'subgenera'Viannia! and'Leishmania!while'Paraleishmania'would'include'the'Leishmania!hertigi,!Leishmania! deanei,! Leishmania! colombiensis,! Leishmania! equatorensis,! Leishmania! herreri! in' addition'to'several'Endotrypanum!strains!(Cupolillo'et'al.,'2000).'This'separation'is'not' consensual' and' traditionally' the' genus' Leishmania! is' divided' only' into' two' distinct' subgenus'Leishmania!(Leishmania)'and'Leishmania!(Viannia)!(Lainson'and'Shaw,'1987).' The'first'contains'the'species'complexes'of'L.!donovani,!Leishmania!tropica,!Leishmania! major! and' Leishmania! aethiopica.' The' sub' genus' Viannia' is' restricted' to' New' World' Subkingdom Order Family Genus Subgenus Complex L.donovani L.tropica L.-major L.-aethiopica L.-mexicana L.-braziliensis L.-guyanensis L.-naiffi L.-lainsoni Species L.-archibaldi L.-killicki L.-major L.-aethiopica L.-amazonensis L.-braziliensis L.-panamensis L.-naiffi L.-lainsoni L.-chagasi L.-tropica L.-garnhami L.-peruviana L.-guyanensis L.-infantum L.-mexicana L.-shawi L.-donovani L.-piffanoi L.-venezuelensis L.-forattinii Protozoa Kinetoplastida Trypanosomatidae Leishmania Leishmania Viannia CHAPTER''.....................................................'I' 4" " species'of'the'Leishmania!guyanensis'and'Leishmania!braziliensis!complexes'and'the'less' known!Leishmania!naiffi!or'Leishmania!lainsoni'(Figure'1).'This'division'of'Leishmania! genus'into'two'subgenera'was'initially'done'using'as'criteria'the'area'of'the'sandfly'gut' (one'of'the'hosts'of'the'protozoan)'that'was'colonized'by'the'parasite'(Lainson'and'Shaw,' 1987).'This'division'was'later'supported'by''DNA'based'phylogenetic'analyses'(Croan'et' al.,'1997).' There' is' also' a' group' of' Leishmania'spp.! related' organisms' designated' as' Sauroleishmania'that'traditionally'are'found'in'lizards.'This'exotic'group'includes'the'non' pathogenic' Leishmania! tarentolae.' Mainstream' authors' place' these' protozoans' in' a' completely'different'genus,'Sauroleishmania!(Lainson,'2010).'Still,'phylogenetical'studies' present'them'as'closely'related'to'the'Leishmania!(Leishmania)!subgenus'(Croan'et'al.,' 1997;' Noyes' et' al.,' 1997).' L.! tarentolae! is' the' most' carefully' studied' Sauroleishmania' species'being'used'as'ubiquitous'non'pathogenic'Leishmania'in'several'studies'(Azizi'et' al.,'2009).'The'recent'sequencing'of'L.!tarentolae'revealed'a'high'level'of'synteny'and' extensive'homology'and'to'other'pathogenic'Leishmania!spp.'(Raymond'et'al.,'2011).'This' protozoan' is' also' considered' as' a' model' organism' for' the' synthesis' of' recombinant' proteins'(Basile'and'Peticca,'2009).' ' The'understanding'of'the'evolution'of'genus'Leishmania'is'hampered'by'the'fact' that'the'phylum'Euglenozoa'lacks'a'good'fossil'record'(Tuon'et'al.,'2008).'Nonetheless,' molecular'studies'place'the'first'direct'ancestor'of' Leishmania'in'the' Ordovician'more' than' 488'million' years'ago' (Mya)' (Kerr,' 2006).' This' was' an' epoch' where' none' of' the' known' host' for' the' parasite' existed,' although' the' origin' of' fish' and' the' subsequent' evolution'of'amphibians'and'reptiles'provided'probably'the'first'hosts'in'an'environment' where'hematophagous'leeches'could'functions'as'the'first'vectors'(Molyneux,'1977).'The' appearance'of'winged'insects'in'the'Carboniferous'(300'Mya)'and'subsequent'radiation' into' Diptera' in' the' Triassic' (200' Mya)' lead' to' the' appearance' of' the' ancestors' of' the' known'insect'vectors'from'the'genus'Phlebotomus'and'Lutzomyia''(Tuon'et'al.,'2008).' The' first' evidence' of' hematophagous' insects' is' recorded' at' 140' Mya' in' the' Cretaceous' (Azar' and' Nel,' 2003)' and' shortly' after' the' first' member' of' the' genus'Leishmania!was' found' in' fossil' record' from' 100' Mya,' Paleoleishmania! proterus' (Poinar' and' Poinar,' 2004).'It'is'just'after'the'extinction'of'the'dinosaurs'and'diversification'of'the'placental' mammals'that'we'find'the'confirmation'in'the'fossil'record'of'the'direct'ancestors'of'the' vectors'Phlebotomus'and'Lutzomyia!and'the'direct'ancestors'of'Leishmania!(Tuon'et'al.,' 2008).!The'global'distribution'of'the'parasites'has'originated'a'heated'debate'concerning' the'geographical'origin'and'the'timing'of'colonization'of'the'different'species'found'today' (Kerr,'2000;'Noyes'et'al.,'2000).'Actually'there'is'more'data'supporting'the'origin'of'the' CHAPTER''......................................................'I' 5" " parasite'in'the'American'tropics'with'migration'to'the'Eurasia'by'the'Bering'land'bridge' (Lukes'et'al.,'2007).'This'theory'also'accounts'for'the'distinct'evolution'of'the'Leishmania! (Viannia)' subgenus' and' for' the' greater' variety' of' species' found' in' south' America' (Lainson,'2010).'' ' ' ' Figure! 2.! Schematic' representation' of' Leishmania' spp.' developmental' forms_ Leishmania'spp.'promastigote'(left)'or'amastigote'(right)'forms.'Adapted'from'(Besteiro' et'al.,'2007).' ' ' ' CHAPTER''.....................................................'I' 12" " genomic' sequences.' These' can' occur' spontaneously' or' happen' as' a' consequence' of' parasite' exposure' to' adverse' conditions' such' as' drug' selection' (Grondin' et' al.,' 1996;' Navarro'et'al.,'1994;'Olmo'et'al.,'1995;'Segovia,'1994;'Ubeda'et'al.,'2008).'As'the'core' genome'of'the'Leishmania'spp.'is'quite'similar'it'is'probable'that'this'genomic'plasticity' contributes' significantly' to' phenomena' such' as' the' distinct' virulence' between' clinical' isolates'and'also'drug'resistance'(Sterkers'et'al.,'2011;'Ubeda'et'al.,'2008).' Several' reports' of' interspecies' Leishmania! spp.!hybrids' (Chargui' et' al.,' 2009;' Odiwuor'et'al.,'2011;'Ravel'et'al.,'2006)'lead'to'the'possibility'of'genetic'exchange'between' parasites.' This' was' highly' controversial' because' it' was' accepted' that' Leishmania! spp.! multiply'by'binary'fission'and'sexual'crossing'under'laboratory'conditions'failed'(Banuls' et' al.,' 2007).' However,' a' 2009' report' clearly' showed' the' formation' of' hybrids' in' the' sandfly'(Akopyants'et'al.,'2009).'More'recently'the'same'phenomena'was'described'with' L.!donovani'(Sadlova'et'al.,'2011)'indicating'that'sexual'reproduction'might'be'a'general' phenomenon' in' Leishmania! spp.!during' the' life' cycle.' This' was' further' supported' by' genetics' analysis' done' in' L.! braziliensis! and'L.! guyanensis! that'demonstrated'that'the' percentage'of'homozygotes'was'much'higher'than'what'expected'for'a'clonal'population' (Rougeron'et'al.,'2011;'Rougeron'et'al.,'2009).'' Like' in' all' eukaryotes,'DNA' content' in'Leishmania'spp.! is' not' restricted' to' the' nucleus.' The' parasite' has' a' single' mitochondrion,' containing' the' mitochondrial' DNA' condensed' in' a' disk_shaped' structure,' the' kinetoplast.' This' structure' is' the' defining' feature'of'these'protozoans'and'was'at'the'origin'of'the'designation'Kinetoplastida'for'the' order!(Shapiro'and'Englund,'1995).'This'second'genomic'organelle'of'the'kinetoplastid' contains'two'classes'of'DNA'rings,'maxicircles'and'minicircles.'The'maxicircles'represent' the'actual'mitochondrial'genome.'Only'a'few'dozen'different'maxicircles'exist,'ranging'in' size'from'35'to'50'kb,'being'present'at'about'10_30'copies'per'cell.'The'gene'products'of' the'maxicircles'are'similar'to'those'of'mitochondrial'DNA'in'higher'eukaryotes,'including' rRNA'and'several'respiratory'chain'components'(Liu'et'al.,'2005).'Each'kinetoplast'also' contain'a'few'thousand'minicircles,'ranging'in'size'from'0.8'to'1.6'kb,'present'at'about' 30.000_50.000'copies'per'cell.'These'minicircles'contain'the'genes'for'guide'RNAs.'These' guide' RNAs' are' necessary' to' edit' the' maxicircle' transcripts' into' functional' messanger' RNA' (mRNA)' (Simpson,' 1997;' Stuart' et' al.,' 1997).' Unlike' higher' eukaryotes,' the' kinetoplastid' mitochondrial' genomes' contain' no' transfer' RNA' constituting' the' single' greatest'difference'in'mitochondrial'genetic'structure'(Schneider'and'Marechal_Drouard,' 2000).' CHAPTER''......................................................'I' 13" " The'mechanism'of'transcription'initiation'in'kinetoplastid'protozoa'is'remarkably' different' from' the' remaining' eukaryotes.' The' near' absence' of' promoters' for' RNA' polymerase'II'is'the'most'notable'difference'in'gene'expression'(Clayton,'2002).'The'most' accepted'theory'for'the'RNA'polymerase'II'activity'in'these'organisms'is'that'transcription' starts' upstream' of' most' 5’) genes) of) each) cluster,) originating) polycistronic) transcripts) (Martinez_Calvillo'et'al.,'2003).'These'polycistronic'transcripts'will'then'be'processed'at' the)5’)end)by)trans_splicing.'A'common'39'nucleotide'methylated'mini_exon'sequence'or' spliced' leader' (SL)' is' trans_spliced' upstream' of' the' ATG' start' codon' of' the' mRNA' replacing' the' capping' system' found' in' the' majority' of' eukaryotic' organisms' (Clayton,' 2002;'Sturm'et'al.,'1999).'The'SL'gene'in'Leishmania!spp.!is'present'as'a'tandem'array'of' about'150'copies'comprising'0.1'%'of'parasite'genome'(Lamontagne'and'Papadopoulou,' 1999;'Miller'et'al.,'1986).'In'contrast'to'most'protein'encoding'genes'in'trypanosomatids,' the'SL'precursor'RNA'has'clearly'identifiable'promoters'with'a'short'consensus'initiator' element' (Luo' et' al.,' 1999)' being' transcribed' by' the' RNA' polymerase' II' (Gilinger' and' Bellofatto,'2001).' As' a' consequence' of' the' almost' complete' absence' of' promoters' for' RNA' polymerase'II'(Clayton,'2002)'there'is'a'loss'of'an'entire'level'of'gene'expression'control' at' the' transcription' level.' This' fact' in' an' organism' that' thrives' in' two' distinct' environments,'with'different'temperatures,'nutrients'and'distinct'survival'requirements' might'be'problematic.'In'fact!Leishmania'does'not'have'any'significant'qualitative'control' at' the' transcription' initiation' level' but' seems' to' have' adopted' a' system' where' post_ transcriptional' mechanisms,' such' as' mRNA' stability' and' processing,' are' the' major' determinants'in'the'regulation'of'mRNA'abundance,'and'ultimately'of'protein'production' (Requena,' 2011).' The' existence' of' stage' specific' proteins,' like' the' amastigote' specific' amastin'family,'are'indicative'of'the'efficiently'of'these'mechanisms'(Moore'et'al.,'1996;' Nourbakhsh'et'al.,'1996;'Souza'et'al.,'1992;'Wu'et'al.,'2000).'In'the'genomic'era,'using'a' combination' of' proteomic' and' genomic' approaches,' it' was' possible' to'confirm' that' an' almost'constitutive'expression'of'the'genome'exits'in'both'promastigote'and'amastigote' stages' with' low' levels' of' stage' specific' variability.' However,' when' using' a' quantitative' proteomic'analysis,'a'higher'heterogeneity'was'found'in'protein'expression'between'both' stages' (Leifso' et' al.,' 2007;' McNicoll' et' al.,' 2006).' Documented' mechanisms' through' which' Leishmania! spp.!regulate' gene' translation' include' changes' in' mRNA' stability' (Brittingham' et' al.,' 2001;' Kelly' et' al.,' 2001;' Mishra' et' al.,' 2003;' Muller' et' al.,' 2010a;' Muller'et'al.,'2010b)'and'rate'of'translation'(Boucher'et'al.,'2002;'Zeiner'et'al.,'2003).' These'gene'regulation'mechanisms'are'remarkably'efficient,'enabling'the'smooth'passage' between'the'different'life'forms'of'the'protozoan.' CHAPTER''.....................................................'I' 14" " 1.5!–!Leishmania!spp.!in!the!lab:!axenic!culture! ! In'the'wild,'a'steady'population'of'Leishmania!spp.'is'maintained'by'the'sylvatic' life' cycle.' To' enable' the' study' of' this' protozoan' in' a' laboratory' environment' a' stable' reproducible'source'of'microorganisms'is'required.'Although'the'use'of'sandflies'for'the' growth'of' the'Leishmania! spp.'is'possible,' issues' related'to'biosecurity'and' the'lack'of' adequate' facilities' negates' the' generalized' utilization' of' the' sand' fly' model' (Volf' and' Volfova,'2011).'As'a'consequence'the'bulk'of'the'available'information'about'Leishmania! spp.'is'obtained'from'the'study'of'axenic'promastigotes.'The'promastigote'form'can'be' easily'cultivated'at'temperatures'below'28ºC'in'different'types'of'media'(Hendricks'et'al.,' 1978;'Sadigursky'and'Brodskyn,'1986).'Years'of'development'of'axenic'culture'permitted'' the'definition'of'generalized'nutritional'requirements'which'include:'heme'(Chang'and' Chang,' 1985);' 6_hydroxymethylpterine' and' related' pteridines' (Bello' et' al.,' 1994);' high' levels' of' folic' acid' (Kar,' 1997);' the' vitamins' thiamine,' nicotinic' acid,' pantothenate,' riboflavin,'and'biotin'(Schuster'and'Sullivan,'2002)'and'also'several'essential'amino'acids' required'by'other'eukaryotes'(Schuster'and'Sullivan,'2002).'The'first'medium'used'for'in! vitro! maintenance' of' Leishmania! spp.!was' Novy_MacNeal_Nicolle' (NNN)' medium' (Nicolle,' 1908),' still' remaining' a' medium' of' choice' for' the' isolation' of' field' strains' (Schuster'and'Sullivan,'2002).'Since'the'development'of'the'NNN'medium'several'other' media'formulations'were'created'for'the'growth'of'Leishmania!spp.'in'a'continuous'effort' to'develop'more'affordable'and'defined'media'(Grekov'et'al.,'2011;'Rodrigues'Ide'et'al.,' 2010;'Schuster'and'Sullivan,'2002;'Sharief'et'al.,'2008).'The'media'formulations'can'be' either'diphasic'or'monophasic.'The'diphasic'media'contains'a'solid/semi_solid'phase'and' a' liquid' phase.'The' solid' phases' are' enriched'with' a' variety' of' supplements,' including' peptone,' beef' infusion,' glucose,' tryptose,' liver' extract,' brain' heart' infusion,' individual' amino'acids,'nutrient'or'trypticase'soy'agars'and'blood'in'concentrations'from'2.5'to'50%' with' the' rabbit' preferred' to'other' sources' of' blood' (Schuster' and' Sullivan,' 2002).' The' liquid'phases'can'be'as'simple'as'water'or'as'complex'as'RPMI'(Schuster'and'Sullivan,' 2002).' These' biphasic' media' are' usually' not' very' amenable' for' routine' lab' utilization' usually'being'cumbersome'for'use.'In'consequence,'their'application'resides'mostly'in'the' isolation' of' field' strains.' Therefore' monophasic' (liquid)' media' are' preferred' for' the' growth'and'maintenance'of'Leishmania!spp.'in'the'laboratory'environment.'These'liquid' media' can' be' undefined' (with' many' of' the' components' found' in' the' solid' phase' of' diphasic'media),'semidefined''(using'commercially'available'insect'and'mammalian'tissue' culture'media,'supplemented'normally'with'fetal'calf'serum)'or'defined'(all'components' are' known).' Undefined' media' are' usually' less' expensive' and' enable' a' broader' use' (Schuster'and'Sullivan,'2002).'Semidefined'media'are'usually'the'media'of'choice'for'lab' CHAPTER''......................................................'I' 15" " use'enabling'a'combination'of'high'yield'and'reproducibility'of'growth.'In'these'media' fetal' calf' serum' (FCS)' is' the' most' used' supplement' in' concentrations' between' 5_20%.' These' defined' media' are' not' amenable' to' use' in' studies' that' involve' the' detection' of' released' proteins' from' the' parasites' because' of' the' dominant' contribution' of' serum' related' proteins' (Silverman' et' al.,' 2008).' In' 1999' a' report,' using' completely' defined' medium,' enabled' a' better' understanding' of' the' nutritional' requirements' of' these' protozoan' (Merlen' et' al.,' 1999).' In' addition,' Leishmania! spp.' grown' in' this' medium' maintained'infectivity,'proteomic'and'antigenic'profiles'of'the'control'semidefined'media' in' a' clear' indication' that' serum' complementation' can' be' replaced' without' apparent' metabolic'cost'to'the'parasites'(Merlen'et'al.,'1999).' The'metacyclic'parasite'is'considered'the'endpoint'of'promastigote'development' inside' the' sandfly' (Sacks' and' Perkins,' 1985).' Metacyclogenesis' can' be' conveniently' reproduced'during'in!vitro!culture:'the'procyclic'forms'correspond'to'promastigotes'in'the' exponential'phase'of'growth,'eventually'these'parasites'enter'into'stationary'phase'and' ultimately'a'fraction'of'these'stationary'parasites'differentiates'into'the'metacyclic'form' (Sacks' and' Perkins,' 1984).' The' in! vitro! characterization' of' the' metacyclic' parasites' is' traditionally' based' on' morphological' information' and' biological' criteria.' Metacyclic' parasites'are'of'a'smaller'size'than'stationary'parasites,'with'a'flagellum'at'least'twice'the' body'length'(Sacks'and'Perkins,'1984).'Moreover'they'are'more'resistant'to'complement' (Zakai'et'al.,'1998)'and'express'stage'specific'genes'like'the'small'hydrophilic'endoplasmic' reticulum_associated'protein'(SHERP)'or'hydrophilic'acylated'surface'protein'B'(HASPB)' (Knuepfer'et'al.,'2001;'Sadlova'et'al.,'2010).'These'characteristics'resemble'those'found'in' sand'fly'promastigotes'(Sacks'and'Perkins,'1984,'1985).'Still'at'the'molecular'level,'a'study' in'L.!major'revealed'some'differences'between'metacyclic'in!vitro'and'in!vivo'with'the' protein'HASPB'showing'a'different'pattern'of'expression'in'the'vector'metacyclic'stage' (Sadlova' et' al.,' 2010).' Albeit,' the' in! vitro! metacyclic' parasites' are' the' reference' promastigote'form'for'most'experimental'procedures'(Zakai'et'al.,'1998).'However,'this' form'of'the'parasite'is'present'in'variable'percentage'in'cultures.'Stationary'phase'cultures' represent'a'heterogeneous'population'with'distinct'virulence'profiles'(da'Silva'and'Sacks,' 1987).' Therefore' several' techniques' were' developed' for' the' purification' of' Leishmania! spp.!metacyclic'forms.'The'oldest'methods'are'based'on'LPG.'LPG'of'procyclic'parasites'is' smaller' than' their' stationary' counterpart,' furthermore' there' are' also' species' specific' differences' related' to' the' sugar' moiety' (Sacks,' 2001).' In' consequence,' the' use' of' LPG' specific' monoclonal' antibodies' has' enabled' the' recovery' of' metacyclics' from' several' species'(Courret'et'al.,'1999;'Lira'et'al.,'1998;'Sacks'et'al.,'1995).'Also,'an'affinity'based' method'that'relies'on'the'lectin'binding'to'the'LPG'of'non'metacyclic'parasites'was'used' CHAPTER''.....................................................'I' 16" " succesfully'for'L.!major'(Pinto_da_Silva'et'al.,'2002;'Soares'et'al.,'2002).'Still,'these'LPG' based'techniques'are'not'applicable'to'several'species'because'they'are'highly'dependent' on' the' knowledge'of' species'specific' changes' in' LPG'composition' (Spath' and' Beverley,' 2001).'In'2001'a'more'general'technique,'based'on'the'buoyant'density'of'the'parasites,' enabled'the'purification'of'metacyclics'using'a'Ficoll'based'gradient'centrifugation'(Spath' and' Beverley,' 2001).' Although' stationary_phase' promastigotes' with' undefined' in! vitro' passages' are' commonly' used' without' limitations,' it' has' been' demonstrated' that' continuous'culture'over'time'induces'loss'of'virulence.'In'fact,'long_term'in!vitro'culture' of'promastigotes'was'one'of'the'first'empirical'approaches'to'development'of'attenuated' strains'enabling'the'study'of'virulence'associated'genes'(Mitchell'et'al.,'1984).'In'fact'it' was' described' that' continuous' culture' of' L.! donovani' induced' virulence' loss' was' associated' with' reduced' expression' of' LPG' and' Kinetoplast' membrane' protein' 11' (Mukhopadhyay'et'al.,'1998).'Also'disadvantageous'adaptations'to'the'media'may'result'in' loss' of' virulence' (Segovia' et' al.,' 1992).' Either' way,' alterations' in' the' physiology' of' the' parasite' induced' by' long_term' growth' in' media' may' lead' to' misinterpretation' and' contradictory'results.'' The' study' of' the' amastigote' form' is' essential' because' this' is' the' stage' whose' proliferation'is'de!facto'responsible'for'the'pathology.'Amastigotes'can'only'be'obtained' from' experimentally' infected' animals' or' host' macrophages' infected' in! vitro.'This' limitation' prevents' the' obtainment' of' large' numbers' of' amastigotes' free' of' host' cell' contaminants,'a'fact'that'has'hampered'the'investigations'of'their'metabolic,'biochemical' and'biological'properties'(Gupta'et'al.,'2001).'An'alternative'model'is'to'keep'amastigote_ like' parasites' in' axenic' cultures.' Several' Leishmania! species' can' be' maintained' as' amastigotes'in'axenic'laboratory'conditions'(Bates'et'al.,'1992;'Doyle'et'al.,'1991;'Eperon' and' McMahon_Pratt,' 1989;' Hodgkinson' and' Soong,' 1997;' Nasereddin' et' al.,' 2010;' Puentes'et'al.,'2000;'Rainey'et'al.,'1991;'Saar'et'al.,'1998;'Sereno'and'Lemesre,'1997).'In' laboratory'conditions,'axenic'amastigotes'can'be'obtained'by'increasing'the'temperature' as' a' trigger,' either' alone' or' in' combination' with' a' reduction' of' pH' (Hodgkinson' and' Soong,' 1997).' However,' careful' optimization' of' culture' conditions' is' strictly' necessary,' since'the'conditions'used'in'one'species/strain'might'not'be'appropriate'for'others'species' (Gupta'et'al.,'2001).'For'L.!infantum!the'differentiation'occurs'within'3_4'days'after'the' transfer'of'stationary_phase'promastigotes'to'a'cell'free'culture'medium'at'an'acidic'pH' and' at' 37ºC' (Sereno' and' Lemesre,' 1997).' The' axenic' amastigotes' have' morphological,' biological,) biochemical) and) immunological) characteristics) that) are) similar) to) “true”) intramacrophagic'amastigotes'(Bates,'1993;'Saar'et'al.,'1998).'Axenic'amastigotes'express' amastigote' specific' genes' like' amastine' and' cysteine' protease' B,' have' specialized' CHAPTER''......................................................'I' 17" " structures' called' megasomes,' thrive' at' temperatures' and' pH' compatible' with' the' macrophage'phagolysosome'and'are'capable'of'productive'infections'(Bates,'1993;'Saar'et' al.,'1998).'In'spite'of'these'similarities,'gene'expression'analysis'in'L.!infantum'axenic' amastigotes' revealed' that' gene' expression' was' distinct' from' intramacrophagic' amastigotes' (Rochette' et' al.,' 2009).' The' differences' in' gene' expression' are' related' to' specific' metabolic' process,' like' fatty' acid' metabolism,' intracellular' transport' and' membrane'vesicular'fusion,'proteolysis,'and'the'response'to'oxidative'stress'(Rochette'et' al.,'2009).'In'general'and'despite'controversy'about'the'biological'properties'(Holzer'et' al.,'2006;'Rochette'et'al.,'2009),'the'axenically'cultured'amastigotes'are'a'useful'tool'in' several' studies' being' commonly' used' in' drug' screening' (Gupta,' 2011).' These' axenic' amastigotes'are'more'likely'to'be'abnormal'promastigotes'than'bona!fide'amastigotes'but' the'lack'of'a'better'model'still'justify'their'use.' " 2!–!Leishmaniasis! ' Leishmaniasis' is' a' typical' example' of' an' anthropozoonosis.' The' majority' of' the' infections' are' originally' zoonotic,' with' dogs,' cats,' lizards,' gerbils,' squirrels' and' other' rodents' as' their' primary' vertebrate' hosts' (Table' 1).' The' exceptions' are' visceral' leishmaniasis'(VL)'caused'by'L.!donovani!and'cutaneous'leishmaniasis'(CL)'caused'by'L.! tropica!in'India'and'Iran,'where'humans'are'the'main'hosts'of'infection.' ! Table!1.!Major'epidemiological'characteristics'of'pathogenic'Leishmania'spp.!Adapted' from'(Dedet,'2008)!! ' ' CHAPTER''.....................................................'I' 18" " Leishmaniasis' is' one' of' the' most' significant' neglected' tropical' diseases,' ' the' estimated' disease' burden' places'it' second' in' mortality' and' fourth' in' morbidity' among' tropical'infections'(Bern'et'al.,'2008).'It'is'prevalent'in'tropical'and'subtropical'regions'of' the'world'and'endemic'in'88'countries'(See'figure'4'for'global'distribution'of'the'disease)' with'a'total'population'at'risk'of'350'million'resulting'in'two'million'new'cases'reported' annually'(Kedzierski,'2010).'Like'many'other'tropical'diseases'leishmaniasis'is'associated' with' poverty' making' the' research' for' better' treatments' or' vaccines' less' attractive' to' industry.' Unlike' other' tropical' diseases' there' has' been' an' increase' on' incidence' of' leishmaniasis' in' the' last' 20' years.' Factors'related'to' population' mobility,'expansion'of' housing' in' highly' endemic' zones' (Gramiccia' and' Gradoni,' 2005)' and' also' increased' resistance'to'first'line'anti_Leishmania'drugs'contribute'to'this'increase'(Ouellette'and' Papadopoulou,'1993).'Also'in'non'endemic'areas'a'steady'rise'on'the'number'of'cases'has' also' been' reported' as' a' consequence' of' global' mobility.' In' fact' the' number' of' British' travelers'with'leishmaniasis'more'than'quadrupled'between'1995'and'2003'(Lawn'et'al.,' 2004)' and' in' just' two' years' between' 2003_2005' more' than' 600' US' soldiers' were' diagnosed'with'leishmaniasis'(Zapor'and'Moran,'2005).'The'appearance'of'opportunistic' co_infections'with'human'immunodeficiency'virus'(HIV)'as'result'of'the'overlap'between' endemic'areas'of'leishmaniasis'and'HIV'foci' in'suburban'areas'also'contributed'to'the' increase'of'the'registered'cases'(Alvar'et'al.,'1997;'Desjeux,'2004;'Tremblay'et'al.,'1996).' The' impact' of' the' co_infection' is' probably' underestimated' due' to' constraints' in' surveillance' and' in' the' actual' detection' of' the' co_infection' (Desjeux,' 2004).' In' south_ western'Europe'more'than'70%'of'all'adult'cases'of'visceral'leishmaniasis'are'associated' with'HIV/AIDS'and'up'to'9%'of'HIV_infected'individuals'are'infected'with'Leishmania' spp.!(Cruz'et'al.,'2006).' ' CHAPTER''......................................................'I' 19" " .' ' Figure!4.'Global'geographic'distribution'of'leishmaniasis,'adapted'from'(Davies'et'al.,' 2003)' ! 2.1!P!Disease!presentation! ' ' Leishmaniasis' can' have' distinct' presentations' (Depicted' in' figure' 5)' generally' depending'of'the'infecting'species'(around'20'are'thought'to'be'pathogenic'–'see'table'1)' and'the'immunological'status'of'the'host.'The'disease'can'present'itself'in'the'form'of' chronic'and'often'scarring'ulcerations'representing'the'non_visceralizing'presentation'of' the' disease:' cutaneaous' leishmaniasis' (CL),' mucocutaneous' leishmaniasis' (MCL)' or' diffuse'cutaneous'leishmaniasis'(DCL).'These'forms'of'the'disease'are'usually'non_fatal,' self'healing'although'socially'stigmatizating'due'to'the'development'of'lesions'in'the'skin' or' mucosal' surfaces.' The' visceralizing' form' of' the' disease' (VL)' has' a' more' severe' presentation,' involving' dissemination' of' the' parasite' to' internal' organs' such' as' bone' marrow,'liver'or'spleen'originating'a'lethal'progressive'disease'if'untreated.'This'form'of' the'disease'is'fatal'if'not'treated'and'is'responsible'for'70.000'yearly'deaths'(Kedzierski,' 2010).'' CHAPTER''.....................................................'I' 20" " ' Figure! 5.! Disease' presentation' and' major' clinical' signs' of' Leishmaniasis.' Pictures' adapted'from'http://emedicine.medscape.com/article/1108860_clinical#a0217! ' 2.1.1!P!Cutaneous!leishmaniasis! ! The' cutaneous' form' of' the' disease' accounts' for' more' than' half' of' the' total' leishmaniasis'cases'in'the'world.'CL'may'result'from'infection'by'any'of'the'Leishmania' spp.'that'infects'humans.'However,'it'is'traditionally'associated'with'several'species'of'the' Leishmania!subgenera'of'both'the'Old'World'(L.!tropica,!L.!major!and'L.!aethiopica)'and' New' World' (L.! mexicana! complex,' especially' L.! mexicana! and' L.! amazonensis).' Also,' several'species'of'the'Viannia!subgenera'(most'notably'L.!braziliensis,'L.!panamensis,!L.! guyanensis!and'L.!!peruviana)'are'also'causative'agents'of'CL'(Murray'et'al.,'2005).'With' an'incubation'period'that'can'last'from'a'few'days'to'months,'the'disease'is'characterized' by'single'or'multiple'localized'lesions'on'exposed'areas'of'the'body,'face,'neck,'arms'and' legs' that' typically' ulcerate' or' persist' as' nodules' or' plaques' (Dowlati,' 1996).' The' initial' lesion' is' often' a' small' and' red' papule' in' the' site' of' inoculation,' showing' localized' cell' infiltration.'Usually,'this'disease'manifestation'will'be'resolved'spontaneously,'with'the' recovery'time'depending'both'on'the'species'and'on'the'immune'status'of'the'individuals.' However,'secondary'infections'are'common,'leading'to'permanent'disfiguration'(Murray' et'al.,'2005).'A'more'severe'form'of'CL'is'DCL'caused'by'L.!aethiopica,'L.!amazonensis! and'L.!mexicana.'This'is'a'particular'form'of'CL,'which'occurs'only'in'hosts'with'deficient' CHAPTER''......................................................'I' 21" " immune'responses.'The'infection'is'characterized'by'a'primary'lesion,'which'spreads'to' involve'multiple'areas'of'the'skin'with'numerous'parasites'present'in'each'lesion'(Roberts' and' Janovy,' 2000).' Another' CL' variation' is' leishmaniasis' recidivans,' a' rare' chronic' infection' caused' essentially' by' L.! tropica.' This' unusual' form' of' leishmaniasis!is' characterized'by'new'lesions'appearing'around'old'healed'ones'(Murray'et'al.,'2005).' ! 2.1.2!P!Mucocutaneous!leishmaniasis! ' Mucocutaneous' leishmaniasis' or' “espundia”' is' a' form' of' CL' in' which' mucosal' dissemination'of'the'infection'occurs.'This'disease'is'usually'caused'by'L.!braziliensis,!L.! panamensis'and'L.!guyanensis'(rarely'the'latter'two'species),'which'are'found'in'Central' and'South'America.''After'a'cutaneous'self_healing'episode'1_10%'of'the'patients'develop' in' the' space' of' 5' years' mucocutaneous' complications' (Machado_Coelho' et' al.,' 2005).' Mucocutaneous'complications'may'also'originate'upon'inadequate'treatment'of'infections' by'some'Leishmania!spp.'such'as'L.!mexicana!(Roberts'and'Janovy,'2000).'In'MCL'the' infection'spreads'to'the'mucosal'system'of'the'nasal'and'oral'cavities,'where'metastatic' lesions'develop'leading'to'partial'or'total'destruction'of'those'membranes.'Unchecked'the' infection' might' originate' severe' disfiguration' or' even' death,' usually' due' to' secondary' bacterial'infections'or'malnutrition'(Osorio'et'al.,'1998;'Santrich'et'al.,'1990).'' ! 2.1.3!P!Visceral!leishmaniasis! ' Species'of'the'L.!donovani!complex'(L.!donovani,'L.!infantum!and'L.!chagasi)'are' the'principal'causative'agents'of'VL,'also'known'as'kala_azar'or'Dum_Dum'fever.'Also,'L.! tropica,' in' the' Old' World' and' L.! amazonensis! in' the' New' World' can' sometimes' be' viscerotropic.'Usually,'VL'has'an'incubation'period'that'varies'from'2'to'4'months'leading' to'several'clinical'presentations:'asymptomatic,'acute'and'chronic'(Murray'et'al.,'2005).' In'highly'endemic'areas,'more'than'60%'of'the'local'population'can'have'circulating'anti_ Leishmania'antibodies,'or'present'a'positive'skin'test'for'the'disease.'However,'only'about' 15%'of'the'population'report'having'the'active'disease'(Bern'et'al.,'2007).'The'remainder' will' retain' a' state' of' subclinical' (asymptomatic)' leishmaniasis' contributing' to' the' perpetuation' of' the' life' cycle' in' anthroponotic' areas.' The' usual' course' of' the' disease' involves'targeting'the'visceral'organs'resulting'in'a'myriad'of'symptoms'that'include'fever,' cachexia,' hepatosplenomegaly' and' polyclonal' hypergammaglobulinemia' (Davidson,' CHAPTER''.....................................................'I' 28" " reaction'(PCR)'based'assays'currently'constitute'the'main'molecular'diagnostic'approach' in' developed' countries.' ' The' PCR' allows' a' highly' sensitive' and' specific' (up' to' 100%)' detection'of'the'Leishmania!spp.'In'fact'a'positive'PCR'is'often'required'for'differential' diagnosis' in'order' to' initiate' therapy' (Reithinger' and' Dujardin,' 2007).'The' PCR' based' parasite'detection'have'consistently'been'shown'to'be'better'than'microscopy'or'parasite' culture,'particularly'in'samples'with'low'parasite'loads'such'as'blood'(Cruz'et'al.,'2002).' The' contribution' of' PCR' also' appears' to' be' particularly' relevant' for' the' diagnosis' of' leishmaniasis'in'patients'co_infected'with'HIV'with'detection'being'done'from'minimally' invasive'blood'samples'of'infected'patients'(De'Doncker'et'al.,'2005).'Also'confirmation'of' successful' therapy' often' involves' PCR' confirmation,' especially' in' VL' (Maurya' et' al.,' 2005).'In'CL'this'confirmation'is'not'required'because'80%'of'patient'scars'remain'PCR' positive'even'several'years'after'clinical'cure'(Schubach'et'al.,'1998).' The'predictive'power'of'the'PCR'based'assays'is'very'high.'They' are'capable'of' detecting'DNA'or'RNA'from'the'parasite'just'a'few'weeks'after'the'appearance'of'the'first' clinical' symptoms' allowing' even' Leishmania! spp.' identification' when' combined' with' restriction' fragment' length' polymorphism' (RFLP)' sequencing.' Different' Leishmania_ specific'DNA'and'RNA'sequences'can'be'targeted.'Commonly'used'regions'include'the' gp63'gene'locus,'telomeric'sequences,'both'conserved'or'variable'regions'in'mitochondrial' minicircles,'mini_exon'derived'DNA'and''rRNA'genes,'among'others'(Osman'et'al.,'1997).' Several'distinct'PCR'based'techniques'are'used.'These'techniques'include:'PCR_restriction' fragment' length' polymorphism' analysis' (PCR_RFLP),' real_time' PCR' (RT_PCR),' PCR_ Single'strand'conformational'polymorphism'(PCR_SSCP)'(for'detection'of'ribosomal'RNA' genes)' and' PCR_ELISA'(Reithinger' and' Dujardin,' 2007).' These' different' techniques' in' combination'or'alone'enable:'the'detection'of'the'parasite;'quantification'of'parasite'in' tissue'(Mary'et'al.,'2004);'detection'of'parasite'viability'using'RNA'instead'of'DNA'(van' der'Meide'et'al.,'2005);'species'identification'(Singh'et'al.,'2005)'and'also'the'detection'of' parasite' specific' features,' such' as' resistant' strains,' depending' on' finding' adequate' phenotypical'genetic'markers'(Reithinger'and'Dujardin,'2007).' However,'the'use'of'PCR'as'a'routine'diagnostic'method'is'a'technique'restricted'to' laboratory' use.' ' In' fact,' for' the' direct' protozoan' detection' in' clinical' laboratories' of' countries'where'the'parasite'is'not'endemic'the'trend'is'to'prefer'molecular'diagnosis.'The' cause' for' this' is' the' lack' of' microscopists' with' extensive' experience' in' detecting' amastigotes'in'microscopy'slides'and'also'the'existence'of'well'equipped'laboratories.'In' contrast,'in'countries'where'the'parasite'is'endemic,'microscopists'skills'are'maintained' due' to' routine' laboratory' practice,' microscopy' tends' to' be' preferred' as' the' first_line' parasite'detection'method'(Reithinger'and'Dujardin,'2007).' CHAPTER''......................................................'I' 29" " 2.2.2!–!Treatment!of!the!disease! ! ' Leishmaniasis'is'consistently'characterized'as'one'of'the'most'neglected'diseases'in' terms'of'coordinated'drug'development,'requiring'new,'safe,'affordable'and'easy_to_use' treatment'options'(Modabber'et'al.,'2007;'Vanlerberghe'et'al.,'2007).'The'first'efficient' drug'treatments'for'leishmaniasis'were'introduced'over'60'years'ago'(Croft'et'al.,'2006).' Nowadays'although'several'treatment'options'are'available'they'present'some'limitations' concerning'toxicity,'cost'and'requirement'of'parenteral'administration'(See'table'2).' ' Table! 2.! Standard' treatment' regimens' for' visceral' leishmaniasis.! Adapted' from' (van' Griensven'and'Boelaert,'2011)' ' ' ' Generic' pentavalent' antimonials' were' introduced' in' the' 1940s' and' still' are' the' recommended'first'line'drugs'for'the'treatment'of'all'forms'of'leishmaniasis'(Frezard'et' al.,'2009;'Vanlerberghe'et'al.,'2007).'Pentavalent'antimonials'include'the'commercially' available' stibogluconate' (Pentostam®)' and' meglumine' antimoniate' (Glucantime®)' preparations.'Pentavalent'antimonials'exact'mechanism'of'action'in'leishmaniasis'is'still' CHAPTER''.....................................................'I' 30" " unknown.'It'is'generally'accepted'that'the'Sbv'acts'as'a'pro_drug'being'converted'to'SbIII,' the' accepted' active' form' of' the' drug' (Frezard' et' al.,' 2009).' The' reduction' of' SbV' can' potentially' happen' in! vivo! in' the' phagolysosome' or' inside' the' parasites.' Glutathione,' cysteine'or'cysteinyl_glycine''thiols'can'actively'reduce'SbV'(Ferreira'Cdos'et'al.,'2003).' Glutathione'is'found'mostly'in'the'cytosol'of'macrophages'while'the'two'latter'thiols'are' mostly' found' inside' the' lysosomes.' The' most' abundant' thiol' in' the' parasite' is' the' glutathione_spermine' conjugate,' trypanothione.' This' thiol' was' shown' to' be' capable' of' reducing' SbV' (Ferreira' Cdos' et' al.,' 2003).' Also' the' parasite' proteins' thiol_dependent' reductase' 1' and' antimoniate' reductase' have' been' involved' in' the' reduction' process' inducing'susceptibility'to'SbV'(Denton'et'al.,'2004;'Zhou'et'al.,'2004).'The'entry'of'SbIII' into'the'parasite'was'shown'to'be'mediated'by'an'aquaglyceroporin'named'AQP1'(Gourbal' et'al.,'2004).'Upon'entry'into'the'parasite,'the'SbIII'will'eventually'lead'to'the'killing'of'the' parasite.'Although'DNA'fragmentation'suggest'a'role'for'apoptosis,'the'exact'mechanisms' of'action'of'the'antimonials'is'still'unclear'(Sereno'et'al.,'2001;'Sudhandiran'and'Shaha,' 2003).'Some'evidence'was'found'to'support'that'possible'molecular'target'for'SbIII'are' trypanothione' reductase' or' zinc_finger' proteins' (Cunningham' and' Fairlamb,' 1995;' Demicheli'et'al.,'2008).'Also'SbV'was'shown'to'have'potential'antiparasitic'properties'in' the' context' of' the' macrophage.' SbV' is' capable' of' forming' stable' complexes' with' ribonucleosides.'These'complexes'might'function'as'inhibitors'to'Leishmania!spp.!purine' transporters'or''interference'directly'with'the'purine'salvage'pathway'(dos'Santos'Ferreira' et'al.,'2006).'Antimonial'resistance'is'a'severe'problem'in'some'endemic'areas'(Lira'et'al.,' 1999).' Antimonial' unresponsive' clinical' isolates' suggest' a' multifactorial' mechanism' of' resistance'(Decuypere'et'al.,'2005;'Singh'et'al.,'2003).'In'fact,'several'specific'antimonial' resistance'mechanisms'are'already'described.'These'include'loss'of'AQP1'activity'(Gourbal' et' al.,' 2004),' extrusion' of' trypanothione' SBIII' complexes' by' ATP_binding' cassette' transporters'or'the'sequestration'of'SBIII'in'vacuoles'(El'Fadili'et'al.,'2005;'Legare'et'al.,' 2001).' Antimonial' use' in' clinical' practice' also'presents' other' important' disadvantages:' long'duration''of'treatment,'parenteral'administration,'frequent'toxic'effects'(Rijal'et'al.,' 2003)' and' also' unpredictable' outcome' of' therapy' in' HIV/VL' co_infected' patients' (Delgado'et'al.,'1999).'Nevertheless,'in'endemic'areas'where'no'resistance'phenomena'to' antimonials' are' reported,' it' still' remains' a' highly' effective' drug' (Vanlerberghe' et' al.,' 2007).'' ' Pentamidine'is'an'aromatic'diamine'used'as'a'second'line'drug'in'leishmaniasis.'It' was'initially'used'for'antimonial'unresponsive'patients'in'India'(Berman,'1997).'Although,' its'precise'mode'of'action'is'not'known,'it'is'reported'that'the'drug'enters'inside'the'L.! donovani! promastigote' through' arginine' and' polyamine' transporters' (Kandpal' and' CHAPTER''......................................................'I' 31" " Tekwani,'1997).'Although'the'pentamidine'mechanism'of'action'remains'unclear,'it'was' reported'that'upon'uptake'it'accumulates'in'the'mitochondrion'and'might'interfere'with' the' mitochondrial' respiratory' chain' (Basselin' et' al.,' 1996).' Although' no' resistant' field' isolates'were'reported,'resistance'related'to'ATP_binding'cassette'(ABC)'transporters'have' already' been' described' (Coelho' et' al.,' 2007).' The' use' of' pentamidine' to' treat' VL' is' hampered' by' the' higher' toxicity' of' pentamidine' when' compared' to' antimonials' and' reports' of' lab' acquired' resistance' phenomena' (Jha,' 1983).' However,' this' drug' is' still' considered'as'an'attractive'alternative'for'CL,'since'it'demonstrates'high'cure'rates'with' short' periods' of' treatment' (Berman,' 1997)' and' is' capable' of' performing' better' than' Glucantime'for'some'species'in'CL''(Soto'et'al.,'1994).' ' In'the'areas'of'antimonial'resistance,''amphotericin'B'is'highly'regarded'for'the' remarkable'efficacy'in'VL'(Thakur'et'al.,'1993).'Amphotericin'B'was'not'developed'as'a' leishmanicidal'drug:'in'fact'it'is'a'common'polyene'antifungal'drug'widely'used'to'treat' systemic'fungal'infections'(Bern'et'al.,'2006).'The'use'of'formulations'of'amphotericin'B' for' the' treatment' of' leishmaniasis' is' biochemically' sound' because' the' target' of' amphotericin'B'in'fungi'are'ergosterol_like'sterols'(Bern'et'al.,'2006)'and'ergosterol'is'the' major' membrane' sterol' of' Leishmania! species' ' (Berman' et' al.,'1986).' Despite' its' high' efficacy,'amphotericin'is'toxic'with'side'effects,'including'nephrotoxicity'(Laniado_Laborin' and' Cabrales_Vargas,' 2009).' Some' adverse' effects' of!amphotericin' B!have' been' circumvented'by'using'liposomal'formulations'like'AmBisome'to'reduce'the'toxicity'and' to' extend' the' plasma' half_life' of' amphotericin' B.' Though,' the' high' cost' of' AmBisome' limits'its'use'in'developing'endemic'countries'(Vanlerberghe'et'al.,'2007).'The'mechanism' of'AmBisome'is'unknown'but'it'is'thought'to'be'related'to'the'interaction'with'sterols.' AmBisome'can'also'actively'prevent'the'entry'of'L.!donovani!in'macrophages'by'binding' with'the'sterols'on'the'macrophage'membrane'(Paila'et'al.,'2010).'Moreover'it'can'also' induce'the'formation'of'aqueous'pores'in'promastigote'cell'membranes'inducing'cell'lysis' (Ramos'et' al.,'1996).'The'acquisition'of'resistance'to'amphotericin'B'is'possible'in' the' laboratory' although' it' was' reported' to' select' against' virulence' (Mbongo' et' al.,' 1998).' Laboratory'acquired'resistance'to'amphotericin'B'is'associated'with'ergosterol'deficiency' in'the'parasite'membrane'(Mbongo'et'al.,'1998).'In'L.!donovani'the'enzyme'S_adenosyl' methionine'transferase'(important' for' the'biosynthesis' of'ergosterol)' is'associated'with' increased'resistance'to'amphotericin'B'(Pourshafie'et'al.,'2004).'Despite'similar'cure'rates' to'antimonials,'amphotericin'B'is'not'the'first'line'treatment'in'highly'endemic'countries' because'it'is'considered'to'be'too'expensive'to'compete'in'cost_effectiveness'with'the'other' regimens.'In'the'treatment'of!VL'caused'by'L.!infantum!in'Southern'Europe,'liposomal' amphotericin'B'is'the'preferred'treatment'regimen'(Vanlerberghe'et'al.,'2007).' CHAPTER''.....................................................'I' 32" " ' Paromomycin,' an' aminoglycoside' antibiotic,' constitutes' another' parenteral' alternative' treatment' to' leishmaniasis' (Thakur' et' al.,' 2000).' ' In' phase' III' studies' performed' in' India' and' Sudan,' paromomycin' demonstrated' to' be' not' inferior' to' amphotericin'B'for'VL'treatment'(Musa'et'al.,'2010;'Sundar'et'al.,'2009).'Moreover,'it' may'even'be'advantageous'because'of'shorter'duration'in'its'administration'and'increased' safety' in' pediatric' patients' (Sundar' et' al.,' 2007).' Topical' application' of' paromomycin' derivatives'also'have'shown'encouraging'results'for'CL'treatment'(Armijos'et'al.,'2004;' Ben'Salah'et'al.,'2009).'The'leishmanicidal'mechanism'of'action'of'paromomycin'is'still' not' clear.' Several' evidences' point' to' an' action' related' to' the' translation' process.' Interference'with'protein'synthesis'and''mitochondrial'interference'were'demonstrated'to' occur' upon' exposure' to' the' aminoglycoside' (Jhingran' et' al.,' 2009).' In' fact,' direct' interference'with'ribosomes'seems'to'occur'preventing'ribosomal'function'and'continuous' recycling' of' ribosomal' machinery' (Hirokawa' et' al.,' 2007;' Jhingran' et' al.,' 2009).' Laboratory'resistance'to'paromomycin'can'be'induced'in'the'laboratory'being'associated' with'a'reduced'uptake'of'the'drug'(Jhingran'et'al.,'2009;'Maarouf'et'al.,'1998).'Despite'the' potential' for' development' of' resistance' associated' with' aminoglycosides,' paromomycin' cures'both,'VL'and'CL'(the'latter'more'effectively)'but'the'limited'availability'of'the'drug' still' restricts' its' use' in' endemic' regions.' Furthermore,' combination' therapy' of' paromomycin'with'antimony'generally'results'in'a'regimen'that'is'highly'efficacious'for' visceral'disease'(Berman,'1997)'reducing'the'side'effects'associated'with'both'drugs.' ' All' the' aforementioned' drugs' present' have' to' be' delivered' in' a' clinical' environment' because' parenteral' administration' is' required.' Therefore' one' of' the' main' goal'of'drug'development'in'leishmaniasis'is'to'find'efficient'oral'agents'(Herwaldt,'1999).' The' imidazole' derivatives' that' inhibit' ergosterol' biosynthesis' were' the' first' oral' agents' used' in' leishmaniasis.' Ketoconazole' was' in' fact' the' first' oral' compound' used' for' leishmaniasis'in'a'controlled'environment'(Urcuyo'and'Zaias,'1982).'Although'it'showed' some' promise' in' some' forms' of' CL' (Navin' et' al.,' 1992)' it' was' demonstrated' to' be' completely' ineffective' for' others' (Navin' et' al.,'1992;' Singh' et' al.,' 1995b).' The' use' in' a' monotherapy' regime' in' VL' is' not' recommended' due' to' the' suboptimal' healing' rates' described'in'several'works'(Rashid'et'al.,'1994;'Sundar'et'al.,'1990).'Also'itraconazole'and' fluconazole'were'tested'with'variable'success'for'CL'with'variable'cure'rates'varying'from' no' effect' to' 100%' (Alrajhi' et' al.,' 2002;' Dogra' and' Saxena,' 1996;' Laffitte' et' al.,' 2005;' Nassiri_Kashani'et'al.,'2005;'Sousa'et'al.,'2011).'The'interest'in'these'imidazole'derivatives' has'been'rekindled'with'reports'of'successful'combination'therapy'in'VL'(Barragan'et'al.,' 2010;' Shakya' et' al.,' 2011).' The' hypoxanthine' analog' allopurinol,' inhibits' the' purine' anabolism'in'Leishmania!spp.'(LaFon'et'al.,'1985).''As'with'the'imidazole'derivatives'it' CHAPTER''......................................................'I' 33" " was' shown' to' have' reduced' value' in' a' monotherapy' setting' (Velez' et' al.,' 1997).' Combination'therapy'with'allopurinol'was'tried'with'some'success'in'CL'(Esfandiarpour' and'Alavi,'2002;'Martinez'and'Marr,'1992;'Momeni'and'Aminjavaheri,'1995;'Momeni'et' al.,' 2002)' but' in' VL' results' are' not' as' promising' showing' reduced' efficacy' even' in' combination' with' other' drugs' (Ramesh' et' al.,' 2010).' Still,' a' report' on' unresponsive' patients'to'antimony'showed'an'improvement'in'pentamidine'performance'when'coupled' with'allopurinol'treatment'(Das'et'al.,'2001).'' ' The' first' major' breakthrough' in' the' use' of' an' oral' agent' was' miltefosine.' This' compound,'originally'developed'as'an'antineoplasic'drug,'presents'a'remarkable'reported' efficiency'(>82%)'in'several'phase'IV'reports'for'VL'(Bhattacharya'et'al.,'2007;'Rahman'et' al.,'2011).'It'also'showed'a'remarkable'efficacy'for'the'treatment'of'different'types'of'CL' and'MCL'(Berman,'2008;'Killingley'et'al.,'2009;'Soto'et'al.,'2009;'Tappe'et'al.,'2010).' Moreover,' it' can' be' safely' used' in' children' (Palumbo,' 2008;' Sundar' et' al.,' 2003)' and' treats'with'high'success'rates,'patients'who'were'refractory'to'antimonial'drugs'(Berman,' 2008).'However,'miltefosine'teratogenicity'constitutes'still'a'major'limitation'to'its'use'for' women'of'reproductive'age'(Berman,'2008).'Furthermore'miltefosine'as'an'oral'drug,'is' more'prone'to'non_supervised'treatment'regimes'that'may'lead'more'easily'to'the'non' compliance'of'treatment'leaving'the'patients'with'sub_therapeutical'doses.'This'latter'fact' in'conjunction'with'miltefosine'long'half_life,'subtherapeuthical'quantities'of'miltefosine' are'still'detected'in'the'patients'blood'5'months'after'the'therapeutical'regime'(Dorlo'et' al.,'2008),'constitutes'a'high'risk'of'development'of'resistance'(Berman,'2008).'In'fact' several' laboratory' isolates' were' obtained' (Moreira' et' al.,' 2011;' Perez_Victoria' et' al.,' 2003a),' but' to' this' date' no' field' isolate' for' miltefosine' resistance' was' recovered.' The' mechanism' of' action' of' miltefosine' and' the' corresponding' molecular' targets' are' still' unknown'(Berman,'2008).'Nonetheless'it'is'clear'that'the'activity'of'miltefosine'is'related' to'intracellular'accumulation'of'the'drug,'which'is'regulated'by'two'transporters,'LdMT' and' its' b_subunit' LdRos3,' a' P_type' ATPase,' belonging' to' the' aminophospholipid' translocase'family'(Perez_Victoria'et'al.,'2003b).'The'accumulation'of'the'drug'leads'to'the' development'of'an'apoptosis'like'death'in'L.!donovani'(Paris'et'al.,'2004).'Resistance'to' miltefosine' is' associated' to' changes' in' the' efflux' of' the' drug.' Mutations' in' the' above' mentioned'transporters'or'over'expression'of'the'glycoprotein'MDR1'leads'to'miltefosine' resistance' (Perez_Victoria' et' al.,' 2003b;' Perez_Victoria' et' al.,' 2001).' In' addition' the' diminution' of' unsaturated' phospholipid' alkyl' chains' in' the' parasite' membrane' was' reported' in' resistant' strains' (Rakotomanga' et' al.,' 2004).' Miltefosine' is' the' most' cost_ effective'option'of'treatment'in'areas'of'detected'antimonial'resistance,'but'its'use'as'a' CHAPTER''.....................................................'I' 34" " first_line'drug'in'monotherapy'is'limited'by'its'teratogenicity'and'the'high'potential'for' resistance'development'(Vanlerberghe'et'al.,'2007).'' ' ' Sitamaquine'(8_aminoquinoline),'is'the'only'drug'that'was'specifically'developed' for'treatment'of'VL'(Yeates,'2002).'The'phase'II'trials'of'sitamaquine'in'India'and'Kenya' demonstrated' its' efficacy' against' VL' (>83%),' but' the' latter' presented' a' case' of' renal' complication'associated'with'sitamaquine'(Jha'et'al.,'2005;'Wasunna'et'al.,'2005).'In'CL' the'drug'presented'a'disappointing'performance'with'a'failure'to'contain'the'disease'even' in' the' mouse' model' (Garnier' et' al.,' 2006).' Its' mechanism' of' action' seems' to' involve' electrostatic'interactions'leading'to'drug'insertion'within'biological'membranes'(Coimbra' et'al.,'2010).'After'binding'to'the'membrane,'sitamaquine'translocation'is'mediated'by'the' action'of'a'still'unknown'transporter.'It'accumulates'in'Leishmania'spp.!cytosolic'acidic' compartments,'acidocalcisomes,'however'correlation'between'its'action'and'this'reported' accumulation'is' not'clear'(Lopez_Martin'et'al.,'2008).'Nonetheless'it' is'clear'that' high' concentration'of'sitamaquine'affects'parasite'motility,'morphology'and'growth'(Duenas_ Romero'et'al.,'2007).'Although'field'resistance'against'this'drug'has'not'been'reported,'in! vitro! resistance' against'the' L.! donovani!promastigote'has' been'reported' (Bories' et' al.,' 2008).'Sitamaquine'still'requires'more'studies'to'evaluate'efficacy,'mode'of'action'and' toxicity'to'become'a'viable'alternative'to'the'already'established'VL'treatments'(Loiseau'et' al.,'2011).' ' A' therapeutical' approach' using' different' combinations' of' some' of' the' above' mentioned'drugs'in'the'treatment'of'leishmaniasis'is'expected'to'decrease'therapy'cost,' reduce'toxicity'and'avoid'resistance'(Meheus'et'al.,'2010;'Shakya'et'al.,'2011).'In'spite'of' several'case'reports'of'success'combination'therapy'in'treatment'for'leishmaniasis'(Collini' et'al.,'2009;'Kumar'et'al.,'2011),'the'lack'of'standardized'clinical'data'makes'monotherapy' still' the' reference' approach.' Only' recently,' significant' steps' have' been' taken' to' standardize'and'evaluate'the'effectiveness'of'combination'therapy.'In'2011'a'published' study'with'more'than'600'VL'patients'in'India'demonstrated'that'combination'treatments' are' efficacious' and' safe,' decreasing' the' duration' of' therapy' (Sundar' et' al.,' 2011).' Therefore,'upon'independent'rigorous'validation,'combination'therapy'might'become'an' important'tool'for'the'control'of'the'disease'(van'Griensven'et'al.,'2010).' ' The' successful' chemotherapy' is' very' much' dependent' on' a' competent' immune' system'(Murray'et'al.,'1989),'therefore'the'treatment'options'for'HIV'co_infection'will'be' dependent' on' the' immune' status' of' the' patient' (Cruz' et' al.,' 2006).' In' general,' these' patients'have'lower'cure'rates,'higher'drug'toxicity'rates,'and'suffer'higher'fatality'level' for'leishmaniasis'than'do'immunocompetent'patients'(Alvar'et'al.,'2008).'The'treatment' CHAPTER''......................................................'I' 35" " of' co_infected' patients' is' made' more' difficult' by' the' lack' of' uniform' clinical' data.' In' consequence'optimal'treatment'regimes'have'not'been'established.'In'fact'relapses'with' complicated'parasitological'and'clinical'data'are'common'making'secondary'prophylaxis' an'option'to'consider'(Pasquau'et'al.,'2005).'In'a'12'month'follow'up'study'with'the'use'of' AmBisome,' secondary' prophylaxis' decreased' the' probability' by' 50%' of' having' relapse' (Lopez_Velez' et' al.,' 2004).' Albeit,' in' settings' where' medical' care' is' insufficient' for' monitoring' toxicity' or' VL' is' anthroponotic,' there' is' the' increase' risk' of' inducing' drug' resistance'with'secondary'prophylaxis'(Croft'et'al.,'2006).'' ! 2.2.3!–!Control!of!vectors!and!reservoirs! ' ' For' the' direct' control' of' leishmaniasis' the' World' Health' Organization' (WHO)' prioritizes' the' use' of' efficient' diagnosis' tools,' the' application' of' adequate' treatment' options'and'the'mandatory'report'of'all'leishmaniasis'cases'as'minimal'control'measures.' In'the'absence'of'vaccines'the'eradication'of'the'disease'includes'broad'disease'control' directives.' These' involve' the' control' of' reservoirs' and' vectors' of' the' disease.' Three' different'approaches'exist'for'the'indirect'control'of'the'disease'transmission:'vector'and' reservoir'control,'and'also'personal'protective'measures.'Within'sylvatic'environments'the' sandflies'generally'rest'in'dark'moist'places'usually'within'a'few'hundred'meters'of'their' breeding' site,' promoting' the' existence' of' very' active' infection' foci.' The' increased' urbanization'on'the'periphery'of'towns,'invading'the'traditional'breeding'grounds'of'the' vector'represents'one'of'the'major'causes'for'increase'of'disease'incidence.'Measures'like' widespread'deforestation' near' urbanized' terrains' or' the' use' of' insecticides' are' feasible' alternatives' to' control' the' vector.' In' fact,' the' extensive' use' of' Dichloro_Diphenyl_ Trichloroethane'(DDT)'had'a'dramatic'effect'on'the'peridomestic'sand'fly'vector'of'VL'in' endemic'areas'of'India,'resulting'in'a'considerable'reduction'in'the'number'of'cases'in' some' localities' (Vanlerberghe' et' al.,' 2007).' Due' to' its' environmental' and' human' hazardous'effects,'DDT'was' substituted'by'synthetic'pyrethroids,'like'deltamethrin'and' cyhalotrin,'which'showed'to'be'effective'against'all'vector'species'(Davies'et'al.,' 2000;' Marcondes'and'Nascimento,'1993).'Another'major'measure'against'both'cutaneous'and' visceral' leishmaniasis' is' the' control' of' reservoir' hosts.' The' control' of' sylvatic' animals' requires'difficult'environmental'decisions,'such'as'forest'cleaning'or'destruction'of'rodent' burrows'around'human'activities.'For'zoonotic'VL,'the'restriction'of'the'canine'domestic' reservoir'is'essential.'The'control'of'transmission'of'canine'leishmaniasis'has'two'major' purposes:' 'protect' the' dogs' themselves' from' VL' and' reduce' the' availability' of' parasite' reservoir.' The' systematic' euthanasia' of' infected' dogs' showed' limited' impact' in' the' CHAPTER''.....................................................'I' 36" " transmission' of' the' parasite' (Gramiccia' and' Gradoni,' 2005).' Several' insecticide_based' preparations' have' been' specifically' registered' for' dog' protection' against' sand' fly' bites,' including' deltamethrin_impregnated' collars' and' topical' application' of' permethrin' or' deltamethrin'(Courtenay'et'al.,'2009;'Halbig'et'al.,'2000;'Maroli'et'al.,'2001;'Reithinger'et' al.,' 2004).' Also' several' vaccine' candidates' are' under' study' for' canine' leishmaniasis' prevention'and'one'has'already'been'registered'for'veterinary'use'in'Brazil'(Borja_Cabrera' et'al.,'2002).'However,'the'efficiency'of'these'preventive'measures'is'dependent'on'their' adoption'by'the'whole'canine'population'and'the'impact'will'be'reduced'if'a'still'unknown' sylvatic'reservoir'maintains'a'population'of'infected'animals'sufficient'to'perpetuate'the' infectious'cycle.'Ultimately,'personal'protective'measures'including'the'use'of'mechanical' barriers' such' as' bed_nets,' sometimes' impregnated' with' insecticides,' the' avoidance' of' outdoor' activities' when' sand' flies' are' most' active' (dusk' to' dawn),' wearing' protective' clothing'and'the'application'of'insect'repellent'to'exposed'skin'are'highly'recommended' WHO'directives'against'leishmaniasis.' ' 3!–!Immunopathogenesis!of!Leishmania!spp.!infection! ' Leishmania! spp.' infections' are' excellent' examples' of' complex' host_parasite' interactions.' The' parasite' developed' a' remarkable' range' of' sophisticated' adaptive' mechanisms,'which'not'only'allow'to'evade'and'inhibit'normal'macrophage'functions,'but' also' subvert' innate' and' acquired' (both' cell' and' humoral)' immunity' to' their' own' advantage.'' ' 3.1!P!Establishment!of!Leishmania!spp.!on!the!host! ' The'early'recognition'and'subsequent'triggering'of'a'proinflammatory'response'to' invading' pathogens' is' associated' with' the' innate' immune' system' (Medzhitov' and' Janeway,' 2000).' The' adaptive' branch' of' the' immune' system' is' responsible' for' the' subsequent'elimination'of'pathogens'and'the'generation'of'immunological'memory.'The' adaptive' immune' response' is' characterized' by' the' specificity' developed' in' clonal' gene' rearrangements'from'a'broad'repertoire'of'antigen_specific'receptors'on'lymphocytes.'The' innate'immune'response,'on'the'other'hand,'is'mediated'mostly'by'phagocytic'cells'and' antigen' presenting' cells' (APCs),' such' as' dendritic' cells' (DCs),' granulocytes' and' macrophages'being'considered'as'somewhat'nonspecific'(Mogensen,'2009).'Traditionally' CHAPTER''......................................................'I' 37" " the'innate'immune'system'was'considered'as'a'primitive,'nonspecific'system'involved'only' in'destroying'and'presenting'antigen'to'cells'of'the'adaptive'immune'system.'Nowadays'it' is' considered' as' a' highly' developed' system' capable' of' discriminating' between' self' and' foreign'with'a'much'greater'specificity'than'previously'thought.'In'fact'it'now'accepted' that' innate' and' adaptive' immune' responses' are' much' more' entwined' than' initially' believed.' Several' important' findings' support' the' idea' that' the' innate' immune' system,' besides'being'essential'for'early'pathogen'recognition,'is'also'involved'in'the'activation' and'shaping'of'adaptive'immunity'(Iwasaki'and'Medzhitov,'2004).'' ' ' Figure! 6.! Cells' involved' in' the' first' steps' of' infection' by'Leishmania! spp.' Metacyclic' promastigotes' are'deposited' in' the' dermis' in' a' mixture' of' immunomodulatory' salivary' secretions' and' parasite_derived' proteophosphoglycans.' Promastigotes' from' the' initial' inoculum' are' phagocytosed' by' tissue_resident' macrophages' and' dermal' dendritic' cells' (DCs).'Inflammatory'monocyte_derived'DCs'(moDCs)'are'recrutited'to'the'local'site'by' local'inflammatory'signaling.'These'infected'inflammatory'moDCs'may'facilitate'parasite' traffic' to' the' draining' lymph' node.' Long_term' replication' and' perpetuation' of' the' pathogen'principally'involves'either'macrophages'or'moDCs,'depending'on'the'parasite' species.'Adapted'from'(Kaye'and'Scott,'2011).' ' Upon'entry'into'a'susceptible'mammalian'host,!Leishmania'promastigotes'must' survive' the' impact' of' the' innate'immune' system.' The' first' immediate' challenge' before' entering'the'first'cells'is'to'survive'the'complement'system.'Metacyclic'parasites'are'highly' resistant' to' complement' mediated' lysis.' This' resistance' is' a'multifactorial' event,' being' associated' with' several' parasite' components,' like' LPG' (Puentes' et' al.,' 1990),' GP63' (Brittingham' et' al.,' 1995)' or' surface' kinases' (Hermoso' et' al.,' 1991).' The' parasites' that' survive'the'complement'system'quickly'interact'with'resident'cells'(Figure'6).'Among'the' first' cells' interacting' with' the' promastigotes' are' the' epidermal' keratinocytes,' that' may' CHAPTER''.....................................................'I' 44" " between'different'classes'of'pathogens.'Major'PAMPs'are'nucleic'acids,'including'DNA,' dsRNA,' ssRNA,' and' 5_triphosphate' RNA,' as' well' as' surface' glycoproteins' (GP),' lipoproteins' (LP),' and' membrane' components' (peptidoglycans' [PG],' lipoteichoic' acid' [LTA],'LPS,'and'GPI'anchors).'Adapted'from'(Mogensen,'2009).! ' ' This' general' division' is' not' strict,' as' some' TLRs' can' recognize' such' divergent' structures'as'LPS,'the'fusion'protein'of'respiratory'syncytial'virus,'and'cellular'heat'shock' proteins'(Akira'et'al.,'2006).'This'capacity'to'recognize'several'structures'using'a'limited' number' of' specific' receptors' can' be' explained' by' the' binding' of' ligands' to' different' regions'of'the'extracellular'portion'of'TLRs'or'the'involvement'of'different'PAMP_binding' molecules,'such'as'the'accessory'molecule'MD2'that'mediates'LPS'binding'to'TLR4'(Kim' et' al.,' 2007).' Ligand_receptor' specific' interactions' can' also' be' achieved' through' the' formation'of'heterodimers'between'TLR2'and'either'TLR1'or'TLR6'(Ozinsky'et'al.,'2000).' TLRs'can'also'be'grouped'according'to'their'cellular'distribution,'TLRs'(TLR1,'_2,'_4,'_5,'_ 6,'and'_10)'are'expressed'at'the'cell'surface,'while'others'(TLR3,'_7,'_8,'and'_9)'are'located' almost'exclusively'in'intracellular'compartments,'such'as'endosomes'and'lysosomes.'This' latter' group' is' specialized' in' recognition' of' nucleic' acids,' with' self' versus' nonself' discrimination'provided'by'the'distinct'localization'of'the'ligands'rather'than'differences' in' molecular' structure' (Iwasaki' and' Medzhitov,' 2004).' Although' TLRs' have' been' identified'in'most'cell'types,'the'most'frequently'studied'TLRs'are'present'in'APCs'such'as' macrophages,'DCs'and'B'lymphocytes'(Iwasaki'and'Medzhitov,'2004).'The'ultimate'result' of' the' TLR' activation' by' a' PAMP' is' the' triggering' of' downstream' signaling' pathways' resulting'in'the'generation'of'an'antimicrobial'proinflammatory'response.'The'binding'to' TLRs'by'individual'PAMPs,'leads'to'the'activation'of'several'different'signaling'pathways.' Signal'transduction'is'initially'mediated'by'adaptor'molecules'that'will'be'also'responsible' for'the'specificity'of'the'response'(O'Neill'and'Bowie,'2007).'The'recruitment'of'adaptor' molecules' to' a' given' TLR' is' followed' by' activation' of' downstream' signal' transduction' pathways' via' phosphorylation,' ubiquitination,' or' protein_protein' interactions,' inducing' the' activation' of' transcription' factors' that' regulate' the' expression' of' genes' involved' in' inflammation' and' antimicrobial' host' defenses' (Akira' et' al.,' 2006)' The' TLR_induced' signaling' pathways' can' be' classified' according' to' the' adaptor' molecules' involved' (See' figure' 9).' Broadly' they' are' divided' into' MyD88' (myeloid' differentiation' factor' 88)' dependent' or' independent' TLRs' (O'Neill' and' Bowie,' 2007).' The' subsequent' signaling' cascade'can'involve'NF_κB,)mitogen_activated'protein'kinases'or'IFN'regulatory'factors' (Akira'et'al.,'2006;'O'Neill'and'Bowie,'2007).'The'first'two'have'a'prominent'role'in'the' induction'of'a'proinflammatory'response'while'the'latter'is'involved'in'IFN'production' (Kawai'and'Akira,'2007;'Mogensen,'2009).'' CHAPTER''......................................................'I' 45" " ' Figure! 9.! TLRs'associated'immune'response.'TLR1/2'and'TLR2/6'utilize'MyD88'and' Mal' as' adaptors.' TLR3' is' dependent' on' TRIF' for' signaling.' In' the' case' of' TLR4,' four' different'adaptors,'i.e.,'MyD88,'Mal,'TRIF,'and'TRAM,'are'involved,'whereas'TLR5,'_7,'_8,' and'_9'utilize'only'MyD88.'The'fifth'adaptor,'SARM,'negatively'regulates'TRIF_dependent' signaling.' Overall,' MyD88_dependent' signaling' induces' proinflammatory' cytokine' production,'whereas'TRIF_dependent'signaling'stimulates'a'type'I'IFN'response.''Adapted' from'(Mogensen,'2009).' ! ' 3.2.1!–!Toll!like!receptors!and!Leishmania! ' ' The'bulk'of'the'knowledge'on'the'role'of'TLRs'during'infection'is'concentrated'on' virus'and'bacteria.'Traditionally'gram_negative'bacteria'are'recognized'by'TLR4'via'the' lipid'A'portion'of'LPS'(Poltorak'et'al.,'1998),'whereas'lipoteichoic'acid,'lipoproteins,'and' peptidoglycan'of'gram_positive'bacteria'are'detected'by'TLR2'(Schwandner'et'al.,'1999;' Yoshimura'et'al.,'1999).'TLRs'activation'by'bacteria'is'not'restricted'to'LPS.'Other'surface' or'intracellular'PAMPs''can'activate'distinct'TLRs'(Mogensen'et'al.,'2006).'A'recurrent' example'in'the'literature'is'flagelin'in'flagellated'bacteria'that'is'specifically'recognized'by' TLR5'inducing'TNF_α)production)in)response)to)flagellated)bacteria)(Hayashi'et'al.,'2001).' Nucleic' acids' in' intracellular' compartments' is' associated' with' viral' infection' and' recognized'by'TLR3'for'double_stranded'RNA,'TLR7'and'TLR8'for'single_stranded'RNA' (Alexopoulou' et' al.,' 2001;' Diebold' et' al.,' 2004;' Heil' et' al.,' 2004).' TLR9' recognizes' unmethylated'CpG'DNA'present'in'viruses'and'bacteria'(Hemmi'et'al.,'2000).'' ' The'importance'of'PRRs'during'infection'with'protozoan'pathogens'is'still'a'matter' of' debate' because,' unlike' virus' and' bacteria' infections,' few' studies' exist' on' protozoan' infections.' Major' PAMPs' identified' in' protozoa' depicted' in' figure' 9,' include' glycosylphosphatidylinositol'(GPI)'anchors'(Almeida'et'al.,'2000;'Almeida'and'Gazzinelli,' CHAPTER''.....................................................'I' 46" " 2001;'Campos'et'al.,'2001),'which'activate'TLR2'and'TLR4,'as'well'as'unmethylated'DNA' activating'TLR9'(Debierre_Grockiego'et'al.,'2003;'Gazzinelli'and'Denkers,'2006;'Shoda'et' al.,'2001).'The'intracellular'protozoan'Toxoplasma!gondii!causes'asymptomatic'infection' in'normal'hosts'but'can'be'fatal'in'immunocompromised'individuals,'particularly'in'the' absence'of'IL_12'production'(73).' In'response'to' T.!gondii!infection,'IL_12'is' produced' through' a' mechanism' dependent' on' MyD88' (Scanga' et' al.,' 2002;' Sukhumavasi' et' al.,' 2008).'This'production'was'shown'to'be'dependent'of'TLR2'and'TLR4'activation'by'GPIs' anchors' (Debierre_Grockiego' et' al.,' 2007).' Also' the' potent' IL_12' inducer' profilin_like' protein' from' T.! gondii! tachyzoites' is' recognized' by' murine' TLR11' (Yarovinsky' et' al.,' 2005),'a'nonfunctional'TLR'in'humans'(Akira'et'al.,'2006).'Also'the'hemozoin,'a'heme' degradation'product' from' Plasmodium! falciparum' infection' was' able' to' activate'TLR9' indirectly' by' becoming' coated' with' parasite' protozoan' DNA' and' targeting' into' the' endosome' (Parroche' et' al.,' 2007).' Also' heat_shock' protein' 70' (HSP' 70)' from' different' trypanosomatids'are'potent'activators'of'the'immune'system'by'myeloid'differentiation' primary' response' gene' 88' (MyD88)' pathway' (Qazi' et' al.,' 2007).' The' opportunistic' intestinal' parasite' Encephalitozoon! cuniculi' can' also' bind' in! vitro' to' TLR2' from' macrophages'inducing' the'production'of'inflammatory'cytokines'(Fischer'et'al.,'2008).' The'TLRs'are'also'known'to'be'essential'for'T.!cruzi'infection'control'as'MyD88'_∕_'and' TRIF'_'∕_'mice'show'increased'susceptibility'to'infection'(Koga'et'al.,'2006).'In'Leishmania' spp.'few'TLR'activators'were'described.'LPG,'but'not'other'surface'glycolipids,'is'a'TLR2' agonist' capable' of' activating' mouse' macrophages' and' human' NK' cells,' in' a' MyD88_ dependent'manner'(Becker'et'al.,'2003;'de'Veer'et'al.,'2003;'Kavoosi'et'al.,'2010).'Also' Leishmania!infantum'Sir2'(silent'information'regulator'2'protein)'was'shown'to'induce' the'maturation'of'DCs'in'a'TLR2_dependent'manner'with'the'secretion'of'IL_12'and'TNF_ α)(Silvestre'et'al.,'2009b).'Evidence'so'far'point'to'a'multiple'TLRs'orchestrated'defense' against' Leishmania! spp.' The' importance' of' TLRs' in' Leishmania' spp.' infection' was' initially'demonstrated'by'the'ability'of'L.!major!parasites'to'activate'the'IL_1'promoter'in' macrophages'via'a'MyD88_dependent'pathway'(Hawn'et'al.,'2002).'Later'it'was'shown' that'mice'lacking'MyD88'have'increased'susceptibility'to'infection'(de'Veer'et'al.,'2003).' Also' TLR4_deficient' mice' had' higher' parasite' burdens' and' were' less' efficient' in' the' resolution'of'cutaneous'lesions' caused'by' L.!major,'suggesting'a'role'for'TLR4'in'host' defense'against'L.!major!(Kropf'et'al.,'2004;'Kropf'et'al.,'2003).'TLR2'and'TLR3'were' also' shown' to' be' necessary' for' NO' and' TNF_α) secretion) in) L.! donovani! infected_ macrophages' (Flandin' et' al.,' 2006).' Paradoxally' TLR_2' _/_' mice' was' shown' to' be' less' susceptible' to' infection' with' reduced' parasite' loads' with' a' diminished' recruitment' of' inflammatory'cells'during'the'first'two'weeks'after'L.'amazonensis'infection'(Guerra'et'al.,' 2010).'Also'TLR_4'mediates'the'effects'of'L.!mexicana'promastigotes'contributing'to'the' CHAPTER''......................................................'I' 47" " inhibition'of'host'macrophage'IL_12'production'(Shweash'et'al.,'2011).'The'expression'of' TLR9'and'IL_12'release'by'myeloid'DCs'was'shown'to'be'required'for'NK'cell'activation'in' mice'infected'with'L.!infantum!(Haeberlein'et'al.,'2010).'Another'remarkable'example'of' TLR'interaction'in'Leishmania!spp.'involves'L.!guyanensis.'This'parasite'is'itself'infected' by' a' virus,' Leishmania! RNA' virus_1' (LRV_1)' (Guilbride' et' al.,' 1992).' The' presence' of' LRV_1' is' associated' with' increased' TLR3' dependent' secretion' of' IFNβ' and' other' pro_ inflammatory' cytokines' from' macrophages.' Moreover,' cutaneous' lesion' is' reduced' in' Tlr3–/–'mice'compared'with'wild_type'mice'when'both'are'infected'with'L.!guyanensis! strains' presenting' high' levels' of' LRV_1,' but' no' difference' is' observed' when' both' are' infected'with'L.!guyanensis!strains'that'present'low'levels'of'or'lacked'LRV_1.'Hence,'the' inflammatory'potential' of' L.! guyanensis! is' dependent' upon' the' virus' load' (Ives' et' al.,' 2011)' in' a' remarkable' example' of' manipulation' of' the' immune' host.' Traditionally,' mounting'an'inflammatory'response'through'PRRs'is'a'prerequisite'for'containment'and' eradication' of' invading' pathogens,' still' it' is' not' clear' the' importance' of' these' PRR_ mediated' responses' in' the' Leishmania! spp.' because' the' inflammatory' environment' promoted'by'their'activation'may'contribute'to'the'immunopathogenesis'by'inducing'the' recruitment'of'neutrophils'and'other'APCs'required'to'establish'the'infection.' ! 3.3!–!APC!deactivation!induced!by!Leishmania!spp.! ! The'capacity'of'the'Leishmania!spp.'to'survive'inside'APCs'is'not'limited'to'the' early'activation'of'TLRs.'It'is'known'that'infected'macrophages'have'a'reduced'capacity'to' kill'the'parasites'upon'activation'(Vannier_Santos'et'al.,'2002).'Much'attention'has'been' given'to'this'remarkable'capacity'of'Leishmania!spp.'to'modulate'and'survive'in'APCs.' This'action'requires'live'and'active'parasites'because'fixed'or'irradiated'parasites'while' inducing' TLR' activation,' they' cannot'proceed' with'the' subsequent' APC' inactivation' as' was'shown'in'DCs'using'L.!infantum'(Neves'et'al.,'2010).'Therefore'agents'for'this'effect' must'be'components'actively'released'from'living'parasites.'In'fact'several'Leishmania! spp.'such'as,'L.!donovani,!L.!major,!L.!mexicana,!and!L.!braziliensis,'induce'NF_kappaB' cleavage,' in' a' GP63' dependent' mechanism,' in' infected' macrophages' leading' to' the' induction' of' chemokine' gene' expression' (Gregory' et' al.,' 2008).' A' similar' phenomena' effect'is'seen'in'DCs'using'L.!infantum'(Neves'et'al.,'2010)'indicating'that'the'mechanisms' for'APC'deactivation'are'probably'overlapping.'The'same'metaloprotease'is'also'capable'of' cleaving' the' protein' tyrosine' phosphatases' leading' to' the' deactivation' of' downsteam' signaling'and'ultimately'leading'to'attenuation'of'host'innate'inflammatory'responses'and' microbicidal'macrophage'functions'(Contreras'et'al.,'2010;'Gomez'et'al.,'2009).'In'2011' CHAPTER''.....................................................'I' 48" " mTOR,'a'serine/threonine'kinase'that'regulates'the'translational'repressor'4E_BP1,'was' also' presented' as' a' target' for' GP63.' The' mTOR' cleavage' leads' to' activation' of' the' translational'repressor'4E_BP1'altering'dramatically'gene'expression'enhancing'parasite' survival.'These'results'were'further'validated'using'4E_BP1'knock'out'mice'which'showed' increased' susceptibility' to' the' parasite,' a' clear' demonstration' of' the' capacity' of' the' parasite'to'manipulate'the'host'machinery'(Jaramillo'et'al.,'2011).'It'has'also'been'shown' that' the' LPG' molecule' attenuates' multiple' macrophage' functions' (54_59)' including' phagosome'maturation'delay,'inhibition'of'oxygen'radical'formation,'and'cell'signaling' through'effects'on'protein'kinase'C'(Dermine'et'al.,'2005;'Descoteaux'et'al.,'1992;'Lodge' and'Descoteaux,'2005).'Also'Leishmania'elongation'factor_1α'was'shown'to'be'capable'of' deactivating'macrophage'functions'by'activating'the'Src'homology'2'domain'containing' tyrosine' phosphatase_1' (SHP_1)' (Nandan' et' al.,' 2002).' These' studies' showed' the' contribution' of' different' parasite' components' to' the' pathogenesis.' Interestingly' also' promastigote'culture'supernatants,'retain'the'characteristics'seen'in'many'of'the'above' mentioned'studies,'in'fact'culture'supernatants'were'shown'to'be'capable'of'reproducing' the'characteristics'of'the'live'parasites'(Contreras'et'al.,'2010).'They'were'also'capable'of' activate'latent'TGF_β'leading'to'enhanced'parasite'persistence'in'infected'macrophages' (Gantt' et' al.,' 2003)' reproducing' what' was' later' seen' for' cathepsin_B' (Somanna' et' al.,' 2002).'In'2009'it'was'shown'that'Leishmania'can'release'exosome_like'vesicles'that'can' also'interact'with'the'host'cells'(Silverman'et'al.,'2010a;'Silverman'et'al.,'2010b).'' These' observations' are' excellent' examples' of' the' complex' host_parasite' interactions' existing' during' the' infection.' The' parasite' seems' to' have' developed' a' remarkable'range'of'sophisticated'adaptive'mechanisms,'which'not'only'allow'to'evade' and'inhibit'normal'macrophage'functions,'but'also'subvert'innate'and'acquired'(both'cell' and'humoral)'immunity'to'their'own'advantage.'Chang'et'al'devised'a'model'to'explain' Leishmania!virulence'that'conceptualizes'two'different'groups'of'parasite'proteins'(Chang' et' al.,' 2003).' Thus,' according' to' this' model,' parasite' surface' and' secreted' molecules' (infection_related'molecules)'are'considered'as'a'prerequisite'for'virulence,'helping'the' parasite' to' successfully' establish' itself' inside' the' host' macrophage' phagolysosome' and' contributing' to' the' maintenance' of' the' infection' by' interfering' with' macrophage' functions.'The'other'group'consists'of'non_secreted'highly'conserved'parasite'intracellular' molecules) classified) as) ‘pathoantigens’) or) ‘panantigens’) (pathology_related' molecules),' which'are'only'visible'upon'amastigote'cytolysis'and'contribute'to'the'immunopathology' associated' with' leishmaniasis' diverting' the' immune' response' towards' intracellular' components,'rendering'them'less'productive'(Chang'et'al.,'2003;'Santarem'et'al.,'2007).' Additional'support'for'such'a'model'came'from'two'studies'in'which'Leishmania'were' CHAPTER''......................................................'I' 49" " shown'to'have'reduced'survival'in'macrophages'when'secretion'was'disrupted.'Expression' of'either'a'mutated'Leishmania!calreticulin,'a'chaperone'of'the'endoplasmic'reticulum' (ER)' which' is' essential'for' a' functional'N_terminal' secretion' system' (Debrabant' et' al.,' 2002),'or'a'dominant'negative'mutant'of'the'AAA'ATPase'Vps4'(Besteiro'et'al.,'2006),' which'has'been'shown'to'disrupt'protein'secretion'from'various'eukaryotic'cells'(Yang'et' al.,'2004),'greatly'reduced'Leishmania'virulence'both'in!vitro!and'in!vivo!(Besteiro'et'al.,' 2006;'Debrabant'et'al.,'2002).! 4!–!Exoproteome! ! 4.1!–!Definition! ' The' collective' extracellular' proteins' released' from' an' organism' under' defined' conditions'is'known'as'secretome'(Hathout,'2007).'Recently,'the'definition'was'further' refined'to'the'use'in'the'context'of'Leishmania!spp.,'secretome'refers'to'proteins'actively' secreted'from'the'cell'using'a'classical'or'a'nonclassical'mechanism'of'secretion,'including' exosome)release.)The)term)‘exoproteome’)is)then)defined)as)all)the)proteins)present)in)the) extracellular' space,' including' extracellular' proteins' released' from' the' surface' or' originating'from'cell'lysis,'and'the'above'mentioned'secretome'(Corrales'et'al.,'2010).' ' 4.2!P!Mechanisms!of!protein!release!in!the!Leishmania!spp.!exoproteome.! ! The'exoproteome'of'Leishmania'spp.'is'composed'of'proteins'released'from'the'surface,' secreted,' and'originating' from' cell' lysis.' For' many' years' proteins' originating' from' cell' lysis' provided' a' plausible' explanation' for' the' presence' of' intracellular' proteins' in' the' exoproteome'(Chang'et'al.,'2003).'Recent'studies'with'L.!donovani'indicated'that'vesicle' structures' with' exosome' like' characteristics' were' also' responsible' for' delivering' intracellular' components' into' the' exoproteome.' Conventional' and' unconventional' secretion'is'also'known'to'contribute'to'the'exoproteome'(see'figure'10'for'details)'with' several'proteins'already'characterized'(Corrales'et'al.,'2010).' ' CHAPTER''.....................................................'I' 50" " ' Figure! 10!P!Schematic' diagram' summarizing' the' different' secretion' mechanisms' in' Leishmania! spp.' (a)' Proteins' secreted' through' the' classical' secretory'pathway,' such' as' gp63'or'chitinase,'are'exported'by'secretory'vesicles'and'released'into'the'extracellular' space' of' the' flagellar' pocket' by' fusion' of' the' vesicles' with' the' plasma' membrane.' Glycosylphosphatidylinositol_anchoring'proteins'are'swept'out'of'the'flagellar'pocket'to' the' cell' body' and' attached' to' the' external' surface' of' the' membrane' by' their' glycosylphosphatidylinositol' moiety.' Gp63' is' released' in' a' secreted' soluble' form' and' a' glycosylphosphatidylinositol_anchored'form.'Some'proteins'devoid'of'a'signal'peptide'are' localized' in' the' flagellar' pocket' (e.g.' LAWD:' Leishmania' antigenic' tryptophan_aspartic' acid.'(b)'HASPB'is'synthesized'on'free'ribosomes'in'the'cytoplasm'and'may'be'transferred' to'the'outer'leaflet'of'the'Golgi'membrane'and'would'use'conventional'vesicular'transport' to' reach' the' plasma' membrane,' where' translocation' could' occur.' The' SH4' domain' of' HASPB'induces'the'production'of'nonapoptotic'membrane'blebbing.'(c)'Proteins'may'be' released'into'the'extracellular'space'through'exosomes'originating'from'either'lysosomes' or' multivesicular' bodies' (MVB)' of' endosomal'origin.' Whether' GRASP,' essential' to' the' unconventional'secretion'pathway'of'Dictyostelium'during'development,'plays'a'role'in' the'nonclassical'secretion'pathway'in'Leishmania'spp.'remains'to'be'clarified.'Adapted' from'(Corrales'et'al.,'2010).' ' 4.2.1!P!The!surface!of!the!promastigote! ' Proteins' released' or' shed' from' the' surface' of' the' promastigote' are' present' in' the' exoproteome.' These' can' be' released' by' the' action' of' proteases' or' phospholipases' that' release' GPI' anchored' proteins' (Yao' et' al.,' 2003).' The' surface' of' Leishmania! spp.' is! composed'of'a'few'dominant'components'that'are'present'in'all'species'(see'figure'11).'' CHAPTER''......................................................'I' 51" " ' Figure!11.'Surface'coats'of'Leishmania'ssp.'The'surface'coats'of'Leishmania'spp.' parasites'are'dominated'by'LPG,'GPI'proteins'and/or'non_protein'linked'GPI'glycolipids.' Adapted'from'(McConville'et'al.,'2002).' ' ' Promastigotes' are' covered' by' glycoproteins' that' are' anchored' to' the' protozoan' membrane'by'a'GPI'anchor.'These'proteins'form'a'glycocalix'that'is'about'7'nm'thick'in' procyclic'promastigotes'and'it'thickens'in'later'stages'of'development'being'at'least'twice' as' thick' in' metacyclic'parasites.' The' dominant'surface' molecule' of' the' promastigote' is' LPG.' It' is' composed' of' repetitive' units' consisting' of' a' disaccharide' and' a' phosphate,' linked'to'the'membrane'by'a'GPI'anchor.'The'LPG'differs'from'species'to'species'by'the' presence'of'glycan'side'chains.'LPG'of'L.!major,'for'example,'is'highly'branched,'whereas' that'of'L.! donovani! is' not' (McConville' et' al.,' 1995).' Furthermore' the' structure'of' LPG' differs'between'procyclic'and'metacyclic'promastigotes,'being'significantly'longer'in'the' latter,'and'is'almost'completely'absent'from'amastigotes'(McConville'et'al.,'1992;'Pimenta' et'al.,'1991).'Studies'using'purified'LPG'or'parasite'strains'defective'in'LPG'production' have'shown'that'LPG'can'play'important'roles'in'parasite'survival'and'modulation'of'the' immune' response.' Another' abundant' surface' molecule' is' the' glycoprotein' gp63' (McConville' and' Ralton,' 1997;' Pimenta' et' al.,' 1991).' This' is' a' zinc_dependent' metalloprotease'with'a'wide'range'of'substrates'(McMaster'et'al.,'1994).'GP63'is'hidden' under' the' 10' fold' more' abundant' LPG' that' actually' towers' over' all' other' surface' components.'GP63'expression'is'down_regulated'in'amastigote'(Schneider'et'al.,'1992).' The'GP63'is'one'of'the'most'prominent'proteins'in'the'parasite'and'is'composed'of'two' distinct'forms'one'that'is'conventionally'secreted'and'another'that'is'shed'(released'from' the'surface)'probably'by'processing'of'the'GPI'anchor.'The'dynamics'of'GP63'release'are' complex'and'known'to'be'responsive'to'environmental'stimuli'(Yao'et'al.,'2007).''Free'or' vesicle'bound'GP63'will'contribute'to'immediate'protection'against'complement'mediated' lysis'(Joshi'et'al.,'2002)'and'might'even'enter'the'host'cell'cytoplasm'through'a'lipid'raft_ CHAPTER''.....................................................'I' 52" " dependent'mechanism'(Gomez'et'al.,'2009),'to'cleave'the'cytosolic'host'protein'tyrosine' phosphatases'(Gomez'et'al.,'2009),'contributing'to'macrophage'energy'(Contreras'et'al.,' 2010).'The'most'abundant'molecules'in'the'promastigote'surface'are'the!glycosylinositol' phospholipid'(GIPL).'These'GPI_linked'glycolipids'are'10'times'more'abundant'than'LPG.' Being' quite' small' compared' to' LPG' they' exist' close' to' the' parasite' membrane.' Unlike' LPG,' which' is' continually' shed,' GIPL' have' a' long' half_life' in' the' membrane' so' it' is' believed'to'have'a'protective'role'at'the'promastigote'surface'(Ferguson,'1997;'Proudfoot' et' al.,' 1995).' In' fact' GIPLs' recovered' from' L.! major! where' capable'of' inhibiting' nitric' oxide'production'in'macrophages'by'an'unknown'mechanism'diminishing'their'capacity' to'eliminate'the'parasites'in!vitro'(Proudfoot'et'al.,'1995).'Nonetheless'it'is'still'a'matter'of' debate' of' what' is' their' exact' role' in' their' interactions' with' the' host' (Ferguson,' 1997;' McConville'and'Ralton,'1997).' ' 4.2.2!P!The!classical!secretory!pathway! ! ' The' use' of' the' secretion' pathway' to' deliver' effector' molecules' by' microbial' pathogens'is'almost'a'trademark'in'pathogenesis.'Still,'little'is'known'concerning'the'exact' secretory' pathway' in' Leishmania' (McConville' et' al.,' 2002).' Most' of' the' organelles' involved'in'secretion'and'the'early'endocytic'pathway'are'organized'around'the'flagellar' pocket.'In'fact'this'organelle'was'identified'to'be'the'endpoint'of'the'secretion'apparatus' as' was' elegantly' shown' for' the' variant' surface' glycoprotein' in' T.! brucei.' Using' immunogold'labeling,'it'was'demonstrated'that'variable'surface'glycoprotein'(VSG)'was' sequentially' present' in' ER,' Golgi' cisternae,' accumulated' in' the' transQGolgi' network,' transport'vesicles,'flagellar'pocket'and'ultimately'cell'surface'(Duszenko'et'al.,'1988).'In' fact,'despite'being'a'very'divergent'eukaryotic'lineage'the'basic'features'of'the'classical' secretory'pathway'are'conserved'(McConville'et'al.,'2002)'with'the'remarkable'exception' of'the'highly'polarized'delivery'system'(Field'et'al.,'2007).'Similarly'to'higher'eukaryotes,' secretory'proteins'are'associated'with'N_terminal'signal'peptides'that'direct'the'proteins' through' the' endoplasmic' reticulum' (ER)' (Schatz' and' Dobberstein,' 1996).' The' ER' comprises'the'nuclear'envelope'and'a'connected'system'of'cisternal'or'tubular'membranes' that'can'be'associated'with'the'plasma'membrane'(Ilgoutz'et'al.,'1999;'Weise'et'al.,'2000).' Depending' on' the' physiological' need' of' the' cell,' the' ER' can' expand' or' diminish,' in' dividing'T.!brucei'the'ER'can'account'for'60%'of'the'internal'membrane'content'(Coppens' et'al.,'1987).'The'ER'network'in'trypanosomatids,'besides'the'traditional'classical'rough' and'smooth'domains,'also'contains'a'specialized'transitional'ER'(tER)'directly'opposite'to' the'single'Golgi'apparatus'that'is'only'present'in'dividing'parasites'(Mullin'et'al.,'2001;' CHAPTER''......................................................'I' 53" " Weise'et'al.,'2000).'The'space'between'the'tER'and'the'CisQGolgi'is'packed'with'transport' vesicles'(Weise'et'al.,'2000).'The'transport'of'these'vesicles'is'maximized'by'the'intimate' association' between' the' Golgi' and' the' tER' (100' nm' apart)' enabling' the' high' level' of' protein'and'lipid'transport'required'by'the'cell'surface'(Mullin'et'al.,'2001;'Weise'et'al.,' 2000).'The'trypanosomatids'have'a'single'Golgi'apparatus'comprised'of'a'stack'of'3'to'10' cisternae'(Figueiredo' and' Soares,' 1995;' Weise' et' al.,' 2000).' Intriguingly' brefeldin' A,' a' compound' traditionally' used' in' the' disruption' of' secretion,' by' preventing' transport' between'ER'and'Golgi,'has'no'effect'on'trypanosomatids'(Figueiredo'and'Soares,'1995;' McConville'et'al.,'2002).'Traditionally'transport'through'the'Golgi'apparatus'is'mediated' by' two' mechanisms:' vesicular' transport' between' cisternae' or' the' maturation' and' progressive'movement'of'cisternae'towards'the'trans'face'of'the'Golgi'(Glick,'2000).'The' latter'seems'to'be'preferred'in'trypanosomatids'because'ultrastructural'evidence'point'to' the'formation'of'new'cisternae'in'the'cisQGolgi'(Weise'et'al.,'2000).'The'Golgi'cisternae' appear'to'be'functionally'differentiated'(McConville'et'al.,'2002),'quite'likely'as'a'result'of' complex'modifications'associated'with'O_linked'glycans'or'phosphoglycans'that'require' specific'sets'of'enzymes'(Ha'et'al.,'1996;'Moss'et'al.,'1999).'The'last'step'of'secretion'is'the' passage'of'proteins'from'the'transQGolgi'to'the'flagellar'pocket.'The'study'of'dominant' glycoproteins'in'the'trypanosomatids'revealed'some'information'related'to,'the'nature'of' the'exact'process'leading'to'this'last'event.'The'variant'surface'glycoprotein'in'T.!brucei'is' transported'to'the'flagellar'pocket'by'a'system'of'cisternal'and'tubovesicular'membranes' (Duszenko'et'al.,'1999;'Webster'and'Grab,'1988).'The'GP63'in'L.!mexicana'is'transported' by'a'distinct'process,'being'delivered'to'the'flagellar'pocket'in'large'vacuoles'(Weise'et'al.,' 2000).'Furthermore'ultrastructural'studies'consistently'show'small'vesicles'between'the' Golgi'and'the'flagellar'pocket'indicating'a'possible'role'for'these'vesicles'(McConville'et' al.,'2002).'It'is'then'possible'that'the'transport'to'the'flagellar'pocket'involves'distinct' mechanisms.' In' fact' the' nature' of' the' main' transport' mechanisms' is' yet' to' be' characterized.' The' flagellar' pocket' is' a' highly' specialized' structure' present' in' all' trypanosomatids.' The' known' functions' for' this' structure' include' secretion' of' proteins,' addition' of' integral' membrane' proteins' and' endocytic' activity' (Landfear' and' Ignatushchenko,'2001).'The'membrane'of'the'flagellar'pocket'represents'between'0,4_3%' of' the' parasite' surface' (Landfear' and' Ignatushchenko,' 2001)' and' is' the' most' active' endocytic'organelle'being'capable'of'internalizing'an'equivalent'membrane'area'every'2' minutes'(Coppens'et'al.,'1987)'(Overath'et'al.,'1997).! ' A' few' studies' exist' on' the' properties' of' individual' secreted' proteins.' They' have' been'shown'to'play'specific'roles'in'the'survival'and'proliferation'of'the'parasite'in'both' the'insect'stage'and'in'the'mammalian'host.'In'the'insect'vector,'it'is'clear'that'several' secreted'elements'have'a'significant'role'in'the'survival'and'proliferation'of'the'parasite.' CHAPTER''.....................................................'I' 60" " The'release'of'microvesicles'(MVs)'by'eukaryotic'microorganisms'was'considered' to'be'biologically'releveant'only'recently.''For'a'long'time'it'was'thought'that'these'vesicles' found' in' culture' supernatant' were' artifacts' resulting' from' indirect' processes' like' mechanical' damage' or' death' (Silverman' and' Reiner,' 2011).' Only' a' few' eukaryotic' pathogens' have' been' shown' to' actively' secrete' MVs.' Microvesicles' were' detected' in' pathogenic' fungus,' like' Cryptococcus! neoformans' (Rodrigues' et' al.,' 2008)' and' Histoplasma!capsulatum!(Albuquerque'et'al.,'2008),!and'in'protozoan'parasites'like'L.! donovani'(Silverman'et'al.,'2010a)'and'Trypanosome!cruzi!(Goncalves'et'al.,'1991).'These' pathogen'derived'vesicles'are'a'new'and'exciting'field,'because'they'have'the'potential'to' mimic'the'characteristics'of'the'host'vesicles.'In'fact'there'are'several'ways'by'which'these' pathogen'derived'vesicles'might'interact'with'the'host'cells.'These'vesicles'are'expected'to' interact'directly'with'surface'receptors'of'target'cells,'functioning'in'fact'as'extensions'of' the'pathogen.'The'potential'action'of'these'vesicles'is'not'limited'to'surface'interactions,' the'direct'delivery'of'virulence'factors'to'the'host'cells'may'play'an'important'role'in'the' pathogenic' process.' Prokaryotes' membrane' vesicles,' a' naturally' secreted' product' of' Gram_negative' bacteria,' have' in' fact' been' found' to' be' the' agent' of' delivery' of' several' toxins' and' other' bacterial' associated' virulence' factors' to' host' cells' (Ellis' and' Kuehn,' 2010).' As' an' example,'Legionella' vesicles' were' shown' to' inhibit' the' fusion' of'bacteria' containing'phagosomes'(Galka'et'al.,'2008),'interestingly'this'seems'reminiscent'of'LPG' driven'delay'in'phagosome'maturation'(Dermine'et'al.,'2005;'Dermine'et'al.,'2000).'For' lower'eukaryotic'MVs'few'studies'were'done.'Nonetheless'some'interesting'reports'exist'in' the'literature.'The'single'most'interesting'fact'is'the'evidence'that'vesicles'recovered'from! L.!donovani'expressing'GFP'are'taken'up'by'macrophages'(Silverman'et'al.,'2010a).'This' fact,'added'to'a'proteomic'analysis'of'the'MVs'of!L.'donovani!that'revealed'the'presence'of' several'known'virulence'factors'(Silverman'et'al.,'2010a)'enhances'the'potential'of'MVs'as' system' for' delivery' of' virulence' factors' (Ellis' and' Kuehn,' 2010).' The' virulence' factors' detected' in' L.! donovani' included' the' prominent' metaloprotease' GP63' whose' immune' modulatory'properties'enabled,'for'example,'direct'modulation'of'host'signaling'(Halle'et' al.,'2009;'Jaramillo'et'al.,'2011).'Also'other'immunomodulatory'proteins'like'elongation' factor'1_α,)aldolase)and)kinetoplastid)membrane)11)(Carvalho'et'al.,'2005;'Nandan'et'al.,' 2007;' Nandan' et' al.,' 2002)' were' found' among' the' cargo' proteins' of' L.! donovani' (Silverman'et'al.,'2010a).'In'fact'the'potential'for'cargo'delivery'into'the'host'cells'may' require'the'reevaluation'of' several' proteins'as'potentially'involved'in'the'pathogenesis.' Actually' all' the'Leishmania' proteins' shown' to' translocate' into' the' macrophage' cytosol' were'shown'to'be'part'of'the'Leishmania!exosome'cargo'(Silverman'et'al.,'2010a).'The' definition' of' the' role' of' these' vesicles' in' the' context' of' infection' is' still' in' its' infancy.' Leishmania!MVs''exhibit'some'anti_inflammatory'properties.'Using'human'monocytes'in' CHAPTER''......................................................'I' 61" " the'context'of' L.!donovani!infection,'MVs' were'shown'to'inhibit'the'pro_inflammatory' cytokine' production' (TNF_α)) while) promoting) immunosuppressive' cytokine' production' (IL_10).' Furthermore' MVs' from' L.! major! also' induced' down' regulation' of' monocytes_ derived'dendritic'cell'maturation'(Silverman'et'al.,'2010b).'Interestingly'this'capacity'was' cargo'dependent'because'exosomes'from'L.!major!HSP100'(_/_)'had'distinct'protein'cargo' and' induced' a' pro_inflammatory' profile' in' the' same' cells' (Silverman' et' al.,' 2010b).' Nevertheless'the'few'reports'that'addressed'the'in!vivo'properties'of'these'vesicles'seem'to' be'consistent'with'a'permissive'infection.'L.'donovani'and'L.!major!MVs'treatment'prior' to' infection' exacerbated' the' infection' and' increased' parasite' load' in' susceptible' and' resistant' mice' (Silverman' et' al.,' 2010b).' A' similar' report' with' T.! cruzi! vesicles' also' resulted'in'increased'parasite'loads'and'exacerbated'infection'(Trocoli'Torrecilhas'et'al.,' 2009).'Little'is'still'known'about'the'biogenesis'of'these'vesicles'in'Leishmania,'although' some'orthologs'to'endosomal'machinery'related'to'exosomal'biogenesis'were'identified' (Ivens'et'al.,'2005).'There'are'still'doubts'relating'to'the'exact'nature'of'these'vesicles,'still' some'general'facts'related'to'vesiculation'are'already'known.'It'seem'to'be'induced'by' physiological'stress'agents'like'temperature'shift'to'37ºC''(Hassani'et'al.,'2011;'Silverman' et'al.,'2010a)'and'acidic'pH'(Silverman'et'al.,'2010a).' ' 4.3!–!Exoproteome!in!Leishmania!spp.!immunopathology! ! 'The) survival) of) the) parasite) is) dependent) on) “convincing”) the) immune) system) components'to'divert'their'activity'towards'a'non'productive'response.'Therefore'if'the' cross' talk' between' the' parasite' and' the' immune' host' could' be' compared' to' actual' communication,' the' surface' and' released' components' would' be' the' actual' language' enabling'specific'interactions'with'their'biological'targets,'like'receptors'or'transcription' factors.' The' Leishmania! exoproteome' is' highly' immunogenic' eliciting' strong' immunity' and'some'protection'against'infection'in'mice'and'dogs'(Lemesre'et'al.,'2007;'Tonui'et'al.,' 2004;' Tonui' and' Titus,' 2007).' Intriguingly' there' was' some' species' specific' cross' protection,' because' L.!major' exoproteome' was' capable' of' protecting' from' L.! donovani' challenge'but'not'from'L.!braziliensis'(Tonui'and'Titus,'2007).'The'high'immunogenicity' of'the'exoproteome'makes'it'an'attractive'source'of'immunogenic'antigens.'In'fact'among' the' components' of' the' possible' recombinant' vaccine' Leish_111f,' ' the' thiol_specific' antioxidant'was'identified'in'the'exoproteome'(Coler'et'al.,'2007).'This'vaccine'has'shown' some' potential' in' the' context' of' human' and' canine' leishmaniasis' (Coler' et' al.,' 2007;' CHAPTER''.....................................................'I' 62" " Nascimento'et'al.,'2010;'Trigo'et'al.,'2010).'Therefore,'the'exoproteome'has'potential'for' the' discovery' of' new' immunogenic' proteins.' In' fact' a' vaccine' approach' for' the' intracellular' pathogen' M.! tuberculosis! is' based' on' the' immunogenicity' of' a' defined' exoproteome'component,'the'30'kDa'major'secretory'protein'(Horwitz,'2005).'Although,' two' recent' reports' from' 2011' showed' a' different' facet' of' the' exoproteome.' L.! major! secreted' antigens' were' shown' to' be' immunosuppressive' inducing' IL_4' production' in' lymphocyte' culture' (Tabatabaee' et' al.,' 2011)' while' L.! mexicana! exoproteome' induced' inhibition'of'macrophage'functions'by'cleavage'and'activation'of'the'host'protein'tyrosine' phosphatases,'specifically'SHP_1'and'PTP1_B'(Hassani'et'al.,'2011).' ' 4.4!–!Approaches!for!the!study!of!the!exoproteome! ! !The'knowledge'on'the'Leishmania!exoproteome'is'in'its'infancy'if'we'compare'it' with'higher'eukaryotes'or'prokaryotes'(Corrales'et'al.,'2010).'' ' In' recent' years' great' progress' in' mass' spectrometry' (MS),' bioinformatics,' and' analytical'techniques'have'enabled'broad'scale'analysis'of'proteins'in'complex'biological' samples,'like'whole'tissue'or'culture'media'(Lane,'2005).'The'quick'development'of'these' techniques' combined' with' a' growing' interest' in' the' exoproteome' of' Leishmania' and' related' trypanosomatids' led' to' several' studies' resulting' in' a' list' of' a' few' hundreds' of' proteins' that' are' found' in' the' exoproteome' from' several' species' (Cuervo' et' al.,' 2009;' Geiger'et'al.,'2010;'Hassani'et'al.,'2011;'Silverman'et'al.,'2008;'Silverman'et'al.,'2010a;' Trocoli' Torrecilhas' et' al.,' 2009).' Although' the' proteomic' techniques' developped' are' highly' efficient,' the' detection' of' exoproteome' components' in' culture' supernatants' presents'specific'challenges.'Standard'culture'media'for'the'growth'of'Leishmania!spp.'is' a' complex' mixture' dominated' by' the' serum' supplement' components.' The' presence' of' serum'proteins'represents'a'specific'problem'for'the'detection'of'the'parasite'exoproteome' because' it' will' mask' the' parasite' derived' components.' Also' proteins' released' into' the' medium' upon' parasite' death' might' contribute' very' significantly' to' the' exoproteome,' masking'the'secretome.'The'low'abundance'of'secreted'proteins'poses'another'challenge' because' concentration' steps' are' required' before' subsequent' proteomic' analysis.' Two' major'techniques'to'resolve'the'concentration'problem'include'selective'precipitation'or' direct'concentration'using'ultrafiltration.'To'overcome'the'above'mentioned'limitations' for' exoproteome' recovery' ' the' standard' procedure' consists' in' washing' the' parasites' to' eliminate' serum' components' and' then' incubate' them' in' a' serum' free' medium' for' a' defined'time'(Corrales'et'al.,'2010).'There'is'a'short'interval'between'the'time'of'culture' CHAPTER''......................................................'I' 63" " required'for'obtaining'detectable'amounts'of'secreted'proteins'and'the'cell'survival'in'a' serum'free'medium'avoiding'cell'lysis,'therefore'short'culture'times'are'preferred.'Studies' with'Leishmania'spp.'tend'to'use'less'than'6'hours' of'incubation'in'serum'free'media' (Cuervo'et'al.,'2009;'Hassani'et'al.,'2011;'Silverman'et'al.,'2008).'' ' The' most' traditional' technique' used' in' proteomic' approaches' to' study' the' exoproteome' is' two_dimentional' gel' electrophoresis' combined' with' MS' (Cuervo' et' al.,' 2009),'but'the'first'true'systematic'study'of'the'Leishmania'exoproteome'was'made'using' stable'isotope'labeling'with'amino' acids'in' cells'_'SILAC.' This'study'by'Silverman'and' colleagues'confirmed'the'specific'enrichment'of'several'intracellular'proteins'that'could' not'be'explained'by'simple'parasite'lysis.'The'above'mentioned'proteomic'study'clearly' demonstrated'that'Leishmania'exoproteome'contains'a'myriad'of'proteins'with'distinct' cellular' origins,' most' lacking' a' clear' amino_terminal' secretion' signal' (Silverman' et' al.,' 2008).''This'fact'was'later'confirmed'in'subsequent'studies'with'other'species'(Cuervo'et' al.,'2009;'Hassani'et'al.,'2011;'Silverman'et'al.,'2010b).'Eventually'the'release'of'exosome' like'vesicles'from'the'L.!donovani'surface'was'identified'as'a'major'contributor'for'non' classical' secretion' in' the' exoproteome' (Hassani' et' al.,' 2011;' Silverman' et' al.,' 2010a).' Genome'based'computational'predictions' have'also' been' used' to' mine'the' Leishmania' spp.' genome' using' computer' algorithms' to' screen' for' conserved' N_terminal' signal' sequences.' Several' bioinformatic' applications'have' been' developed' for' the' detection' of' secreted'proteins'from'eukaryotes'(Klee'and'Sosa,'2007).'These'programs'are'based'on' different' algorithms' and' lead' often' to' distinct' predictions.' To' further' increase' the' difficulty' associated' with' in! silico' prediction' none' of' these' tools' has' been' created' specifically'for'Leishmania'spp.'In'fact'there'is'only'one'report'on'the'validation'of'some' of'these'bioinformatics'tools'in'the'related'trypanosomatid'T.!cruzi'(Ferella'et'al.,'2008).' The' value'of' these' individual' approaches' was' limited' but' combining' several' prediction' servers'allowed'to'have'almost'50%'prediction'rate'when'compared'to'the'detection'of'the' proteins'in'specific'sub_cellular'fractions'(Ferella'et'al.,'2008).'Therefore'to'improve'the'in! silico' prediction' power' several' distinct' tools' can' be' combined:' presence' of' transmembrane' domains,' glycosylphosphatidylinositol_anchoring' signal' and' mitochondrial' targeting' signals' can' be' combined' to' screen' for' classically' secreted' proteins.'To'date'there'was'only'one'major'study'done'for'in!silico'prediction'of'potential' secreted'proteins'in'L.!donovani'(Silverman'et'al.,'2008).'Silverman'et!al.'analyzed'the' secretome'searching'for'protein'sequences'with'a'signal'peptide,'lacking'a'transmembrane' domain' and' glycosylphosphatidylinositol_anchoring' signal,' and' found' 217' potentially' secreted' proteins.' Among' these' only' 14' were' detected' in' the' subsequent' proteomic' approach'(Silverman'et'al.,'2008).'In'fact'the'study'of'the'Leishmania!spp.'secretome'is' CHAPTER''.....................................................'I' 64" " impaired' by' the' complexity' of' the' exoproteome' therefore' experimental' validation' is' required' in' order' to' evaluate' actual' secretion' of' the' predicted' proteins.' Furthermore,' taking'into'consideration'the'relative'minor'differences'in'gene' content'and'RNA'gene' expression'among'the'different'Leishmania'spp.'(Lynn'and'McMaster,'2008),'the'value'of' these' in! silico! approaches' is' always' relative' because' an' applied' proteomic' study' will' always'be'necessary'when'performing'comparative'or'stage'specific'studies.' ' Due' to' the' importance' of' the' extracellular' components' of' the' parasite' in' the' interaction' with' surrounding' environment,' it' is' of' vital' importance' to' improve' and' validate' appropriate' guidelines' for' exoproteome' recovery.' This' should' lead' to' the' understanding'of'the'exoproteome'dynamics'through'the'parasites'life'cycle'and'to'define' the'mechanisms'associated'with'protein'release'in'the'different'developmental'forms.'This' should' allow' a' better' understanding' of' the' parasite' biology' and' the' mechanisms' that' enable'it'to'survive.' ' CHAPTER'.....................................................'II' 65" " ! ! ! ! ! ! ! Chapter!II! Results' CHAPTER''.....................................................'II' 66" " ' ' CHAPTER'.....................................................'II' 67" " 1.!Objectives! ' This' project' was' originally' focused' on' studying' the' Leishmania' secretome' with' the' objective' of' understanding' the' contribution' of' individual' secreted' proteins' towards' virulence.' To' identify' secreted' proteins' responsible' for' virulence' we' proposed' using' virulent'and'avirulent'strains/species'and'comparing'their'secretomes.'The'complexity'of' the'secretome'prevented'the'use'of'different'strains/species'to'look'for'secreted'proteins' in' a' basic' comparative' approach' between' virulent' and' avirulent' strains.' Therefore' we' refocused'the'objective'of'the'project'into'the'development'and'validation'of'an'approach' that'would'enable'the'study'of'the'exoproteome'components'of'stationary'and'logarithmic' parasites.'To'achieve'this'we'proposed'to:' ‐ Define'the'time'frame'that'we'could'work'with'parasites'with'optimal'virulence;' ‐ Develop'a'protein'free'medium'that'would'enable'the'study'of'the'secretome;' ‐ Validate'and'characterize'the'exoproteome'from'stationary'and'logarithmic'parasites;' ‐ Evaluate'the'effect'of'the'exoproteome'in'the'DCs.' The'accomplishment'of'these'goals'will'enable'the'creation'of'new'tools'for'the'study'of' the' exoproteome' components' ultimately' contributing' to' a' better' understanding' of' Leishmania'spp.'pathogenesis.' ' ' ' ' ' ' CHAPTER''.....................................................'II' 68" " ' ' CHAPTER'.....................................................'II' 69" " 2.!Article!name:!Impact!of!continuous!axenic!cultivation!in!Leishmania! infantum!virulence!! ' Diana' Moreira#,' Nuno'Santarém#,' Inês' Loureiro,' Joana' Tavares,' Ana' Marta' Silva,' Ana' Marina'Amorim,'Ali'Ouaissi,'Anabela'Cordeiro_da_Silvaǂ'&'Ricardo'Silvestreǂ' #'and'ǂ'Both'authors'contributed'equally'to'this'work' ' Accepted'for'publication'in'Plos'Neglected'Tropical'Disease' ' In' the' present' study' we' evaluated' the' phenomena' of' virulence' lost' associated' with' continuous'growth'in'RPMI.! ' Main!Results:! _Metacyclic'parasites'are'highly'immunomodulatory.' _Virulence'loss'is'associated'with'continuous'subpassages'appears'as'early'as'10'passages.' _The'virulence'loss'is'associated'to'a'defect'in'differentiation'into'amastigotes'and'can'be' reverted'upon'differentiation'into'amastigotes,'both'in!vitro!and!in!vivo.' ' Conclusions:' L.! infantum! upon' continuous' subculture' rapidly' looses' virulence' through' a' process' related'to'the'incapacity'to'differentiate'into'amastigotes.' ' ' ' ' ' ' CHAPTER''.....................................................'II' 76" " promastigotes,' identified' by' morphological' criteria,' i.e.,' short' and' slender' with' a' long' flagellum'twice'the'body'length'using'phase'contrast'on'a'Nikon'Eclipse'80i.'' ' qPCR'analysis' ! Total' RNA' was' isolated' from'cells' with' the' Trizol®' reagent' (Invitrogen,' Barcelona,' Spain),'according'to'the'manufacturer's'instructions.'Briefly,'parasites'were'washed'with' ice_cold'phosphate_buffered'saline'(PBS),'harvested'and'homogenized'in'800'µl'of'Trizol' by)pipetting)vigorously.)After)addition)of)160)μl)of)chloroform,)the)samples)were)vortexed,) incubated'for'2'min'at'room'temperature'and'centrifuged'at'12.000'g,'for'15'min,'at'4ºC.' The'aqueous'phase'containing'RNA'was'transferred'to'a'new'tube'and'RNA'precipitated' with'400'µl'of'isopropanol'for'at'least'10'min'at'room'temperature.'Following'a'10'min' centrifugation' at' 12.000' g,' the' pellet' was' washed' with' 1' ml' of' 75%' ethanol' and' resuspended) in) 10) μl) of) 60ºC) heated) RNase) free) water.) The) RNA) concentration' was' determined'by'using'a'Nanodrop'spectrophotometer'(Wilmington,'DE,'USA)'and'quality' was'inspected'for'absence'of'RNA'degradation'or'genomic'DNA'contamination,'using'the' Experion'RNA'StdSens'Chips'in'the'ExperionTM'automated'microfluidic'electrophoresis' system' (BioRad' Hercules,' CA,' USA).' RNA' was' stored' at' ' ' _80ºC' until' use.' RT' was' performed) with) equal) amounts) of) total) extracted) RNA) (1) μg)) obtained) from) parasites) recovered'from'different'experimental'conditions'by'using'Superscript'II'RT'(Gibco'BRL)' and'random'primers'(Stratagene).'Real_Time'quantitative'PCR'(qPCR)'reactions'were'run' in'duplicate'for'each'sample'on'a'Bio_Rad'My'Cycler'iQ5'(BioRad,'Hercules,'CA,'USA).' Primers'sequences'were'obtained'from'Stabvida'(Portugal)'and'thoroughly'tested.'qPCR' was'performed'in'a'20'µl'volume'containing'5'µl'of'complementary'cDNA'(50'ng),'10'µl'of' 2x'Syber'Green'Supermix'(BioRad,'Hercules,'CA,'USA),'2'µl'of'each'primer'(250'nM)'and' 1'µl'H2O)PCR)grade.)Specific)primers)for)histone)H4)(forward:)5’)ACACCGAGTATGCG)_3’;) reverse:)5’_'TAGCCGTAGAGGATG_3’;)LinJ35.1400)histone)H4:)Gene)ID)5073031),)Small) Hydrophilic' Endoplasmic' Reticulum_associated) Protein) (SHERP)) (forward:) 5’) CAATGCGCACAACAAGAT' _3’;) reverse:) 5’_' TACGAGCCGCCGCTTA_3’;) LinJ23.1190) SHERP:'Gene'ID'5069222)'and'rRNA45)(forward:)5’CCTACCATGCCGTGTCCTTCTA)_3’;) reverse:) 5’_' AACGACCCCTGCAGCAATAC' _3’)) (Ouakad' et' al.,' 2007)' were' used' for' amplification.'After'amplification,'a'threshold'was'set'for'each'gene'and'cycle'threshold_ values'(Ct_values)'were'calculated'for'all'samples.'Gene'expression'changes'were'analyzed' using'the'built_in'iQ5'Optical'system'software'v2.1'(Bio_Rad'laboratories,'Inc).'The'results' were' normalized' using' as' reference' gene' the' rRNA45' rRNA' sequence' (Ouakad' et' al.,' 2007).' CHAPTER'.....................................................'II' 77" " ! Viability'analysis' Purified' and' non_purified' promastigotes' at' a' density' of' 105/ml' were' washed' and' suspended'in'Annexin'V'binding'buffer.'Parasites'were'incubated'at'room'temperature'for' 15' minutes' with' AnnexinV_Cy5' (BD' Pharmingen,' San' Diego,' CA)' and' 7_AAD' (Sigma).' Parasites'subjected'to'Ultra'Violet'light'during'30'minutes'and'kept'in'culture'for'4'hours' were' used' as' positive' control.' In' amastigote' differentiation,' 2x106' cells' with' 1' µM' of' propidium' iodide' (PI)' were' used.' Data' were' collected' in' a' BD' FACScalibur' cytometer' (20.000'gated'events)'and'analyzed'by'FlowJo'software'(Ashland,'OR).! ' Promastigote'CFSE_labeling' Purified' and' non_purified' promastigotes' (6x107/ml)' were' washed' two' times,' suspended' in' PBS' containing' 5' µM' of' carboxyfluorescein' succinimidyl' ester' (CFSE)' (Invitrogen' Molecular' probes,' Eugene,' Oregon)' and' incubated' at' 37oC' for' 10' minutes.' Labeled'parasites'were'washed,'incubated'at' 4oC'for' 5'minutes.'Parasites'were' washed' again'to'remove'the'exceed'CFSE'dye'and'suspended'in'culture'medium'before'proceeding' to'macrophage'infections.'For'promastigote'to'amastigote'differentiation'and'proliferation' analysis,'107'CFSE_labeled'promastigotes'were'placed'on'1'ml'of'MAA20.'Each'day,'100'µl' of' culture' added' with'1' µM' of'PI' was' analyzed'by' flow' cytometry.' Axenic' amastigotes,' identified'by'the'absence'of'visible'flagella'and'oval'shape'body,'were'observed'in'phase' contrast'on'a'Nikon'Eclipse'80i.'' '' In!vitro'macrophage'infection' Cell'suspension'of'bone'marrow'was'obtained'by'flushing'the'femurs'of'susceptible' Balb/c) mice.) The) cell) suspension) was) cultured) in) Dulbecco’s) modified) Eagle’s) medium) (DMEM)' (Lonza,' Switzerland),' supplemented' with' 10%' heat_inactivated' FBS' (Lonza,' Switzerland),'2'mM'L_glutamine,'100'U/ml'penistreptomycin'and'1'mM'sodium'pyruvate.' After'overnight'incubation'at'37ºC,'non_adherent'cells'were'recovered'(300'g'for'10'min,' at' room' temperature)' and' cultured' in' 24_well' culture' dishes' at' 2x105cells/ml' in' supplemented'DMEM.'For'bone_marrow'derived'macrophages'(BMMø)''differentiation' 10%' L_929' cell' conditioned' medium' (LCCM)' was' added' at' days' 0' and' 4.' At' day' 7' of' culture,'CFSE'labeled'promastigotes'were'incubated'with'the'BMMø'at'a'10:1'ratio.'After'4' hours,'infection'was'stopped'the'infection'rates'were'determined'at'4,'24'and'48'hours' CHAPTER''.....................................................'II' 78" " post_infection'by'a'BD'FACScalibur'cytometer'and'analyzed'by'FlowJo'software.'In'some' experiments,'BMMø'were'infected'and'submitted'to'lipopolysaccharide'(LPS)'stimulus.' Briefly,' four' hours' after' infection,' infection' was' stopped' and' 1' µg/ml' of' LPS' (Sigma)' added.'Twenty_four'hours'post_infection,'BMMø'culture'supernatants'were'collected'for' cytokine' quantification' by' Enzyme_Linked' Immunosorbent' Assay' _' ELISA' (TNF_α,) IL_ 12p40,'IL_6'and'IL_10),'using'commercial'sandwich'immunoassay'kits'(Biolegend'and'BD,' San'Diego,'CA).'Also,'BMMø'were'recovered'at'24'hours'post_infection'for'surface'co_ stimulatory'markers'analysis.'Thus,'BMMø'were'stained'with'CD40_PE'and'MHCII_APC' at'4oC,'during'30'minutes'in'the'dark.'The'cells'were'then'washed'in'PBS'and'suspended' in' 200' µl' of' PBS_2%' FBS.' Data' were' collected' by' a' BD' FACScalibur' cytometer' and' analyzed'by'FlowJo'software.' ! ' Animal'experiments'and'parasite'quantification' Promastigotes'recovered'from'stationary'culture'with'4,'21'and'31'in!vitro!passages' stationary_culture'were'collected,'washed'and'suspended'in'sterile'PBS.'A'volume'of'200' µl'of'PBS'containing'108'parasites'was'injected'intraperitoneally.'Mice'of'each'group'were' sacrificed' at' 56' days' post_infection.' The' parasite' burden' in' the' spleen' and' liver' was' determined'by'limiting'dilution'as'previously'described'(Silvestre'et'al.,'2007).' ! ! Statistical'analysis' The' data' was' analyzed' using' the' non_parametric' Kruskal_Wallis' test' followed' by' Dunn'posttest'for'multiple'comparisons'when'necessary.' CHAPTER'.....................................................'II' 79" " Results! ! The'maintenance'of'L.!infantum'promastigotes'in'axenic'cultures'results'in'diminished' virulence'' ' We'started'by'clarifying'our'in!vitro!model'of'L.!infantum!infection'in'relation'to'the' parasite'development'stage.'L.!infantum!parasites'recovered'from'the'spleen'of'infected' Balb/c'mice'were'used'to'start'axenic'cultures'at'a'106'parasites/ml.'The'first'task'was'to' clearly' define' the' culture' time' frame' in' which' we' can' recover' stationary' parasites.' Performing'basic'cell'cycle'analysis'we'excluded'the'use'of'the'parasites'until'2'days'of' culture'because'there'was'still'significant'active'division'(Fig.'S1A).'In'order'to'evaluate' the'infectivity'of'stationary'L.!infantum,'we'used'CFSE_labeled'stationary'promastigotes' recovered'at'3,'5'and'9'days'of'in!vitro!growth'and'BMMø'as'infection'cellular'target.'Our' data'demonstrate'that'3rd'culture_day'L.!infantum'promastigotes'were'significantly'less' infectious'when'compared'with'the'5th'and'9th'days'of'culture'(Fig.'S1B).'These'differences' were'already'observed'at'4'hours'post_infection'indicating'a'deficient'parasite'uptake'with' 3rd'culture_day'L.!infantum'promastigotes.'Intraphagolysosomal'adaptation'mechanisms' do' not' appear' to' be' involved' in' the' infection' differences' since' similar' infection' percentages'reductions'were'found'between'4'and'24'hours'(15.6'±'0.4'for'3rd'culture_day;' 15.0'±'1.2'for'5th'culture_day;'18.1'±'1.1'for'9th'culture_day,'when'comparing'4'with'24' hours'post_infection).'Many'reports'now'relate'virulence'with'parasites'culture'viability' (Wanderley'et'al.,'2006;'Wanderley'et'al.,'2009).'In'order'to'lay'down'the'hypothesis'that' differences' in' infectivity' could' be' attributed' to' non_viable' parasites,' we' evaluated' the' percentages' of' apoptotic' or' necrotic' parasites' by' AnnV/7AAD' labeling' (El_Fadili' et' al.,' 2010).'Nevertheless,'no'significant'differences'were'found'between'all'culture'days'(data' not'shown).' Several'groups'have'already'reported'that'long_term'in!vitro'cultivation'(more'than'12' months)'of'Leishmania!spp.'leads'to'a'totally'avirulent'promastigote'population'(Grimm' et'al.,'1991;'Segovia'et'al.,'1992).'According'to'these'findings,'we'decided'to'evaluate'if'the' sustained' maintenance' of' L.! infantum' promastigotes' in' axenic' culture' at' shorter' time' periods' lead' to' distinct' BMMø' in! vitro! infection' rates' with' distinctive' immunologic' phenotypes.' In' order' to' accomplish' this,' we' maintained' L.! infantum! promastigotes' recovered'from'the'spleen'of'infected'mice'for'4,'21'and'31'passages,'which'are'equivalent' to'20,'105'and'155'division'events,'considering'simple'exponential'growth.'The'long_term' maintenance'of'L.!infantum!in'culture'did'not'modify'the'promastigote'growth'behavior' (Fig.'1A)'neither'their'viability'that'was'always'superior'to'90%'(data'not'shown).'Taking' into'account'the'distinct'infection'profiles'depicted'in'Fig.'S1B,'we'chose'5_day'culture' CHAPTER''.....................................................'II' 80" " promastigotes'to'compare'infectivity.'When'non_purified'parasite'cultures'were'used'to'in! vitro'infect'BMMø,'a'marginal'but'significant'loss'of'infectivity'at'48'hours'for'P21'and' P31'when'compared'to'P4'was'observed'(Fig.'1B).'Metacyclic'forms'have'been'understood' to'be'the'most'infective'parasite'form'(Louassini'et'al.,'1998;'Yao'et'al.,'2008).'Therefore,' we' enriched' the' promastigote' culture' recovered' from' P4,' P21' and' P31' in' metacyclics' recovered' by' Ficoll' density' gradient,' herein' referred' as' Ficoll_purified' promastigotes' (Spath'and'Beverley,'2001),'and'analyzed'their'infectivity'in'primary'BMMø'cells'(Fig.'1C).' When'Ficoll_purified'metacyclic'promastigotes'from'these'cultures'were'used,'differences' were'abrogated'irrespective'of'the'passage'used'(Fig.'1C).'To'confirm'the'enrichment'of' metacyclic'promastigotes'in'this'fraction'and'as'an'internal'control'of'our'experimental' conditions,'we'analyzed'the'expression'of'two'genes,'SHERP'and'histone'H4'that'can'be' used'to'evaluate'metacyclogenesis.'SHERP'gene'is'found'to'be'up_regulated'in'infective' metacyclic'promastigotes'(Knuepfer'et'al.,'2001).'On'the'other'hand,'higher'expression'of' histone'H4'is'associated'with'exponential'phase'promastigotes'(Soto'et'al.,'1997).'Indeed,' the'qPCR'analysis'demonstrated'a'significant'increase'in'the'SHERP'mRNA'transcripts'in' Ficoll_purified' promastigotes' when' compared' with' non_purified' parasite' cultures' (Fig.' S2).' These' results' suggested' that' the' maintenance' of' L.! infantum! in' axenic' cultures' resulted'in'a'virulence'leakage'affecting'their'infectivity'probably'by'the'loss'of'metacyclic' parasites.'Nevertheless,'no'significant'differences'were'found'between'the'percentage'of' Ficoll_purified'promastigote'recovery'from'each'culture'(P4:'6.6'±'0.1%;'P21:'3.4'±'1.6%;' P31:'4.0'±'0.9%).'' ' ' Maintenance' of' long_term' axenic' L.! infantum' cultures' decrease' parasite' capacity' to' modulate'host'cell'functions'in'an'inflammatory'context''' ! We'have'above'demonstrated'that'the'BMMø'infection'by'L.!infantum!promastigotes' depends'not'only'upon'the'days'of'culture'but'is'significantly'modulated'by'their'axenic' culture' period.' However,' we' were' unable' to' detect' any' major' changes' on' macrophage' activation'status'when'submitted'to'L.!infantum!infection'(data'not'shown),'which'can'be' explained'by'the'Leishmania!silent'entry'mechanism'(Silvestre'et'al.,'2009a).'Therefore,' we'hypothesized'that,'when'facing'an'inflammatory'stimulus,'axenic!cultures'with'high' passage' number' should' be' less' successful' in' subverting' macrophage' effector' functions' being' less' capable' of' promoting' infection.' In' order' to' investigate' this' hypothesis,' we' incubated'BMMø'cells'with'Ficoll_purified'or'non_purified'parasites'from'distinct'culture' periods,'which'were'4'hours'later'submitted'to'LPS'stimulation.'As'before,'we'observed'a' decrease'of'the'infection'rate'with'the'augmentation'of'parasite'in!vitro'passages'(Fig.'2A).' CHAPTER'.....................................................'II' 81" " This' difference' was' minimized' if' Ficoll_purified' promastigotes' were' used' instead,' although' it' was' still' statistically' significant' at' 48' hours' post_infection' (Fig.' 2B).' LPS' stimulation' rapidly' induces' a' surface' up_regulation' of' MHCII' molecules' and' co_ stimulatory'marker'CD40.'The'analysis'of'these'markers'demonstrated'that'high'passage' number'parasites'had'lower'capacities'to'counteract'the'LPS'activation'stimulus'(Fig.'2C).' Once'again,'these'differences'were'abrogated'when'metacyclic_enriched'populations'were' used'(Fig.'2C).'We'have'also'evaluated'the'levels'of'secreted'IL_6,'IL_12p40'and'TNF_α) and'of'the'anti_inflammatory'IL_10'cytokine.'We'found'that'the'capacity'to'control'LPS_ induced' cytokines' was' variable' depending' on' the' number' of' parasite' passages,' likely' reflecting'its'distinct'virulence.'While'significant'differences'were'found'with'P31'parasites' in' the' BMMø' secretion' levels' of' IL_6' and' TNF_α) (Fig.) S3A) and) S3B,) respectively),) the) major'modifications'were'observed'at'the'IL_12p40'and'IL_10'levels'(Fig.'S3C'and'S3D,' respectively).' Indeed,' P4' parasites' were' more' capable' to' down_regulate' IL_12p40' secretion' induced' by' LPS' stimulus,' while' increasing' IL_10' cytokine' secretion.' This' demonstrates' that' high' passage' parasites' failed' to' counteract' the' secretion' of' pro_ inflammatory' cytokines' induced' by' LPS' in' a' similar' manner.' Moreover,' if' a' pro_ inflammatory/IL_10' ratio' is' constructed,' a' strong' correlation' was' observed' between' shorter' axenic' culture' maintenance' periods' and' lower' pro_inflammatory/IL_10' ratios' (Table'1).'Since'the'metacyclic'enrichment'diminished'the'differences'observed'in'parasite' infection'rate'and'co_stimulatory'markers'found'with'stationary_phase'promastigotes'in' different'passages,'we'further'investigated'whether'the'cytokine'bias'was'similarly'altered.' Indeed,' the' use' of' Ficoll_purified' metacyclic' enriched' promastigotes,' whatever' their' source,'shifted'the'cytokine'environment'towards'an'anti_inflammatory'ratio.'Although' some' statistical' differences' were' found' between' Ficoll_purified' promastigotes' from' different' passages,' all' displayed' lower' pro_inflammatory/IL_10' ratios' when' compared' with' LPS' stimulation' (Table' 1).' Overall,' these' data' suggests' that' when' Leishmania_ infected'BMMø'are'faced'with'an'inflammatory'stimulus,'there'is'a'specific'overall'loss'of' modulatory'capacity'that'seems'to'be'related'to'the'highly'immunoregulatory'population' of'metacyclic'parasites.' '' ' Sustained'culture'of'L.!infantum'promastigotes'results'in'an'in!vivo'loss'of'virulence' ' We'have'above'demonstrated'that'sustained'axenic'parasite'culture'results'in'a'rapid' loss'of'in!vitro!virulence.'Previous'reports'demonstrating'an'in!vivo!loss'of'virulence'were' based'on'long_term,'usually'more'than'1'year,'parasite'maintenance'(Grimm'et'al.,'1991;' Segovia' et' al.,' 1992).' However,' we' have' observed' a' clear' in! vitro! defect' after' only' 21' CHAPTER''.....................................................'II' 82" " passages.'Therefore,'we'decided'to'validate'the'observed'phenotype'by'performing'in!vivo! infections' using' the' susceptible' Balb/c' mice' model.' Six' weeks' after' the' infectious' challenge'with'non_purified'stationary_phase'promastigotes'recovered'from'P4,'P21'or'P31' cultures,'a'significant'difference'was'found'between'P4'and'high'passage'number'parasite' infections'in'the'liver'(Fig.'3A).'Similarly,'we'have'observed'a'significant'lower'parasite' burden' in' the' spleen' of' P31' infected' mice' that' P4' infections' (Fig.' 3B).' These' results' confirm'the'observed'in!vitro'loss'of'virulence'with'parasite'culture'maintenance.'' ' ' Loss' of' virulence' originates' from' inadequate' capacity' to' differentiate' into' amastigote' forms' ! To'elucidate'the'biological'mechanisms'that'account'for'the'loss'of'virulence'due'to' long' term' parasite' culture,' we' started' by' hypothesizing' two' major' potential' reasons:' decrease' number' of' metacyclic' promastigotes' or' inadequate' differentiation' into' amastigote'forms.'The'quantification'of'Ficoll_purified'promastigotes'described'above'did' not' show' any' significant' differences' among' the' passages' suggesting' similar' metacyclic' quantities.'However,'the'Ficoll'density'gradient'assay'is'not'a'specific'and'sensible'test'for' the' quantification' of' metacyclic' promastigotes' in' a' culture' but' rather' a' method' for' its' enrichment.' Thus,' to' evaluate' the' hypothetical' deficit' on' the' generation' of' metacyclic' promastigotes,'we'have'performed'in!vitro!infections'using'BMMø'as'targets,'where'we' substitute'5%'or'10%'of'non_purified'stationary_phase'promastigotes'from'each'passage' with' similar'percentages' of' Ficoll_purified' fractions'of' P4' cultures' to' increase'the' total' percentage' of' metacyclic' promastigote.' As' a' positive' control,' we' used' a' naturally' attenuated'L.!infantum!from'which'we'were'unable'to'recover'metacyclic'promastigotes.' Indeed,'although'the'promastigotes'of'this'L.!infantum'strain'presents'a'similar'axenic' growth' curve' (Fig.' S4),' we' were' always' unable' to' recover' by' Ficoll' density' gradient' relevant' number' of' promastigotes' (lower' that' 0.1%' of' initial' culture)' from' stationary_ phase'cultures.'The'quantification'of'CFSE_positive'BMMø'demonstrated'that'increasing' the'percentage'of'Ficoll_purified'promastigotes'did'not'significantly'enhance,'at'any'time' point,'the'percentage'of'infected'BMMø'for'P4'(Fig.'4A),'P21'(Fig.'4B)'or'P31'(Fig.'4C)' promastigotes.'However,'the'opposite'was'observed'with'the'naturally'attenuated'strain,' where' a' significant' increase' of' infected' macrophages' was' observed' at' 48' hours' post_ infection'(Fig.'4D).'These'results'demonstrated'that'the'lack'of'virulence'originated'from' sustained' parasite' culture' cannot' be' reverted' by' the' addition' of' enriched_metacyclic' fractions.'This'excludes'a'defect'in'the'capacity'to'generate'metacyclic'promastigotes'as' the' inherent' biological' cause' for' the' virulence' loss.' Therefore,' we' investigated' the' CHAPTER'.....................................................'II' 83" " potential'role'of'inadequate'capacity'to'differentiate'in'the'amastigote'form.'CFSE_labeled' stationary_phase' promastigotes' recovered' from' each' passage' were' placed' on' MAA20' (Sereno'and'Lemesre,'1997)'and'followed'for'three'days.'We'evaluated'the'promastigote' differentiation'by'light'microscopy,'axenic'amastigotes'proliferation'by'CFSE'labeling'and' overall'viability'by'PI'staining.'All'cultures'presented'axenic'amastigotes_like'cells'after'3' days'of'differentiation'(data'not'shown).'However,'high'passage'number'promastigotes' displayed'a'striking'decrease'of'differentiated'cells.'To'quantify'these'differences,'we'have' assessed' the' progressive' diminution' in' the' intensity' of' CFSE' staining' after' the' differentiation' process.' In' fact,' while' P4' promastigotes' progressively' diminished' CFSE' fluorescence'(Fig.'5A'and'B),'high'passage'number'promastigotes'exhibit'a'severe'defect' to'proliferate'as'amastigotes'forms,'as'observed'in'both'histogram'curves'(Fig.'5A)'and' quantification'of'mean'fluorescence'CFSE'intensity'(Fig.'5B).'Moreover,'this'defect'was' not'correlated'with'a'difference'on'cell'death,'since'similar'percentages'of'viable'parasites' were'found'for'all'cultures'during'the'differentiation'process'(Fig.'5C).'Interestingly,'the' naturally'attenuated'strain'did' not'display'any'significant'change'to'differentiate'when' compared'with'P4'promastigotes,'suggesting'a'distinct'mechanism'of'loss'of'virulence'that' is'not'related'with'the'capacity'to'generate'axenic'amastigotes.'' ' ' L.!infantum'virulence'is'restored'after'full'differentiation'to'amastigote'forms! ! !It' is' a' current' empirical' methodology' to' pass' Leishmania! spp.' promastigotes' in' experimental'models'to'maintain'virulence.'We'have'hypothesized'that'the'differentiation' process'from'promastigotes'to'amastigotes'forms'would'select'the'most'virulent'parasites' in'a'heterogeneous'culture'assuring'the'continuity'of'competent'and'adapted'parasites.' Therefore,' to' explore' this' assumption' we' have' differentiated' promastigotes' from' each' passage'number'in'amastigotes'both'in'axenic'and'in!vivo!conditions.'Axenic'amastigotes' were'obtained'by'differentiating'promastigotes'in'MAA20'medium'(Sereno'and'Lemesre,' 1997)'for'a'period'of'3'days.'The'viable'axenic'amastigotes'were'maintained'axenically'in' culture' for' 10' days,' after' which' were' re_differentiated' to' promastigotes' forms.' In' alternative,'we'recovered'L.!infantum!parasites'from'the'spleen'of'infected'Balb/c'mice'by' allowing' amastigote' to' promastigote' differentiation' for' a' period' of' 7' days.' All' these' promastigotes'were'sub_cultured'for'4'passages'and'used'to'infect'BMMø.'Remarkably,'we' did'not'observe'any'difference'between'infections'whatever'the'initial'parasite'source'used' (Fig.'6A'and'B).'Again,'we'used'as'a'control'the'naturally'attenuated'L.!infantum!strain.' Although'an'increase'of'virulence'was'observed'after'the'differentiation'protocol'(Fig.'6A),' when' compared' to' non_differentiated'parasites' (Fig.' 4D),' we' observed' a' general' lower' CHAPTER''.....................................................'II' 84" " infection'percentage'with'the'exception'of'4'hours'post_infection.'Overall,'these'results' demonstrate'that'the'defect'on'virulence'due'to'sustained'parasite'maintenance'can'be' recovered' either' by' in! vitro! or' in! vivo! full' differentiation' to' amastigote' and' back' to' promastigotes'forms.'' CHAPTER'.....................................................'II' 85" " Discussion! ! Visceral' Leishmania' infections' studies' have' been' the' center' of' some' controversy' which'can'occasionally'be'traced'back'to'the'use'of'distinct'in!vitro'promastigotes'culture' conditions.'The'plasticity'of'the'Leishmania!genome'(Bastien'et'al.,'1992a)'is'an'important' variable'to'consider'when'axenic'promastigotes'are'used'for'in!vitro!or'in!vivo!studies.' Thus,' parasite' phenotypic' plasticity' allows' it' to' adapt' to' the' environment' generating' discrepancies' between' studies' in' different' laboratories' even' when' using' the' same' Leishmania!strain.' In' the' current' work,' we' have' started' by' investigating' in' our' in! vitro! model' of' L.! infantum!infection'the'relation'between'the'parasite'development'stage'and'its'infectivity.' The' first' step' was' to' discard' logarithmic' parasites' because' they' are' not' ultimately' responsible' for' the' infection' (Sacks' and' Perkins,' 1985),' so' we' used' a' basic' cell' cycle' analysis'to'discard'multiplying'parasites'(less'than'10%'of'total'population'are'in'S/G2' phase'after'the'third'day'of'culture).'This'data'correlated'clearly'with'basic'morphological' visualization.' Stationary' L.! infantum! cultures' in' day' 5' and' 9' induced' higher' BMMø' infection'rates'than'day'3'parasites.'This'difference'in'infectivity'might'translate'the'time' frame'required'for'becoming'truly'metacyclic'parasites'(Sadlova'et'al.,'2010),'which'was' also'corroborated'by'the'less'amount'of'metacyclic'recovered'(data'not'shown).'Since'the' presence' of' apoptotic' parasites' is' essential' for' a' virulent' inoculum' of' Leishmania! promastigotes'(van'Zandbergen'et' al.,'2006),'we'decided' to'quantify'the'percentage'of' apoptotic'and'dead'parasites'in'each'case'to'remove'this'possible'bias'from'our'analysis.' In' fact,' for' all' time' frames' tested,' the' differences' in' infectivity' were' not' related' to' apoptotic'or'dead'parasites'in'the'non_purified'or'Ficoll_purified'populations,'with'culture' viability'always'higher'than'90%.'' Some' authors' described' that' in! vitro' maintenance' for' long' periods' constitute' an' important'factor'for'the'loss'of'virulence'in'L.!infantum'(Grimm'et'al.,'1991)'and'L.!major! (Segovia' et' al.,' 1992)' promastigotes.' Nonetheless,' this' loss' of' virulence' is' a' reversible' phenomenon,' since' serial' passages' on' susceptible' mice' allow' the' parasite' to' recover' a' virulence' phenotype' (Katakura' and' Kobayashi,' 1985).' In' the' present' study,' we' complemented'the'previous'observations'by'comparing'the'impact'of'continuous'in!vitro! culture' on' Leishmania! promastigote!virulence' and' also' into' the' capacity' of' host' macrophage'manipulation.'Our'data'clearly'demonstrated'a'loss'of'L.!infantum'virulence' related' to' the' augmentation' of' in! vitro' culture' periods' although' no' modification' was' observed'in'the'axenic'promastigote'growth'behavior.'This'significant'loss'of'infectivity' was'observed'as'soon'as'105'days'of'successive'(21'passages)'culture'and'worsened'with' parasite'maintenance'in'culture.'In'fact,'20'passages'was'the'soonest'time'point'where'we' CHAPTER''.....................................................'II' 92" " 37.'Barak'E,'Amin_Spector'S,'Gerliak'E,'Goyard'S,'Holland'N,'et'al.'(2005)'Differentiation' of' Leishmania! donovani' in' host_free' system:' analysis' of' signal' perception' and' response.'Mol'Biochem'Parasitol'141:'99_108.' 38.' Goyard' S,' Segawa' H,' Gordon'J,' Showalter' M,' Duncan' R,' et' al.' (2003)' An' in' vitro' system' for' developmental' and' genetic' studies' of' Leishmania! donovani! phosphoglycans.'Mol'Biochem'Parasitol'130:'31_42.' 39.'Debrabant'A,'Joshi'MB,'Pimenta'PF,'Dwyer'DM'(2004)'Generation'of' Leishmania! donovani' axenic' amastigotes:' their' growth' and' biological' characteristics.' Int' J' Parasitol'34:'205_217.' 40.' Saar' Y,' Ransford' A,' Waldman' E,' Mazareb' S,' Amin_Spector' S,' et' al.' (1998)' Characterization'of'developmentally_regulated'activities'in'axenic'amastigotes'of' Leishmania!donovani.'Mol'Biochem'Parasitol'95:'9_20.' ' ' CHAPTER'.....................................................'II' 93" " Figure!1.!LongPterm!in!vitro!maintenance!of!L.!infantum!promastigotes!does! not! alter! parasite! growth.'L.! infantum! growth' curves'were' performed' by' neubauer' chamber' counting' (A).' BMMø' were' infected!at' a' 1:10' (cell/parasite)' ratio' with' non_ purified'(B)'and'Ficoll_purified'(C)'promastigotes'labeled'with'CFSE.'Data'were'acquired' by' FACScalibur' cytometer' and' analyzed' by' FlowJo' software.' Three' independent' experiments' were' performed;' one' representative' experiment' is' shown.' The' mean' and' standard'deviation'are'shown.'*P!<'0,05,'**P!<'0,01,'***P!<'0,001'statistical'significance' relatively'to'P4.'! ! Figure! 2.! LongPterm! in! vitro! maintenance! of! L.! infantum! promastigotes! results! in! loss! of! virulence! in! vitro.' BMMø' were' submitted' to' LPS' stimulation' 4' hours' after' infection' with' non_purified' (A)' or' Ficoll_purified' (B)' CFSE_labeled' promastigotes' at' a' 1:10' (cell/parasite)' ratio.' The' percentage' of' infected' cells' was' determined'by'the'number'of'CFSE_positive'cells'in'a'FACScalibur'cytometer.'Expression' of'MHCII'and'co_stimulatory'molecule'CD40'at'24'hours'post_infection.'Thick'line'–'P31,' dotted' line' _' P4,' thin' line' _' LPS' and' shadded' hystogram' _' isotype' control' (C).' Two' independent'experiments'were'performed;'one'representative'experiment'is'shown.'The' mean' and' standard' deviation' are' shown.' *P! <' 0,05;' **P! <' 0,01' statistical' significance' relatively'to'P4.'! ! Figure! 3.! LongPterm! in! vitro! maintenance! of! L.! infantum! promastigotes! results!in!!loss!of!virulence!in!vivo.'Balb/c'mice'were'infected'with'stationary'phase' promastigotes'submitted'to'4,'21'or'31'successive'in!vitro!passages.'After'6'weeks'post_ infection,'the'parasite'load'was'determined'in'liver'(A)'and'spleen'(B)'by'limiting'dilution.' The'mean'and'standard'deviation'are'shown.'*P!<'0,05;'**P!<'0,01;'***P!<'0,001' ! Figure! 4.! Decreased! capacity! to! infect! is! not! correlated! with! a! loss! of! metacyclic! promastigotes.' BMMø' were' infected!at' a' 1:10' (cell/parasite)' ratio' with' non_purified' promastigotes' submitted' to' 4' (A),' 21' (B)' or' 31' (C)' successive' in! vitro! passages' with' 5%' or' 10%' of' Ficoll_purified' parasites' or' without' (Mock).' As' a' control,' BMMø'were'infected'with'a'naturally'attenuated'strain'in'the'same'conditions'(D).'Data' were' acquired' by' FACScalibur' cytometer' and' analyzed' by' FlowJo' software.' Two' independent'experiments'were'performed;'one'representative'experiment'is'shown.'The' mean'and'standard'deviation'are'shown.'**P!<'0,01'' ' CHAPTER''.....................................................'II' 94" " Figure! 5.! Diminished! capacity! of! longPterm! cultured! L.! infantum! promastigotes! to! differentiate! and! proliferate! as! axenic! amastigotes.' CFSE_ labeled'non_purified'promastigotes'submitted'to'4,'21'or'31'successive'in!vitro!passages'or' from'a'field'recovered'naturally'attenuated'strain'(Aten)'were'cultured'in'MAA20'for'3' days'to'induce'differentiation'into'the'amastigote'form.'(A)'Parasite'multiplication'was' followed'by'FACScalibur'quantification'of'CFSE'fluorescence.'(B)'For'each'time,'the'mean' fluorescence'intensity'(MFI)'was'calculated.'The'mean'and'standard'deviation'are'shown.' **P! <' 0,01;' ***P! <' 0,001' (C)' At' the' same' time,' parasite' viability' was' followed' by' propidium'iodide'(PI)'incorporation.'' ! ! Figure! 6.! Virulence! is! recovered! after! in! vitro! or! in! vivo! promastigoteP amastigote! differentiation! process.' BMMø' were' infected!at' a' 1:10' (cell/parasite)' ratio'with'non_purified'promastigotes'differentiated'from'in!vitro!axenic'amastigotes'(A)' or'exQvivo'intracellular'amastigotes'(B).'As'a'control,'a'naturally'attenuated'strain'(Aten)' was'submitted'to'the'same'experimental'conditions.'Data'were'acquired'by'FACScalibur' cytometer' and' analyzed' by' FlowJo' software.' Three' independent' experiments' were' performed;'one'representative'experiment'is'shown.'The'mean'and'standard'deviation'are' shown.'*P!<'0,05;'**P!<'0,01.' ! Figure!S1.!Cell!cycle!analysis!and!in!vitro!virulence!of!L.!infantum!recovered! in!distinct!culture!days.'L.!infantum!promastigotes'were'cultured'at'a'106/ml.'Each' day,'2x106' promastigotes'were'recovered'and'the'cell'cycle'analyzed'by'PI'staining'(A).' BMMø'were'incubated'with'non_purified'CFSE'labeled'L.! infantum!promastigotes'at'a' ratio'of'1:10'(cell/parasite).'The'percentage'of'infected'cells'was'obtained'by'quantifying' the' number' of' CFSE_positive' cells' (B).' Data' were' acquired' at' 4' and' 24' hours' post_ infection' in' a' FACScalibur' cytometer' and' analysed' by' FlowJo' software.' Three' independent'experiments'were'performed;'one'representative'experiment'is'shown.'The' mean' and' standard' deviation' are' shown.' *P! <' 0,05,' **P! <' 0,01' statistical' significance' relatively'to'3rd'day'of'parasite'growth.! ! Figure! S2.! Indirect! quantification! of! metacyclic! promastigotes! in! heterogenous! and! FicollPpurified! cultures! by! gene! transcription! analysis.' Transcription'profile'of'SHERP'and'Histone'H4'genes'obtained'by'qPCR,'for'non_purified' and'Ficoll_purified'promastigotes.'Normalizations'were'made'against'the'reference'gene' rRNA45.'Three'independent'experiments'were'performed,'each'performed'in'duplicate;' CHAPTER'.....................................................'II' 95" " one'representative'experiment'is'shown.'The'mean'and'standard'deviation'are'shown.'*P! <'0,05.' ! Figure! S3.! LongPterm! cultured! L.! infantum! promastigotes! show! decrease! capacity!to!modulate!an!inflammatory!stimulus!in!vitro.'BMMø'were'submitted' to'LPS'stimulation'4'hours'after'infection'with'non_purified'promastigotes.'The'levels'of' IL_6'(A),'TNF_α)(B),'IL_12p40'(C)'and'IL_10'(D)'were'quantified'24'hours'post_infection' on'BMMø'supernatants'by'ELISA.'Three'independent'experiments'were'performed;'one' representative'experiment'is'shown.'The'mean'and'standard'deviation'are'shown.'*P!<' 0,05;'**P!<'0,01;'***P!<'0,001''statistical'differences'relative'to'LPS''unless'depicted'by'a' bar.'' ! Figure! S4.! Naturally! attenuated! L.! infantum! strain! has! a! similar! axenic! growth!in!comparison!to!WT!strain.!L.!infantum'growth'curves'were'performed'by' neubauer'chamber'counting.'The'mean'and'standard'deviation'are'shown.! !' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' CHAPTER''.....................................................'II' 96" " Figure!1! ' CHAPTER'.....................................................'II' 97" " Figure!2! ' ' ' CHAPTER''.....................................................'II' 98" " Figure!3! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' CHAPTER'.....................................................'II' 99" " ' Figure!4! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' CHAPTER''.....................................................'II' 100" " Figure!5! ' CHAPTER'.....................................................'II' 101" " Figure!6! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' CHAPTER''.....................................................'II' 108" " ' ' ' CHAPTER'.....................................................'II' 109" " 3.!Article!name:!The!development!of!a!semiPdefined!media!for!growth!of! Leishmania!infantum! ' Nuno'Santarém,'Ricardo'Silvestre,'Diana'Moreira,'Marc'Ouellette'&'Anabela'Cordeiro_da_ Silva' ' In'the'present'study'we'developed'a'protein'free'medium'for'the'cultivation'of'L.!infantum! ' Main!Results:! _The'low'molecular'weight'components'of'FCS'components'could'be'substituted'by'the' SDM'base.' _The'parasites'grown'in'cRPMI'were'able'to'sustain'the'growth'of'L.!infantum'and'retain' similar' characteristics' to' standard' RPMI' (morphology,' cell' cycle,' viability,' metacyclogenesis).' _The' parasites' grown' in' cRPMI' were' able' to' infect' to' the' same' extent' as' parasites' in' standard'medium'both'in!vitro!and'in!vivo.' ' Conclusions:' The'medium'cRPMI'is'a'viable'alternative'to'grow'L.!infantum'retaining'basic'biological' characteristics'like'morphology'and'virulence' ' ! ! ! ! ! ! CHAPTER''.....................................................'II' 110" " Article!title:! The'development'of'a'semi_defined'media'for'growth'of'Leishmania'infantum' ' Authors:! Nuno' Santaréma,b,' Ricardo' Silvestrea,' Diana' Moreiraa,' Marc' OuelletteC' &' Anabela' Cordeiro_da_Silvaa,b' ' a'Parasite'Disease'Group,'IBMC'_'Instituto'de'Biologia'Molecular'e'Celular,'Universidade' do'Porto,'Portugal' b'Centre'de'Recherche'en'Infectiologie'du'Centre'de'Recherche'du'CHUL,'Quebec,'Canada' c'Departamento de Ciências Biológicas,'Faculdade'de'Farmácia,'Universidade'do'Porto,' Portugal' ' ' Corresponding'author:'' Nuno'Santarém,'Parasite'Disease'Group,'Biology'of'Infection'and'Immunology,'Instituto' de'Biologia'Molecular'e' Celular'da' Universidade'do'Porto,'Rua'do'Campo'Alegre,'823,' 4150_180'Porto,'Portugal' ' ' ' Keywords:!Leishmania!infantum;'Axenic'in!vitro!cultures;'Virulence'loss,'Culture'Media! ' ' ' ' ' CHAPTER'.....................................................'II' 111" " Abstract! The'elimination'of'serum'or'of'serum_derived'macromolecules'from'Leishmania!culture' media' could' decrease' culture' variability' and' enable' the' development' of' experimental' approaches'that'require'the'absence'of'proteins'in'the'culture'media.'We'report'a'defined' medium,'derived'from'RPMI'1640'and'SDM'79'without'serum_derived'proteins'and/or' macromolecules'that'can'be'used'as'a'substitute'of'serum_supplemented'media'for'the' continuous' in! vitro! cultivation' of' promastigote' forms' of' Leishmania.' This' medium,' cRPMI'enables'continuous'subculture'of'Leishmania!maintaining'a'typical'promastigote' morphology,'viability,'cell'cycle'profile,'metacyclic'profile'and'the'capacity'to'differentiate' into'amastigotes.'The'absence'of'serum'and'macromolecules'in'the'culture'medium'did' not'markedly'change'the'in!vitro'infectivity'in'bone'marrow'derived'macrophages'and' their' virulence' in' animals' compared' with' parasites' cultivated' in' standard' nondefined' medium.' Serum' free' culture' will' be' increasingly' important' in' providing' stability' and' reproducibility' in' promastigote' culture' enabling' applications' such' as' exoproteome' recovery'and'metabolic'studies.' ' ' ' CHAPTER''.....................................................'II' 112" " Introduction! Leishmania' spp.' are' parasites' with' a' digenetic' life' cycle.' Extracellular' promastigotes' proliferate' within' the' digestive' tract' of' sandflies' until' a' subsequent'blood' meal,' which' enables'the'transmission'of'the'parasite'to'a'mammalian'host.'In'the'host,'the'parasite' differentiates'into'the'intracellular'amastigote'form'that'thrives'in'the'phagolysosome'of' macrophages'[1,2].'More'than'20'species'of'these'parasitic'protozoa'are'responsible'for'a' group'of'vector'borne'neglected'tropical'diseases'collectively'known'as'leishmaniasis.'The' disease'is'responsible'for'2'million'new'cases'each'year'and'350'million'people'distributed' throughout'88'countries'are'at'risk.'Leishmaniasis' is'characterized'by'different'clinical' manifestations' that' can' range' from' self' healing' cutaneous' presentations' to' a' fatal' visceralizing'ailments'[3].'Moreover,'the'clinical'manifestations'are'often'species'specific' and'influenced'by'the'immune'status'of'the'host,'becoming'a'serious'issue'in'the'case'of' HIV'co_infections'[1].'No'effective'human'vaccine'exists'and'disease'treatment'is'often' toxic,' prone' to' developing' resistance' or' expensive' for' the' majority' of' the' infected' population'[3].'There'is'thus'a'growing'need'for'better'understanding'of'these'protozoa'to' allow'the'development'of'better'and'affordable'therapeutical'approaches.' Most'of'the'available'information'about'Leishmania'spp.'has'been'obtained'from'studying' axenic'promastigotes'cultures.'The'promastigote'forms'can'be'easily'cultivated'in'different' types'of'media'under'laboratory'conditions'[6].'The'unrestricted'use'of'these'media'has'an' inherent' biological' cost.' It' was' described' by' several' authors' that' continuous' in' vitro' growth'of'parasites'induces'virulence'loss'[7].'This'decrease'in'virulence'can'be'due'to' several' factors:' a' growing' incapacity' to' differentiate' into' amastigotes' (Diana' et' al,' submited),' ' or' through' hypothetical' ' adaptations' to' the' medium' that' lead' to' loss' of' infecting'capacity'mediated'by'the'loss'of'virulence'factors'[7].'Conventional'media'are'not' appropriate' for' several' biological' studies' such' as' exoproteome' recovery' or' metabolic' studies' because' most' media' are' complemented' with' fetal' calf' serum' (FCS)' or' other' protein' supplements.' FCS' is' a' complex,' highly' variable,' and' difficult' to' characterize' reagent.'It'is'particularly'expensive'when'used'to'produce'large'culture'of'cells'for'protein' purification.'An'ideal'culture'medium'must'enable'indefinite'parasite'maintenance'upon' subculturing,' retain' the' basic' characteristics' of' promastigotes' (morphology' and' infectivity),' be' affordable,' have' a' simple' preparation' and' ultimately' be' as' defined' as' possible' to' enable' metabolic' studies' [6].' With' these' criteria' in' mind,' we' developed' a' functional' and' inexpensive' medium' based' on' two' distinct' media' routinely' used' for' growing.,'namely'RPMI'and'SDM'79'[8,9].'The'cRPMI'is'a'FCS_free'medium,'or'free'of' proteins'and'peptides'that'readily'supports'the'growth'and'maintenance'of'promastigote' forms' of' various' Leishmania' infantum' strains' and' also' other' species' of' Leishmania.' CHAPTER'.....................................................'II' 113" " Morphologic'and'biologic'properties'of'cRPMI'grown'promastigote'forms'are'presented' and'their'virulence'was'evaluated.' ! ! Matherials!and!methods:! Parasites'and'cell'culture' A'cloned'line'of'virulent'L.'infantum'(MHOM/MA/67/ITMAP_263)'was'grown'at'26°C'in' SDM_79' [9]' supplemented' with' 10%' fetal' calf' serum' –' FCS' _' (Lonza),' 5' μg/ml' hemin' (Sigma),'and'5'μM'biopterin'(Sigma)'at'pH'7.0'_'standard'SDM'_'or'RPMI'1640'medium' (Lonza)' supplemented' with' 10%' FCS,' 2mM' L_glutamine' (Lonza),' 100' U/ml' penicillin' (Lonza),'100'mg/ml'streptomycin'(Lonza)'and'20'mM'HEPES''buffer'(Lonza)'–'standard' RPMI'or'cRPM'(RPMI'base'complemented'with'10%'SDM'base'and'2,5'µg/ml'of'hemin).' To' minimize' the' possibility' of' clonal' bias,' we' have' performed' three' independent' recoveries' of' the' parasite' from' Balb/c' mice' for' these' experiments.' All' cultures' were' initiated'at'either'106'parasites/ml'(standard'RPMI'and'cRPMI)'or'2,5x105'for'standard' SDM'and'passed'every'5'days.'Promastigote'to'amastigote'differentiation'was'achieved'by' culturing' 1x107' stationary' phase' promastigotes/ml' in' a' cell' free' culture' medium' at' an' acidic'pH'and'37ºC'(MAA20)'[10].'' Complementation'experiments' FCS'was'passed'through'a'centriprep'Ultracel'YM_3'filtering'unit'(Millipore)'to'remove' the'bulk'of'the'protein'content'from'the'filtrate.'The'filtrate'was'then'passed'through'the' filtering' device' once' again' and' then' sterilized' by' filtration' through' a' 0,2' µm' filter' (Milipore)'and'stored'at'4ºC,'until'use'(this'fraction'will'be'herein'referred'as'<3'kDa).' The' retentate' was' dialyzed' twice' against' phosphate' buffer' saline' (PBS)' to' completely' remove'the'low'molecular'weight'components'and'stored'at'4ºC'until'use'(this'fraction' will' be' herein' referred' as' >3' kDa).' For' the' complementation' experiments' L.! infantum' promastigotes'growing'in'complete'RPMI'or'SDM79'were'washed'twice'in'PBS'and'used' at' a' density' of' 1x106/ml' in' the' respective' base' medium' without' hemin.' These' were' subcultured'for'2'passages'complemented'with'none'or'one'of'the'following'supplements:' 10%'FCS;'10%'>3kDa;'10%'<3kDa;'2.5'µg/ml'hemin;'5'ng/ml'hemin'and'10%'>3Kda;'5' ng/ml' hemin' and' 10%' <3Kda.' After' two' passages' the' growth' enabling' situations' were' placed'at'a'density'of'1x106/ml'and'evaluated'for'growth'after'4'days'using'a'Neubauer' chamber.' Morphology' of' the' parasites' was' registered' using' phase' contrast' on' a' Nikon' Eclipse'80i.' CHAPTER''.....................................................'II' 114" " Supplementation'experiments' For' the' supplementation' experiments' L.! infantum' promastigotes' growing' in' complete' RPMI'were'washed'twice'in'PBS'and'used'at'a'density'of'1x106/ml'in'the'respective'base' with'2.5'µg/ml'of'hemin.'These'parasites'were'then'complemented'with'one'or'none'of'the' following'SDM'79'supplements:'10%'SDM;'20%'SDM;'50%'SDM'or'100%'SDM.'After'4' days,'growth'was'determined'using'a'Neubauer'chamber.' Growth'curves' Promastigotes' in' cRPMI,' standard' RPMI' or' standard' SDM' growing' for' 24' hours' in' a' startup' culture' with' the' above' defined' starting' inoculums' were' used' as' starter' for' the' growth' curves.' At' defined' time' points' the' parasites' were' counted' using' a' Neubauer' chamber.' Cell'cycle'analysis' Promastigotes,' 2x106,' were' recovered'from' cultures' in' the' required' medium' at' defined' time'points,'washed'twice'and'resuspended'in'1'ml'of'PBS'2%'FCS.'This'was'followed'by' the'addition'of'3'ml'of'cold'absolute'ethanol'(Panreac)'with'continuous'vortexing.'Cells' were'fixed'for'at'least'1'hour'at'4ºC'and'then'washed'twice'in'PBS,'1200'g'for'5'minutes.' Before'analysis,'cells'were'resuspended'in'50'μg/ml'propidium'iodide'(Sigma)'staining' solution'with'0.5'ng/ml'RNase'A'(Sigma)'and'incubated'30'min'at'4ºC.'Data'was'collected' in'a'BD'FACScalibur'cytometer'(20.000'gated'events)'and'analyzed'by'FlowJo'software' (Ashland).' Viability'analysis' Promastigotes'were'recovered'washed'and'resuspended'at'105/ml'in'Annexin'V'binding' buffer' (BD' Pharmingen).' Parasites' were' then' incubated' at' room' temperature' for' 15' minutes'with'AnnexinV_Cy5'(BD'Pharmingen)'and'7_AAD'(Sigma).'Parasites'subjected'to' ultra'violet'light'during'30'minutes'and'kept'in'culture'for'4'hours'were'used'as'a'positive' control.'Data'were'collected'in'a'BD'FACScalibur'cytometer'(20.000'gated'events)'and' analyzed'by'FlowJo'software.' Ficoll'density'purification'assay' Metacyclic' promastigotes' were' purified' after' 3,' 5' or' 9' days' of' culture' or' from' 5_day' cultures'with'4,'and'21'in'vitro'passages'by'Ficoll'density'gradient,'as'previously'described' [11].'Briefly,'6'ml'of'40%'Ficoll'was'overlaid'by'6'ml'of'10%'Ficoll'in'RPMI.'Then,'6'ml'of' media'containing'1.2x109'parasites'was'placed'at'the'top'of'the'Ficoll'gradient.'The'step' CHAPTER'.....................................................'II' 115" " gradient'was'centrifuged'for'10'minutes'at'370g'at'room'temperature'without'brake.'The' metacyclics' promastigotes' were' recovered' from' the' layer' between' 0%' and' 10%' Ficoll' solution.' Metacyclic' promastigotes' were' identified' by' morphological' criteria,' i.e.,' short' and'slender'with'a'long'flagellum'twice'the'body'length'using'phase'contrast'on'a'Nikon' Eclipse'80i'microscope.'' Real'time'RT_PCR'' Total'RNA'was'isolated'from'cells'with'the'Trizol®'reagent'(Invitrogen,'Barcelona,'Spain),' according'to'the'manufacturer's'instructions.'Briefly,'parasites'were'washed'with'ice_cold' phosphate_buffered' saline' (PBS),' harvested' and' homogenized' in' 800' µl' of' Trizol' by' pipetting'vigourously.'After'addition'of'160'μl'of'chloroform,'the'samples'were'vortexed,' incubated'for'2'min'at'room'temperature'and'centrifuged'at'12.000'g,'for'15'min,'at'4ºC.' The'aqueous'phase'containing'RNA'was'transferred'to'a'new'tube'and'RNA'precipitated' with'400'µl'of'isopropanol'for'at'least'10'min'at'room'temperature.'Following'a'10'min' centrifugation' at' 12.000' g,' the' pellet' was' washed' with' 1' ml' of' 75%' ethanol' and' resuspended' in' 10' μl' of' 60ºC' heated' RNase' free' water.' The' RNA' concentration' was' determined'by'using'a'Nanodrop'spectrophotometer'(Wilmington,'DE,'USA)'and'quality' was' inspected' for' absence' of' degradation' or' genomic' DNA' contamination,' using' the' Experion'RNA'StdSens'Chips'in'the'ExperionTM'automated'microfluidic'electrophoresis' system' (BioRad' Hercules,' CA,' USA).' RNA' was' stored' at' _80ºC' until' use.' RT' was' performed' with' equal' amounts' of' total' extracted' RNA' (1' μg)' obtained' from' parasites' recovered'from'different'experimental'conditions'by'using'Superscript'II'RT'(Gibco'BRL)' and'random'primers'(Stratagene).'Real_Time'quantitative'PCR'(qPCR)'reactions'were'run' in'duplicate'for'each'sample'on'a'Bio_Rad'My'Cycler'iQ5'(BioRad,'Hercules,'CA,'USA).' Primers'sequences'were'obtained'from'Stabvida'(Portugal)'and'thoroughly'tested.'qPCR' was'performed'in'a'20'µl'volume'containing'5'µl'of'complementary'cDNA'(50'ng),'10'µl'of' 2x'Syber'Green'Supermix'(BioRad,'Hercules,'CA,'USA),'2'µl'of'each'primer'(250'nM)'and' 1)µl)H2O)PCR)grade.)Specific)primers)for)histone)H4)(forward:)5’)ACACCGAGTATGCG)_3’;) reverse:) 5’_' TAGCCGTAGAGGATG_3’),) Small) Hydrophilic) Endoplasmic) Reticulum_ associated' Protein' (SHERP)) (forward:) 5’) CAATGCGCACAACAAGAT) _3’;) reverse:) 5’_' TACGAGCCGCCGCTTA_3’))and)rRNA45)(forward:)5’CCTACCATGCCGTGTCCTTCTA)_3’;) reverse:) 5’_' AACGACCCCTGCAGCAATAC' _3’)) [12]) were) used) for) amplification.) After) amplification,' a' threshold' was' set' for' each' gene' and' cycle' threshold_values' (Ct_values)' were'calculated'for'all'samples.'Gene'expression'changes'were'analyzed'using'the'built_in' iQ5'Optical'system'software'v2.1'(Bio_Rad'laboratories,'Inc).'The'results'were'normalized' using'as'reference'gene'the'rRNA45'rRNA'sequence'[12].' CHAPTER''.....................................................'II' 116" " Staining'of'parasite'promastigotes'with'CFSE' Stationary_phase' promastigotes' from' each' culture' medium' at' a' concentration' of' 1.2*107/ml' were' used' for' carboxyfluorescein' succinimidyl' ester' (CFSE)' labeling.'' Leishmania'promastigotes'were'washed'twice'with'PBS'and'labeled'with'5µM'of'CFSE'for' 10'minutes'at'37ºC'with'shaking'for'3'minutes.'Then,'9mL'of'complete'DMEM'was'added' and'tubes'centrifuged'for'10'minutes'at'1200g.'The'supernatant'was'discarded'and'the' pellet'was'resuspended'in'5mL'of'complete'DMEM'and'incubated'at'4ºC'for'5'minutes.' Finally,' promastigotes' were' resuspended' in' complete' DMEM' before' proceeding' to' macrophage'infections.' ' In'vitro'macrophage'infection' Cell'suspension'of'bone'marrow'was'obtained'by'flushing'the'femurs'of'susceptible'Balb/c' mice.)The)cell)suspension)was)cultured)in)Dulbecco’s)modified)Eagle’s)medium)(DMEM)) (Lonza,'Switzerland),'supplemented'with'10'%'heat_inactivated'FBS'(Lonza,'Switzerland),' 2' mM' L_glutamine,' 100' U/ml' penistreptomycin' and' 1' mM' sodium' pyruvate.' After' overnight' incubation' at' 37ºC,' non_adherent' cells' were' recovered' (300g' for' 10' min,' at' room' temperature)' and' cultured' in' 24_well' culture' dishes' at' 2x105cells/ml' in' supplemented' DMEM.' For' macrophage' differentiation' 10%' L_929' cell' conditioned' medium' (LCCM)' was' added' at' days' 0' and' 4.' At' day' 7' of' culture,' CFSE' labeled' promastigotes' were' incubated' with' the' adherent' bone_marrow' derived' macrophages' (BMMø)'at'a'10:1'ratio.'After'four'hours,'infection'was'stopped' and'the'infection'rates' were'determined'at'4,'24'and'48'hours'post_infection'by'a'BD'FACScalibur'cytometer'and' analyzed'by'FlowJo'software.'' Animal'experiments'and'parasite'quantification' Ten' to' twelve_week_old' female' Balb/c' mice' were' obtained' from' Instituto' de' Biologia' Molecular' e' Celular' (IBMC;' Porto,' Portugal)' animal' facilities.' Under' laboratory' conditions,'the'animals'were'maintained'in'sterile'cabinets'and'allowed'sterile'food'and' water' ad' libitum.' Animal' care' and' procedures' were' in' accordance' with' institutional' guidelines.' All' experiments' were' approved' by' and' conducted' in' accordance' with' the' IBMC.INEB' Animal' Ethics' Committee' and' the' Portuguese' Veterinary' Director' General' guidelines.'RS'has'an'accreditation'for'animal'research'given'from'Portuguese'Veterinary' Direction' (Ministerial' Directive' 1005/92).' Promastigotes' recovered' from' stationary' culture'with'4'in'vitro'passages'stationary_culture'were'collected,'washed'and'suspended' in' sterile' PBS.' A' volume' of' 200' µl' of' PBS' containing' 108' parasites' was' injected' CHAPTER'.....................................................'II' 117" " intraperitoneally.' Mice' of' each' group' were' sacrificed' at' 56' days' post_infection.' The' parasite'burden'in'the'spleen'and'liver'was'determined'by'limiting'dilution'as'previously' described'[13].' ' Statistical'analysis' The' data'was' analyzed' using' the'non_parametric'Kruskal_Wallis'test' followed' by' Dunn' posttest'for'multiple'comparisons'when'necessary.' ! ! Results:! Development'of'a'FCS'free'medium' ' The'use'of'FCS'as'a'supplement'in'most'standard'media,'makes'them'unfit'for'studies' involving'the'parasite'exoproteome'or'basic'metabolic'pathways.'Therefore,'our'strategy' was'to'create'a'protein'free'medium'that'enabled'the'continuous'growth'of'the'parasites' conserving'basic'biological'characteristics'and'infectivity.' The'first'objective'was'to'determine'the'importance'of'the'10%'FCS'complementation'in' two'different'media'(SDM'and'RPMI).'To'achieve'this'we'used'ultracentrifugation'devices' with' 3kDa' membranes' to' separate' the' FCS' into' two' distinct' fractions:' the' retentate' composed'of'protein'associated'components'with'a'molecular'weight'higher'than'3'kDa' (>3'kDa)'and'the'filtrate'composed'of'components'with'less'than'3'kDa'(<3'kDa).'These' two'distinct'fractions'were'then'used'to'complement'the'defined'SDM'and'RPMI'bases' (bSDM'and'bRMPI).'The'growth'of'parasites'in'these'media'was'evaluated'after'4'days'of' culture'(Fig'1'A_B).'Parasite'growth'was'determined'after'3'passages'in'the'same'medium' to'enable'adaptation'to'the'media'and'evaluate'the'true'capacity'of'sustaining'culture.'All' growth' capable' situations' enabled' subsequent' subpassages' (data' not' shown).' Without' hemin'complementation,'bSDM'and'bRPMI,'were'not'able'to'promote'growth'even'when' complemented'with'<3kDa.'This'lack'of'growth'was'shown'to'be'partially'reverted'by'the' addition' of' hemin.' The' concentration' of'hemin' was' adjusted' to' 2,5' ug/ml,' since' lower' amounts' were'growth' limiting'(data' not' shown).' The'bSDM' when' complemented' with' hemin' presented' 60%' of' normal' growth' while' bRPMI' did' not'grow' significantly' when' complemented' with' hemin' though' the' parasites' were' alive' and' presented' normal' morphology.'The'complementation'of'bSDM'or'bRPMI'with'>3kDa'enabled'total'recovery' of'growth'for'both'media'(Fig'1A_B).'The'complementation'of'both'bases'with'the'>3kDa' CHAPTER''.....................................................'II' 124" " 14.'Volf'P,'Volfova'V'(2011)'Establishment'and'maintenance'of'sand'fly'colonies.'J'Vector' Ecol'36'Suppl'1:'S1_9.' 15.'Lemesre'JL,'Darcy'F,'Kweider'M,'Capron'A,'Santoro'F'(1988)'Requirements'of'defined' cultivation' conditions' for' standard' growth' of' Leishmania' promastigotes' in' vitro.' Acta' Trop'45:'99_108.' 16.'Sadigursky'M,'Brodskyn'CI'(1986)'A'new'liquid'medium'without'blood'and'serum'for' culture'of'hemoflagellates.'Am'J'Trop'Med'Hyg'35:'942_944.' 17.'Chang'CS,'Chang'KP'(1985)'Heme'requirement'and'acquisition'by'extracellular'and' intracellular'stages'of'Leishmania'mexicana'amazonensis.'Mol'Biochem'Parasitol'16:'267_ 276.' 18.'Carvalho'S,'Cruz'T,'Santarem'N,'Castro'H,'Costa'V,'et'al.'(2009)'Heme'as'a'source'of' iron'to'Leishmania'infantum'amastigotes.'Acta'Trop'109:'131_135.' 19.'Krishnamurthy'G,'Vikram'R,'Singh'SB,'Patel'N,'Agarwal'S,'et'al.'(2005)'Hemoglobin' receptor'in'Leishmania'is'a'hexokinase'located'in'the'flagellar'pocket.'J'Biol'Chem'280:' 5884_5891.' 20.' Srivastava' P,' Sharma' GD,' Kamboj' KK,' Rastogi' AK,' Pandey' VC' (1997)' Heme' metabolism'in'promastigotes'of'Leishmania'donovani.'Mol'Cell'Biochem'171:'65_68.' 21.' Gholamhosseinian' A,' Vassef' A' (1988)' Superiority' of' hemoglobin' to' hemin' for' cultivation'of'Leishmania'tropica'promastigotes'in'serum_free'media.'J'Protozool'35:'446_ 449.' 22.'Azzouz'S,'Maache'M,'Sarciron'ME,'Petavy'AF,'Osuna'A'(2009)'Study'of'the'stress' proteins'secreted'by'Leishmania'donovani'after'treatment'with'edelfosine,'mitelfosine'and' ilmofosine,'and'morphological'alterations'analyzed'by'electronic'microscopy.'Parasite'16:' 215_221.' ' ' ' ' ' CHAPTER'.....................................................'II' 125" " Figures:! Figure!1!–!Complementation!studies!with!bSDM!and!bRPMI! 1A! _' Growth' of' L.' infantum' promastigotes' after' four' days' of' culture' in' bSDM' complemented'with':''no'supplement'(NS);'10%'of'the'lower'fraction'of'the'serum'(<3' kDa);' 10%' of' high' molecular' components' of' serum' (>3kDa);' 10%' of' high' molecular' components'of'serum'with'2.5'µg/ml'hemin'(Hem;'>'3'kDa);'2.5'µg/ml'hemin'(Hem)'and' 10%'of'lower'molecular'components'of'serum'with'2.5'µg/ml'hemin'(Hem;'<3'kDa).'The' number' of' parasites' was' normalized' in' relation' to' standard' SDM.' One' representative' experiment'of'five.' 1B! P'Relative'growth'of'L.'infantum'promastigotes'after'four'days'of'culture'in'bRPMI' complemented'with:' 'no'supplement'(NS);'10%'of' the'lower'fraction'of'the'serum'(<3' kDa);' 10%' of' high' molecular' components' of' serum' (>3kDa);' 10%' of' high' molecular' components'of'serum'with'2.'5'µg/ml'hemin'(Hem;'>'3'kDa);''2.5'µg/ml'hemin'(Hem)' and'10%'of'lower'molecular'components'of'serum'with'2.5'µg/ml'hemin'(Hem;'<3'kDa).' The'number'of'parasites'was'normalized'in'relation'to'standard'RPMI.'One'representative' experiment'of'three.' 1C! P' Growth' of' L.' infantum' promastigotes' after' four' days' of' culture' in' bRPMI' complemented' with' 2.5' µg/ml' of' hemin' and' different' percentages' of' bSDM.' One' representative'experiment'of'five'made.' ' Figure!2!–!Growth!curves!of!L.!infantum!in!the!different!media! Promastigotes' were' cultured' with' an' initial' concentration' of' 1x10' 6' /ml' parasites' in' standard' RPMI' (RPMI)' or' cRMPÌ' (cRPMI)' and' 2,5x105' in' standard'SDM' (SDM).' The' growth' curve' was' done' by' counting' the' parasites' at' different' time' points' in' a' hemocytometer.'One'out'of'at'least'three'independent'experiments'is'shown.' ' Figure!3!–!Dominant!morphology!of!stationary!L.!infantum! Promastigotes' were' cultured' with' an' initial' concentration' of' 1x10' 6' /ml' parasites' in' standard'RPMI'or'cRMPÌ'and'2,5x105'in'standard'SDM'.'After'5'days'the'general'aspect' of'the'cultures'was'registered.'Panel'(A)'SDM;'panel'B'(RPMI);'panel'C'(cRPMI).' ' CHAPTER''.....................................................'II' 126" " Figure!4!–!Viability!and!cell!cycle!analysis! A'–'Promastigotes'viability,'at'the'defined'time'points,'as'defined'by'Annexin'V'(Ann'V)' and' 7_aminoactinomycin' D' (7_AAD)' negative' parasites' growing' in' standard' RPMI' (RPMI),'cRMPÌ'(cRPMI)'or'standard'SDM'(SDM).'The'viability'analysis'was'done'using' FACSCalibur'cytometer'and'analyzed'by'FlowJo'software.'One'out'of'at'three'independent' experiments'is'shown.'' B_D'_'Percentage'of'parasites'in'G1,'G2'or'S'at'the'defined'time'points'in'standard'RPMI' (B),' standard' SDM' (C)' and' cRPMI' (D).' The' cell' cycle' analysis' was' done' using' FACSCalibur'cytometer'and'analyzed'by'FlowJo'software.'One'out'of'at'least'three'' ' Figure!5!P!Indirect!quantification!of!metacyclic!promastigotes!in!the!different! media.! Promastigote'relative'gene'expression'of'SHERP'and'META1'at'the'defined'time'points,'as' determined'qPCR,'when'compared'to'histone'h4.'Basic'normalizations'for'the'three'genes' were' made' using' the' reference' gene' rRNA45.' Three' independent' experiments' were' executed,'each'performed'in'duplicate;'one'representative'experiment'is'shown.' ' Figure!6!–!In!vitro!and!in!vivo!virulence!of!promastigote!growing!in!cRPMI! BMMø' were' infected' at' a' 1:10' (cell/parasite)' ratio' with' non_purified' promastigotes' submitted'to'4'(A)'or'21'(B)'successive'in'vitro'passages'in'cRPMI,'standard'RPMI'and' standard'SDM.''Data'were'acquired'by'FACScalibur'cytometer'and'analyzed'by'FlowJo' software.'The'mean'and'standard'deviation'are'shown.'Three'independent'experiments' were'performed;'one'representative'experiment'is'shown.'Balb/c'mice'were'infected'with' stationary' phase' promastigotes' submitted' to' 4' successive' in' vitro' passages' in' cRPMI,' standard'RPMI'and'standard'SDM.'After'6' weeks'post_infection,'the'parasite'load'was' determined' in' liver' (D)' and' spleen' (C)' by' limiting' dilution.' The' mean' and' standard' deviation'are'shown.'Two'independent'experiments'were'performed;'one'representative' experiment'is'shown.*P'<'0,05;'**P'<'0,01;'***P'<'0,001' ' ' ' CHAPTER'.....................................................'II' 127" " Supplemental!data:! Supplemental! figure! 1! P! Growth! curves! of! L.! infantum! in! cRPMI! after! 20! passages.'' Promastigotes'with'4'or'20'passages'were'cultured'with'an'initial'concentration'of'1x10'6' /ml' parasites'in' standard' cRPMI' (RPMI).' The'growth' curve' was'done'by' counting'the' parasites' at' different' time' points' in' a' hemocytometer.' One' out' of' at' least' three' independent'experiments'is'shown.' ' Supplemental!table!1!–!Composition!and!cost!of!standard!SDM! ' Supplemental!table!2!–!Composition!and!cost!of!standard!RPMI! ' Supplemental!table!3!–!Composition!and!cost!of!cRPMI! ' Supplemental!table!4!–!Comparative!cost!of!the!media!used!in!the!study! ' ' ' CHAPTER''.....................................................'II' 128" " Figure!1! ' ' ' ' CHAPTER'.....................................................'II' 129" " Figure!2! ' ' ' ' ' ' ' ' ' ' CHAPTER''.....................................................'II' 130" " Figure!3! ' ' ' ' ' ' CHAPTER'.....................................................'II' 131" " Figure!4! ' CHAPTER''.....................................................'II' 132" " Figure!5! ' ' ' ' ' ' ' CHAPTER'.....................................................'II' 133" " Figure!6! 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