Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Thermotolerance

of

Histoplasma

capsulatum

at

40

◦

C

predominates

among

clinical

isolates

from

different

Latin

American

regions

Jorge

H.

Sahaza

a,b

_,

_Gabriela

_{Rodríguez-Arellanez}

a

_,

_Cristina

_E.

_Canteros

c

_,

María

del

Rocío

Reyes-Montes

a,∗

_,

_Maria

_Lucia

_Taylor

a,∗

a_{UniversidadNacionalAutónomadeMéxico(UNAM),FacultaddeMedicina,DepartamentodeMicrobiología-Parasitología,Ciudadde} México,Mexico

b_{UnidaddeMicologíaMédicayExperimental,CorporaciónParaInvestigacionesBiológicas,Medellín,Colombia} c_{DepartamentoMicolog´ia,INEI-ANLIS“Dr.CarlosG.Malbrán”,BuenosAires,Argentina}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1August2019 Accepted28December2019 Availableonline25January2020

Keywords: LatinAmerica H.capsulatum Yeasts Thermotolerance Thermosensitivity

a

b

s

t

r

a

c

t

Theyeast phaseof 22 Histoplasmacapsulatum clinicalisolatesfrom Mexico,Argentina, Colombia,andGuatemalaandthreereferencestrains,onefromPanamaandtwofromthe UnitedStatesofAmerica(USA),werescreenedforthermosensitivitycharacteristicsusing differentanalyses.Growthcurvesat0,3,6,12,24,and30hofincubationat37and40◦C,the growthinhibitionpercentageat40◦C,andthedoublingtimeat37and40◦Cweredetermined forallyeastsstudied.Mostoftheisolatesexaminedexhibitedthermotolerantphenotypes at40◦C,whereasathermosensitivephenotypeat40◦CwasonlydetectedintheDowns referencestrainfromtheUSA.Growthinhibitionvalueslowerthan33.8%supportedthe predominanceofthethermotolerantphenotypeat40◦C.Thedoublingtimemeansfound forthedifferentisolateswere5.14h±1.47hat37◦Cand5.55h±1.87hat40◦C.Thisisthe firstreporttounderscorethepredominanceofthermotolerantanddelayeddoublingtime phenotypesinH.capsulatumclinicalisolatesfromdifferentregionsofLatinAmerica.

©2020PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileirade Infectologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The saprobe multicellular mycelial morphotype (M-phase) of the dimorphic fungus Histoplasma capsulatum develops in special environments at 25–28◦C. Aerosolized infective propagulesofthe M-phase,mainlymicroconidia andsmall hyphalfragments,canproducearespiratoryinfectionwhen

∗ _{Correspondingauthors.}

E-mailaddresses:remoa@unam.mx(M.d.Reyes-Montes),emello@unam.mx(M.L.Taylor).

inhaled byhumansandothermammals.Dependingonthe immunocompetenceofthehost,inoculumsize,virulence,and phylogeneticspeciesoftheH.capsulatumisolate,theinfection canleadtosevereillness.1,2

The unicellular yeast morphotype (Y-phase) of this pathogen is related to intracellular parasitism in sus-ceptible hosts (mainly immunocompromised individuals). The dimorphic transition of H. capsulatum is of particular

https://doi.org/10.1016/j.bjid.2019.12.007

1413-8670/©2020PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileiradeInfectologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

interestbecauseitisnecessaryforthemanifestationoffungal virulence.3_{Underlaboratoryconditions,theM-toY-transition} canbereversiblyinducedbytemperatureswitchesfrom25to 28◦C(M-phase)to35–37◦C(Y-phase).

Critical studies about H. capsulatum phenotypes have focused on its morphology,4,5 _{thermosensitivity,}6 _and bio-chemical properties.7–9 _{Gass & Kobayashi}10 _{described an} atypical H. capsulatumstrain, namedDowns, isolated from anelderlypatientwithdisseminatedhistoplasmosis,which showedthermosensitive phenotypeat40◦C andavirulence inmice. Spitzer et al.11 _{reported that fungal isolates from} patientswithAids-associatedhistoplasmosisfromSt.Louis, Missouri,UnitedStatesofAmerica(USA),sharedthe same thermosensitivity characteristics ofthe Downs strain. This association was confirmed by molecular methods, which revealed that St. Louis H. capsulatum isolates had a poly-morphicmtDNAprofilesimilartothatoftheDownsstrain. Accordingtothe earlierclassificationofH. capsulatum pro-posedbyVincentetal.,12_{allSt.Louisisolateswereincluded} inasinglegroupnamedClass1.Later,thefungalstrainsfrom Class1werereclassifiedasNorthAmericanClass1(NAm1) phylogeneticspeciesbyKasugaet al.,13 _{andNAm 1strains} havenowbeen renamedbySepúlveda etal.14 _asH.

missis-sippiensesp.nov.Formerly,NAm1strainswereconsideredto havelowvirulence,thoughrecentrecordshavedocumented anotherNAm1straindevelopingavirulentphenotypeunder experimentalassays.2

Overall,mostphenotypicstatementsaboutH.capsulatum haveresultedfromtheuseofalimitednumberofisolatesand consideringasreferencethesameovermanipulatedstrains: Downs(low virulence)10 _{and G-217B (highvirulence)}5 _from theUSAandG-186B(highvirulence)15 _{fromPanama. Given} that H. capsulatum is distributed between the 54◦N16 _and 38◦S17_{latitudes,itsdistinctphenotypesmaybeusedto} dis-criminatebetweenseveralisolatesfromdifferentgeographic distributions,whichwillcontributetotheunderstandingof thispathogendiversity.

Theaimofthepresentstudywastophenotypeclinical iso-latesofH.capsulatumfromdifferentregionsofLatinAmerica consideringtheirY-phasethermosensitivityat40◦C,through thedetectionoftheirgrowthcurve at37 and40◦C,growth inhibitionpercentage(GI%)at40◦C,anddoublingtime(Dt)at 37and40◦C.

Material

and

methods

Histoplasmacapsulatum

Twenty-twoclinicalisolatesfromfourLatinAmerican coun-trieswereprocessed,11fromMexico,fivefromArgentina,five fromColombia,andonefromGuatemala,togetherwiththree clinicalreferencestrains,G-186BfromPanama(ATCC-26030), G-217B(ATCC-26032)andDowns(ATCC-38904)fromtheUSA. Thephylogeneticspeciesandadditionaldetailsforthese clin-icalisolatesandreferencestrainsarelistedinTable1.

All isolates studied were well characterized prior to their deposition in the H. capsulatum Culture Collection of the Fungal Immunology Laboratory of the Department of Microbiology and Parasitology, School of Medicine, UNAM

(LIH-UNAM).H.capsulatumspecimensinthecollectionwere well preservedinsteriledistilledwaterand on Sabouraud-agar withmineraloil,fromthetime oftheirfirst isolation. This collection is registered in the World Data Center on Microorganisms(WDCM)databaseoftheWorldFederationfor CultureCollectionsasWDCM817LIH-UNAMandisavailable athttp://www.wfcc.info/ccinfo/index.php/strain/display/817/ fungi/.

Morphotypetransition

TheM-phaseofeachH.capsulatumisolateandreferencestrain wasinitiallyculturedat25–28◦Conmycobiotic-agar(Bioxon, Becton-Dickinson,MexicoCity,MX).Myceliawereharvested bycentrifugingthecultureat800gfor15minandthenwere growninasyntheticmedium18at37◦Cinanorbitalshaker at200rpm. Thismediumwas replacedevery 72hforeach cultureuntiltheY-transitionwasreachedinallcultures,in approximatelyonetotwoweeks.Oncethedimorphic transi-tionwascomplete,eachyeastculturewasincubatedat37◦C for24–48hinbrain-heartinfusion(BHI)-broth(Bioxon) supple-mentedwith0.1%L-cysteineand1%glucose.Theyeastcells were harvested bycentrifugingat800g for15min, washed twicewithfreshBHI-broth,andstoredat–80◦Cinthe pres-ence offetalcalfserum (GIBCO,GrandIslandBiologicalCo. NY,USA)anddimethylsulfoxideata9:1ratiountilrequired.

Yeast-morphotypethermosensitivity

After gradualdefrosting, the yeastswere grown in supple-mentedBHI-broth.Eachyeastculturewasincubatedat37◦C for 24–48h, which isthe estimated time requiredto reach the logarithmic growth phase (log-phase). Each yeast cul-tureinthelog-phasewastransferredtofreshsupplemented BHI-broth and incubated at37◦C for24h. Theyeastswere centrifuged at800g for15min,andthepelletwasusedfor assays.Toyeastsweresuspendedin10mLofsupplemented BHI-brothtoreachthedesiredinoculumof0.2optical densi-ties(OD).Eachyeastsuspensionwasinitiallydiluted1:10and subsequently seriallydilutedin200␮LofBHI-brothofeach 96-wellmicroplate (Nunc,Roskilde,Denmark). Becausethe firstmicroplatecolumnwasfilledonlywith200␮Lof supple-mented BHI-broth(blank), yeastserialdilutionswere made from the second to the lastcolumns. TheOD valueswere measuredat405nmusingaLabsystemsMultiskanMSreader (Labsystems,Helsinki,Finland),andeachvaluewas automat-icallyadjustedwiththeblank.Toperformthegrowthcurve assay foreach isolateand reference strain, eightwells per microplatecolumnwerefilledwith200␮Lofa0.2ODyeast dilution.Eachgrowthcurvewassetupintriplicateeitherat 37or40◦C.Opticaldensityreadingsweretakenat0,3,6,12, 24,and30hofincubationatbothtemperatures,andtheOD valueswereaveragedusingn=12perisolateorstrainvalue foreachtimetested.TheresultswereplottedasODversus incubationtimesatbothtemperaturestested.

Thegrowthinhibitionpercentage(GI%)at40◦CofeachH. capsulatumyeastculturewasestimatedfromthedataofthe growthcurvebasedonthefollowingequation:GI%=1-(ODtx –ODt0at40◦C/ODtx–ODt0at37◦C)x100,wheretxisthe ODvalueateachtimetestedandt0representstimezero.

Table1–Histoplasmacapsulatumclinicalisolatesandreferencestrainsstudied. Isolate/strain Yearof Isolation Phylogenetic Species/Lineage Geographic origin Histoplasmosis clinicalform Patient immune-compromise EH-316 1993 – MX D HIV+ EH-317 1992 – MX D HIV+ EH-319 1991 – MX D HIV+

EH-323 1993 LAmA MX D HIV+

EH-324 1994 LAmA MX D HIV+

EH-325 1996 – MX D HIV+

EH-326 1996 LAmA MX D HIV+

EH-328 1991 LAmA(LAmA1) MX D HIV+

EH-355 1996 LAmA MX D HIV+

EH-356 1996 LAmA MX D HIV+

EH-359 1995 – MX DML Unknown

DMic993444 1999 LAmB(LAmB1) AR DML Unknown

DMic993445 1999 LAmB(LAmB1) AR DML Unknown

DMic993446 1999 LAmB(LAmB1) AR DML HIV+

DMic993267 1999 LAmB(LAmB1) AR DML HIV+

DMic01739 2001 LAmB AR DML HIV+

LA 1996 LAmA(LAmA2) CO D HIV+

AP – LAmA(LAmA1) CO D Unknown

WE 1996 – CO P HIV+

MZ2 1995 LAmA(LAmA2) CO DML SLE

GeM 2001 LAmA(LAmA1) CO Unknown Unknown

H.1.02.W 1995 LAmA(LAmA2) GT P Unknown

G-186Bd,a _{1967 H81lineage PA P Unknown}

G-217Be,b _{1973 NAm2 USA UnknownUnknown}

Downsf,c _{1969 NAm1 USA DML RA}

AR,Argentina;CO,Colombia;GT,Guatemala;MX,Mexico;PA,Panama;USA,UnitedStatesofAmerica;D,disseminated;DML,disseminated

withmucosallesions;P,pulmonary;HIV,humanimmunodeficiencyvirus;SLE,systemiclupuserythematosus;RA,rheumatoidarthritis.

ThelistedH.capsulatumphylogeneticspecieswerebasedontheclassificationbyKasugaetal.;13_{inparenthesis,thoselatelyrenamedbyTeixeira}

etal.25

a _{H.capsulatumsensustrictoDarling1906(H81Panamalineage).} b _{H.ohiensesp.nov.(NAm2).}

c _{H.mississippiensesp.nov.(NAm1)phylogeneticspeciesbasedontheclassificationbySepúlvedaetal.}14

d_Berliner.15

e _{Berliner&Biundo.}5

f _{Gass&Kobayashi.}10

Thedoubling time(Dt)valuesatboth37and40◦Cwere calculatedaccordingtothefollowingequation:Dt=ODt0/␮, where␮=OD tx onthe slopeofthe growth curve foreach isolateandreferencestrainstudied.19

Statistics

Thermosensitivitydatawerestatisticallyanalyzedusing Stu-dent’s t test (Microsoft Excel 2013 for Windows). For all analyses,means±standarddeviations(SD)weredetermined. Asignificantdifferencewasconsideredwhenp-value≤0.01.

Results

Most of the studied H. capsulatum clinical isolates were obtainedfrom1991to2001andbelongtotheLatinAmerican groupA(LAmA1andLAmA2)andLatinAmericangroupB (LAmB1)phylogeneticspecies(Table1).Thereferencestrains G-186B,G-217B,and Downswere isolated inthe1960sand 1970sandbelongtotheH81Panamalineage,North Ameri-canClass2(NAm2),andNorth AmericanClass1(NAm1) phylogeneticspecies,respectively(Table1).

Of the 25 H. capsulatum yeast culture samples studied, 20 were from patients withthe disseminated clinicalform of histoplasmosis (eight were from patients with mucosal lesions),threederivedfrompatientswithlocalizedpulmonary histoplasmosis,andtheGeMisolatefromColombiaaswellas theG-217BreferencestrainfromtheUSAwerenotassociated withanyspecialdataregardingtheclinicalformofthe respec-tivepatients.Inaddition,15isolatesfromLatinAmerica(10 fromMexico,threefromArgentina,andtwofromColombia) camefrompatientsinfectedwithhumanimmunodeficiency virus,oneisolatewasfromaColombianpatientwithsystemic lupuserythematosus,andtheotherwastheDownsreference strain fromtheUSA originallyisolated froman86-year-old patient with rheumatoid arthritis. Finally, six isolates (one fromMexico,twofromArgentina,twofromColombia,andone fromGuatemala)andtworeferencestrains(G-217-BfromUSA andG-186BfromPanama)werederivedfrompatientswithout anyclinicalrecordssuggestinganimmunosuppressive condi-tion(seeTable1).

Regarding thermosensitivitydetection, theH.capsulatum Y-phase ofeach isolateand referencestrain was analyzed according toits growthcurve at37and 40◦C,GI%at40◦C, andDtat37and40◦C.

1.1

A

C

D

O. D . O. D . O. D . O. D .

B

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1.2 1.1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1 0 3 6 12 0 3 6 Time (h) Time (h) Time (h) Time (h) 12 24 24 24 30 DMic993444 DMic993444 DMic993267 DMic993446 DMic1739 30 AP LA WE MZ 2 GeM 0 0 3 6 12 3 6 12 24 EH-316 EH-319 EH-324 EH-317 EH-323 EH-325 EH-328 EH-356 H.1.02.W EH-326 EH-355 EH-359 30 G-186B G-217B Downs 30 37°C

Fig.1–GrowthcurvesofHistoplasmacapsulatumyeastsat37◦C.TheODat405nmforeachincubationtimewasmeasured usingaMultiskanreader;theyeastgrowthconditionsandgrowthcurveconstructionaredescribedintheMaterialand Methodssection.(A)IsolatesfromArgentina;(B)isolatesfromColombia;(C)isolatesfromMexicoandGuatemala;and(D) referencestrains.Onthebasisofthreeindependentassays(n=12perODvalue),significantdifferencesingrowthcurves wererecordedforp≤0.01alongtheincubationtimepointstested.

1 0.9 0.8 0.7 0.7 0.6 0.5 0.4 0.3 0.1 0 3 6 12 24 30 0 3 6 12 24 30 LA EH-316 G-186B Downs EH-319 EH-324 EH-326 EH-355 EH-359 EH-317 EH-323 EH-325 EH-328 EH-356 H.1.02.w Dmic993444 Dmic993445 Dmic993267 Dmic993446 Dmic1739 AP WE MZ 2 G-217B GeM 1.2 1.1 1 0.9 0.8 0.6 0.5 0.4 0.3 0.2 0.1 0 3 6 12 24 30 0 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 3 6 12 24 30 Time (h) O. D . O. D . O. D . O. D . Time (h) Time (h) Time (h) 0.2

A

B

40°C

D

C

Fig.2–GrowthcurvesofHistoplasmacapsulatumyeastsat40◦C.TheODat405nmforeachincubationtimewasmeasured usingaMultiskanreader;theyeastgrowthconditionsandgrowthcurveconstructionaredescribedintheMaterialand Methodssection.TheisolatesandreferencestrainsanalyzedwerethesameasdescribedinFig.1.

For the growth curve (Fig. 1A–D and Fig. 2A–D), all OD dataat0hofincubation(t0)wererecorded inthe rangeof 0.164±0.002and0.286±0.001at37◦Cand0.129±0.006and 0.288±0.001at40◦C(datanotshown).Thisensuredthatthe initialyeast-populationatt0forallH.capsulatumisolatesand referencestrainsstudiedwereashomogeneousaspossible. TheSDconfirmedtheminimalvariationofreadingswithin thesamepopulation.

Differencesinlag(latency), log (exponential),stationary, anddeclineH.capsulatumgrowthcurvephasesweredetected at37and 40◦Cforthe incubationtimepointsassessed, as showninFig.1A–DandFig.2A–D,respectively.

Mostoftheisolatesandreferencestrainsexhibited simi-largrowthcurvesatboth37(Fig.1A–D)and40◦C(Fig.2A–D). Several isolatesfrom Mexico, Argentina,and Colombia did notexhibitalag-phaseat37◦C(Fig.1A–D),reachingthe log-phasebeforethreehoursandthestationary-phasebetween sixand24h. Incontrast,thelag-phasewas welldefinedin the DMic993444 isolate from Argentina, four isolates from Mexico(EH-317,EH-324,EH-328,andEH-356),andtheDowns strainfromthe USA(Fig. 1A,C,D).TheDMic993444isolate fromArgentinaandtheDownsstrainreachedthelog-phase aftera12hlag-phase(Fig.1A,D).Mostisolatesreachedthe stationary-phase at 30h of culture, without evidence of a declineinthepopulation.

Basedon growth curves at37 and 40◦C (Fig. 1A–D and Fig.2A–D),allisolatesandtworeferencestrains(G-217Band G-186B)were consideredthermotolerant,irrespectiveofthe clinicalformandimmuneconditionofthepatientfromwhom theywereisolated.TheDowns strainwasconsidered ther-mosensitive becauseit didnot growat40◦C,as shown in Fig.2D.Differencesingrowthcurvesat37◦Cbetweenthe ref-erencestrainsG-217B(thermotolerantprototypeat40◦C)and Downs(thermosensitiveprototypeat40◦C)weresignificant (p≤0.01)at6,12,24,and30hofincubation,whereas differ-encesinthegrowthcurvesofthesestrainsat40◦Cwerenot applicableduetolackofgrowth ofthe latterstrainatthis temperature.

Thegrowthcurveat37◦CfortheisolateDMic993444from anArgentineanpatientisnoteworthy,becauseitshowed sig-nificantdifferences(p≤0.01)fromtheG-217Bstrainat6–30h and from the Downs strain at 24 and 30 h of incubation (Fig.1A,D).IncontrasttotheDownsreferencestrain,isolate DMic993444alsoexhibitedgrowthat40◦Cafter24and30hof incubation;thus,itwasconsideredtohaveathermotolerant phenotype.However,thisisolatepresentedsignificant differ-ences(p≤0.01)inODvaluesfromthreeto30hofincubation at40◦ Cwhencomparedwiththethermotolerantprototype G-217Breferencestrain(Fig.2A,D).

TwelveH.capsulatumisolates(threefromMexico,fivefrom Argentina,threefromColombia,andonefromGuatemala) dis-playedGIvaluesfrom15.6to33.8%at30hofincubationat 40◦C (Table2).TheestimationofGI%at40◦Ccorroborated thethermosensitivitycharacteristicsoftheisolatesand refer-encestrainsstudied.WiththeexceptionoftheDownsstrain, athermotolerantphenotypewasconfirmedforalltheH. cap-sulatumisolatesandremainingreferencestrainsbecausethey exhibitedlowGIvalues,below33.8%(Table2).

Table2–Growthinhibitionpercentage(GI%)of

Histoplasmacapsulatumyeastsat40◦C.

Isolate/Strain GI%a 3h 6h 12h 24h 30h EH-316 2.5 0.7 12.7 5.7 15.6 EH-317 0.0 0.0 1.4 5.0 6.2 EH-319 2.8 17.1 11.4 15.7 33.8 EH-323 0.0 0.0 0.0 2.7 0.7 EH-324 0.0 0.0 0.0 5.9 4.3 EH-325 0.0 0.0 0.0 2.8 2.8 EH-326 0.0 0.0 0.0 6.4 5.6 EH-328 0.0 0.0 0.0 4.7 0.9 EH-355 0.0 0.0 0.3 4.8 4.8 EH-356 0.0 0.0 0.0 0.0 0.0 EH-359 0.0 0.7 14.1 31.9 28.7 DMic993444 NA NA NA 16.5 28.6 DMic993445 5.0 0.0 25.7 15.2 24.2 DMic993446 0.0 18.5 28.3 28.8 26.6 DMic993267 3.1 12.3 16.5 19.0 15.9 DMic01739 0.0 0.4 18.0 25.3 21.2 LA 0.0 0.0 10.8 12.5 23.2 AP 0.0 0.0 0.0 6.7 2.2 WE 0.0 0.0 0.0 2.5 1.8 MZ2 0.0 0.0 17.7 25.1 27.9 GeM 0.0 5.1 16.9 18.6 17.7 H.1.02.W 0.0 0.0 6.7 12.4 17.5 G-186B 0.0 0.0 5.7 5.2 1.9 G-217B 0.0 0.0 0.0 8.2 5.3 Downs NA NA NA NA NA

NA,notapplicable.

a _{GI%wasestimatedaccordingtotheequationgivenintheMaterial}

andMethodssection.

TheDtvalues,calculatedatboth37and40◦CforeachH. capsulatumisolateandreferencestrain,variedat37◦Cfrom 1.36h(EH-319,Mexicanisolate)to8.30h(MZ2,Colombian iso-late)withameanof5.14h±1.47h,andat40◦Cfrom1.54h (EH-319,Mexicanisolate)to9.42h(DMic01739,Argentinean isolate)withameanof5.55h±1.87h(Table3).

Two isolates(EH-319andEH-326)fromMexicanpatients withAids-associateddisseminatedhistoplasmosispresented thefastestDt,withvalueslowerthantheaveragesofallDt valuesat37and40◦C.Incontrast,fourisolatesfrompatients withmucosallesions(EH-359fromMexico,DMic993446and DMic01739fromArgentina,andMZ2fromColombia)andone isolateassociatedwithapulmonaryclinicalform(H.1.02.W fromGuatemala)wereconsideredtopresentthemostdelayed Dt,showingDtmeanvalueshigherthanthosefoundat37 and40◦Cformostoftheisolatesandreferencestrains stud-ied(Table3).Notably,isolateDMic993444(Argentina)exhibited DtvaluesclosetotheDtmeansobtainedforH.capsulatumat bothtemperatures(Table3), despiteitsvery longlag-phase (Figs.1Aand2A).TheDtcalculatedfortheDownsstrainat 37◦Cwas amongthelongest valuesobtainedinthis study (Table3);duetoitsthermosensitivephenotypedataat40◦C werenotobtainedfortheDownsstrain.

Ofthe25 isolatesandreferencestrainsofH.capsulatum studied,15isolatesandtworeferencestrainsshowedincrease in Dtfrom 37 to40◦C,six isolatesexhibiteddecreased Dt, andoneisolatemaintainedthesameDtvalueatboth

tem-Table3–Doublingtimes(Dt)ofHistoplasmacapsulatum

yeastsat37and40◦C.

Isolate/Strain Dt(h)a _{ChangesofDtfrom}

37to40◦_C 37◦C 40◦C EH-316 4.36 5.00 Increase EH-317 5.12 6.00 Increase EH-319 1.36 1.54 Increase EH-323 4.06 5.18 Increase EH-324 4.30 4.36 Increase EH-325 5.00 6.24 Increase EH-326 3.18 3.18 NC EH-328 4.42 4.48 Increase EH-355 4.24 5.36 Increase EH-356 5.12 4.48 Decrease EH-359 6.54 8.54 Increase DMic993444 6.06 6.42 Increase DMic993445 5.18 4.54 Decrease DMic993446 6.36 7.24 Increase DMic993267 5.36 7.36 Increase DMic01739 6.42 9.42 Increase LA 5.18 4.42 Decrease AP 4.24 3.18 Decrease WE 5.42 4.24 Decrease MZ2 8.30 7.42 Decrease GeM 5.00 7.06 Increase H.1.02.W 7.24 8.00 Increase G-186B 5.42 5.48 Increase G-217B 3.30 4.24 Increase Downs 7.36 NA NA Meansb _{5.14±1.47 5.55±1.87}

NC,Nochanges;NA,Notapplicable.

a _{Dtestimationwasperformedaccordingtotheequationdescribed}

intheMaterialandMethodssection.

b _{Dataat37and40}◦_{CarepresentedasthemeansofH.capsulatum}

yeastDtvalues±SD,onthebasisofthreeindependentassays,

usingn=12perODvalue.

peratures,whereastheDownsstraindidnotgrowat40◦Cas mentionedabove(seedetailsinTable3).

Discussion

ItisimportanttonotethatallH.capsulatumclinicalisolates selectedforthisstudyhavealimitednumberofsubculturesin suitablemediaaftertheirfirstisolation.Inaddition,because theyweretakenfromtheiroriginalcultures,asexplainedin theMaterialandMethodssection,theydidnotdevelopcritical phenotypicchanges.Conversely,thereferencestrainsG-186B, G-217B,andDownshavebeenculturedrepeatedly.

Somephenotypic characteristics(e.g.,serotypes, chemo-types, isozymes and fatty acid profiles) of the two H. capsulatummorphotypeshavebeenexploredtogroupand/or classifyfungalstrains.7,9,20,21_{Inafewcases,thesedatawere} analyzedbyoverlookingimportantassociationswithcritical aspectsoftheinteractionbetweenthefungusandits envi-ronment.StudiesofH.capsulatumassessingthermosensitivity associatedwithfungalvirulencewereconductedbyMedoff et al.,6 _{Keath et al.,}22 _{Spitzer et al.,}11 _{and Gargano et al.}23 Recently,Garfootetal.24_{suggestedthatthethermotolerance} ofthe HistoplasmaY-phase tothe body temperature ofthe

mammalianhostisassociatedwiththeO-mannosylationof itsproteins.However,mostofthesereportsevaluatedascarce number ofH.capsulatumisolatesorstrainsthatcamefrom circumscribedgeographicareasfromNorthAmerica,which arenotrepresentativeofthegeneticandphenotypicfungal changesthatmaybeoccurringindifferentregionsassociated with the phylogeographic distribution ofthe H. capsulatum complex inthe Americas,as statedbyKasuga etal.13 _and Teixeiraetal.25

Thepresentworkrepresentsaninnovativereportbecause allthestudiedH.capsulatumclinicalisolateswerefromLatin American countries, wherethis type ofphenotype evalua-tion had not been described until now. Based on limited subculturesoftheseisolates,theirthermo-phenotypic char-acteristicscanbeconsideredtobenaive.Thedataobtained forthe22isolatesandthreestrainsstudiedarereliableasthey incorporatedtheresultsofmultiplemeasurementsin repeti-tiveassays.AllH.capsulatumisolatesandtworeferencestrains studiedwerethermotolerantat40◦C.Theexceptionwasthe Downsreferencestrain,confirmingitsthermosensitive phe-notypeatthis temperature. Interestingly,theDowns strain is alsoassociatedwith avirulence ina murine model, and accordingtoSpitzeretal.,11_{apotentialassociationbetween} virulenceandthermotoleranceintheH.capsulatumY-phase can be inferred. However, no associations between H. cap-sulatum temperature sensitivity and the clinical forms of histoplasmosis or the immune statusof the patientswere found.

RegardingDtestimation,wefounddelayedDtsat40◦Cfor mostof thestudied H.capsulatum samples,indicating that fungal exposuretothis temperaturedelayed the growthof thermotolerant yeasts,but didnotkill them.Under invivo infectionconditions,thisfindingsuggeststhathostdefense mechanismsmightincludetherestrictionoffungal dissem-inationviafebrileinflammatoryprocesses.TheDtvaluesat 37◦Cfoundformoststudied isolatesandreference strains werecompatibleforpathogenswithalowrateofgrowthand consistentwithpreviousdescriptionsforH.capsulatum.5,15

Interestingly, the isolate DMic993444 obtained from an Argentinean histoplasmosis patient with mucosal lesions associatedwithadisseminatedclinicalform,presentedalong lag-phaseatboth37and40◦C,suggestingaprobabledelayin adaptationtotheexperimentalconditions.

Despite the involuntary biases that must be considered inthegatheringandhandingofpatientrecords,thepresent findingsproviderelevantinformationregardingthe thermo-phenotypic characteristics of the H. capsulatum Y-phase, irrespective ofassociationswithclinical disease formsand different geographic origins inthe Americas. An appropri-ate number of fungal isolates and reference strains were tested,and,undoubtedly,thistypeofstudywilladvancethe knowledgeonthepossiblemanifestationsofthepathogenin susceptiblehosts.

Contributions

MLTandMRRMwereinvolvedinthestudydesign,analyzed andinterpretedtheresultsanddraftedthemanuscript.JHS performed the experiments, conducted the data analyses

andparticipatedindraftingthemanuscript.GRAmaintained the fungal cultures. CC provided a critical review of the manuscript.MLTconceptualizedandcoordinatedtheproject. Allauthorsreadandapprovedthefinalmanuscript.

Funding

ThisresearchwasfundedbyagrantfromtheNationalCouncil of Science and Technology (CONACYT) from Mexico (Ref-166052).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thispaperconstitutes collateral fulfillment ofthe require-mentsoftheGraduatePrograminBiologicalSciencesofthe UNAM.JHS thankstheGraduate Programin Biological Sci-enceoftheUNAMandthescholarshipofCONACYT-Mexico (Ref-245151).

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