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FEMS Immunology and Medical Microbiology 45 (2005) 423–428

Electrophoresis karyotype and chromosome-length polymorphismof Histoplasma capsulatum clinical isolates from Latin America

Cristina Elena Canteros a,*, Marıa Fernanda Zuiani a, Viviana Ritacco a,b,Diego E. Perrotta a, Marıa Rocıo Reyes-Montes c, Julio Granados d,

Gerardo Zuniga e, Maria Lucia Taylor c, Graciela Davel a

a Departamento Micologıa, INEI ANLIS ‘‘Dr. Carlos G. Malbran’’, Av. Velez Sarsfield 563, 1281 Buenos Aires, Argentinab Consejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET), Buenos Aires, Argentina

c Departamento de Microbiologıa-Parasitologıa, Facultad de Medicina, Universidad Nacional Autonoma Mexico, Mexico DF, 04510, Mexicod Instituto Nacional de Ciencias Medicas y Nutricion ‘‘Salvador Zubiran’’, Mexico DF, 14000, Mexico

e Departamento de Zoologıa, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Mexico DF, 11340, Mexico

Received 27 April 2005; accepted 27 May 2005

First published online 19 July 2005

Abstract

Intact chromosomes of 19 clinical isolates ofHistoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala andthe laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal bandingpatterns of the human isolates revealed 5–7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approx-imately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsu-

latum varies widely in nature, as observed previously in laboratory strains. No definite association was found betweenelectrokaryotype and geographical or clinical source.� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Histoplasma capsulatum; Karyotyping; Pulsed-field gel electrophoresis; Chromosomes

1. Introduction

Histoplasma capsulatum is a dimorphic fungus thathas been recognized as an important worldwide patho-gen, agent of histoplasmosis. The disease, which in someLatin American regions should be considered a publichealth threat, presents a wide diversity of clinical mani-festations. However, in most individuals the infection isasymptomatic or manifests itself as a self-limiting flu-like syndrome [1]. Lately, the risk of progressive formsof histoplasmosis has increased due to HIV infection,

0928-8244/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2005.05.015

* Corresponding author. Tel./fax: +54 11 4302 5066.E-mail address: ccanteros@anlis.gov.ar (C.E. Canteros).

malignancy, or immunosuppressive treatment. Althoughthe immunological status of the susceptible host is criti-cal to define the clinical outcome of the infection, the ge-netic traits of different fungal strains might alsodetermine differences in pathogenesis [2]. The knowledgeon the chromosomal band profile of clinical isolatesmight therefore shed light on the role of fungal geneticdiversity in the evolution of different clinical forms ofthe disease [3,4].

In the past and even now, chromosomes have beenstudied by traditional methods, such as cytogenetics,but when these methods were applied to characterizefungal chromosomes, difficulties arose due to their smallsize. In addition, these methodologies are laborious foranalyzing a great number of strains [3,4]. The separation

. Published by Elsevier B.V. All rights reserved.

424 C.E. Canteros et al. / FEMS Immunology and Medical Microbiology 45 (2005) 423–428

of intact chromosomes by pulsed-field gel electrophore-sis (PFGE) has become a well-established technique tostudy the molecular karyotype of several fungal species.Besides, the resolution of chromosomal DNA sizes byPFGE has many other applications, including intra-spe-cies strain identification, phylogenetic analysis, andpreparation of DNA for further genomic studies. Thetechnique permits to evidence chromosome-length poly-morphism and to resolve chromosomal bands of smallsize [3–5].

To our knowledge, the karyotype of H. capsulatum

has not been sufficiently explored. The only study usingPFGE techniques was reported by Steele et al. [6], andshowed variability in band size and migration amongthree laboratory reference strains of H. capsulatum bycontour-clamped homogeneous electric field (CHEF)and field inversion gel electrophoresis (FIGE). Later,Carr and Shearer [7] used renaturation kinetics, genomicreconstruction and flow cytometry to determine thewhole genome size, complexity and ploidy of two H.

capsulatum laboratory reference strains with a long his-tory of in vitro manipulation.

As only a few laboratory strains have been karyo-typed to date, the genome variability of H. capsulatum

has not been sufficiently explored. In order to enhancethe knowledge on chromosome-length polymorphismof this pathogen, we compared the electrophoretic kary-otypes of recent H. capsulatum human isolates from dif-ferent bodily sites and geographical origins in LatinAmerica.

Table 1Characteristics of studied H. capsulatum clinical isolates and a reference stra

Patient Isolate Country oforigin

Year ofisolation

Specimen Chrsize

1 01738 Argentina 2001 Mucose 2.7–01739 Argentina 2001 Blood 2.7–

2 92590 Argentina 1992 Bone marrow 2.7–3 993267 Argentina 1999 ND 2.7–

4 00205 Argentina 2000 Blood 3.0–00335 Argentina 2000 Skin 3.0–

5 01870 Argentina 2001 Bone marrow 1.8–01869 Argentina 2001 Sputum 1.8–

6 H.1.12.96 Guatemala 1996 Skin 2.0–7 993446 Argentina 1999 Skin 1.5–8 EH-316 Mexico 1993 Blood 2.7–9 EH-317 Mexico 1992 Blood 2.7–10 01558 Argentina 2001 Skin 1.8–11 EH-326 Mexico 1996 ND 2.7–12 EH-325 Mexico 1996 Peritoneal fluid 1.3–13 EH-327 Mexico 1991 ND 3.0–14 00334 Argentina 2000 CSF 2.5–15 EH-324 Mexico 1994 ND 3.5–16 90455 Argentina 1990 Mucose 2.5–

Ref. strain G186B Panama 1967 ND 1.1–

ND, not determined and CSF, cerebrospinal fluid.

2. Materials and methods

2.1. Isolates

Nineteen clinical isolates of H. capsulatum obtainedfrom 16 Latin America patients and reference strainG186B (ATCC 26030) were converted to yeast-phaseand maintained by culturing in GYE-agar (2% glucose,1% yeast-extract and 1.5% Bacto-agar) at 37 �C in 5%CO2 atmosphere. After primary isolation, subcultureswere kept to a minimum. The year, country and speci-men source of isolates are described in Table 1. Speciesidentification was based on morphologic characteristics,thermal dimorphism and exoantigen production [1,8].

2.2. Chromosomal DNA plugs

The method described by Perrotta et al. [9] was ap-plied with slight modifications. After 72 h growth at37 �C and 5% CO2 in slant-cultures, yeast-cells were sus-pended in 100 ml GYE broth and incubated at 37 �C for72 h in a reciprocal shaker at 120 rpm. Cells were har-vested after centrifuging at 800g–10 min, washed in ster-ile distilled water and resuspended in 230 ll of 50 mMEDTA, pH 8.0. Per each 100 mg of yeast-cells, 6.5 ll(84 U) of lyticase (Promega Biosciences, Inc., San LuisObispo, CA) and 83 ll of Trichoderma harzianum lyticenzyme (6 lg ll�1) (Promega) were added. The yeastcells were immobilized in 2% (wt/vol) low melting-temperature agarose (Gibco, Grand Island Biological

in

omosomal bandrange (Mbp)

Number ofchromosomal bands

Pof PFGE

bands (Mbp)Karyotypeprofile

8.5 5 24 I8.5 5 24 I

8.5 5 25 I8.5 5 26 I

8.5 5 26 I8.5 5 26 I

9.8 7 36 II9.8 7 36 II

8.5 6 28 III9.5 7 36 IV10.0 5 32 V10.0 5 32 V9.5 6 32 VI9.7 5 28 VII9.7 6 29 VIII10.0 5 29 IX9.0 5 27 X9.5 5 29 XI9.7 6 37 XII

9.7 5 22 XIII

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Co., NY) in 125 mM EDTA, pH 8.0, and dispensed intomolds to form the plugs. Plugs were incubated 8 h at28 �C in 50 mM EDTA, pH 8.0, under gentle shaking.Spheroplasts immobilized within plugs were disruptedwith 10 ml lysis solution (500 mM EDTA, 1% [wt/vol]N-laurylsarcosine (Sigma Chemical Co., St. Louis,MO) and 0.5 mg ml�1 proteinase K (Gibco), for 18 hat 50–55 �C. Plugs were then washed three times at50 �C and twice at 28 �C with TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0) and stored at 4 �C in50 mM EDTA pH 8.0 until required.

2.3. Pulsed-field gel electrophoresis

Separation of chromosomal bands was performedunder PFGE conditions in a CHEF DR III apparatus(Bio-Rad Laboratories, Richmond, CA). In preliminaryexperiments, different agarose concentration, voltage,pulse rate, and run time were assayed. The best chromo-somal band resolution was achieved by using 0.7% aga-rose (Pulsed-Field Agarose, Bio-Rad) in 1· TAE buffer(40 mM Tris–HCl, 40 mM acetate and 2 mM EDTA,pH 8.0) and 72 h electrophoresis in three block-phases(phase 1 pulse 4500 s, 1.5 V cm�1, 24 h; phase 2 pulsesramped from 3600 to 2700 s, 1.5 V cm�1 30 h; phase 3pulses ramped from 2700 to 750 s, 1.8 V cm�1 18 h). Insome cases, the presence of smaller chromosomal bandswas resolved by raising agarose concentration to 1%.Size standards were Schizosacharomyces pombe (5.7–3.5 Mbp) and Hansenula wingeii (3.13–1.05 Mbp) (Bio-Rad). After electrophoresis, the gel was stained with0.5 lg ml�1 ethidium bromide (Sigma) for 30 min andwashed in distilled water for 1 h [10]. All the experimentswere performed by triplicate in different runs. Digitalimages of gels were obtained with a Bio-Rad Gel Doc1000.

2.4. Analysis

The software Kodak Digital Science ID Image Anal-ysis (Kodak, NY) was employed to estimate chromo-somal band sizes. The size (Mbp) of each H.

capsulatum chromosomal band was calculated basedon migration compared to that of the size standard.The size of the whole genome of each tested H. capsula-

tum isolate was calculated by the sum of each individualchromosomal band size, generated in PFGE conditions[11,12].

The relatedness of strains was determined by analyz-ing the PFGE banding patterns with the software Bio-Numerics (Applied Maths, Kortrijk, Belgium). S.

pombe was run in the first and last lanes of each gel asan external standard for intra and inter-experiment nor-malization. A reference system was defined based onband positions of this strain in the first gel entered tothe software and all subsequent gel images were normal-

ized according to the reference system. The Dice coeffi-cient was used to analyze banding pattern similaritiesand the unweighted pair group method with arithmeticaverages to analyze clustering. As chromosomal imageswere broad and diffuse, a fairly high band position tol-erance (2.5%) was chosen to match visual inspection.The cophenetic correlation was calculated to evaluatethe robustness of clusters. Strains were defined to belongto the same karyotype when showing 100% similarityand identical number of chromosomal bands in suchanalysis conditions.

3. Results and discussion

The present study is the first attempt of using PFGEto explore the genome size, the chromosomal band num-ber and the genetic linkage of human H. capsulatum iso-lates obtained from different bodily sites andgeographical sources. This method had not been appliedto H. capsulatum karyotyping since the early study onlaboratory strains by Steele et al. [6]. In general, we con-firmed the results of that report regarding intra-speciesgenomic variability.

Table 1 describes the characteristics of the isolatesand their karyotypes. The dendrogram, gel images andschematic PFGE patterns of H. capsulatum clinical iso-lates from Argentina, Mexico and Guatemala are pre-sented in Fig. 1 together with that of the referencestrain G186B from Panama.

As reported for other fungal species, some H. capsu-

latum chromosomal bands in our study exceeded therange size of the external reference strain S. pombe

and therefore their sizes had to be estimated tentativelyby extrapolation [11,13]. Besides, certain bandsdisplayed high fluorescence intensity after ethidium bro-mide staining, which could be attributed to co-migrationof chromosomes [11,14]. By changing PFGE switchingintervals and gel concentrations as suggested by Chavezet al. [15], we succeeded in resolving the doublets insome cases, like strains 01869 and 01870 (see the distinctlow-size pair of bands). In other cases (the smallest bandin strains 90455, 993267) no evidence of duplication wasfound under the different electrophoretic conditionstried (data not shown). We considered these bands assingle in the analysis but the presence of doublets cannotbe totally ruled out.

According to our results, the karyotype of strainG186B showed five chromosomal bands, being thesmallest 1.1 Mbp in size. Using similar, but not identi-cal, PFGE techniques, Steele et al. [6] ascribed fourchromosomal bands to this strain, including an excep-tionally small one of 0.5 Mbp. These discrepancies in re-sults might be due to differences in experimentalconditions and analysis accuracy. They also show thatit is necessary to adhere to a rigorous standardization

Fig. 1. Dendrogram, normalized gel images and schematic representation of PFGE patterns of 19 recent of H. capsulatum and the reference strainG186B. Dendrogram constructed using Dice coefficient and the unweighted pair group method of arithmetic averages. Cophenetic values are shownon each node. MX: Mexico, PA: Panama, GT: Guatemala, AR: Argentina. Estimated chromosome band sizes are expressed in megabase pairs(Mbp). h Isolates from different clinical sources of the same patient.

426 C.E. Canteros et al. / FEMS Immunology and Medical Microbiology 45 (2005) 423–428

in order to build a database with reproducible and inter-changeable information on H. capsulatum PFGE karyo-types. And yet, it should be stressed that the strength ofPFGE lies not in the precise assessment of chromosomalband number but in the discrimination of whole band-ing patterns among genetically different isolates pro-vided electrophoretic conditions are fixed.

To our knowledge, Carr and Shearer [7] were the onlyauthors that assessed the whole genome size of H. cap-

sulatum and they did it in a few laboratory strains. Weestimated in 22 Mbp the whole genome size of the refer-ence strain G186B, matching closely the size attributedby these authors to the same strain using more accuratetechniques. This agreement supports the use of PFGE asa tool for calculating tentative genome size.

In our study, the observed number/size of bands andthe whole genome size were not homogeneous among re-cent human isolates, but ranged around values reportedfor common laboratory strains. The origin of suchdivergences among wild strains of the same species isstill unclear and might be related with chromosomalrearrangement involving gain or loss of genomic mate-rial in the natural environment and/or the infected host.This hypothesis has been postulated by Carr andShearer for H. capsulatum [7] and already demonstratedfor other fungal species by other authors [3,4].

According to our results, at least three isolates fromArgentina showed as many as seven chromosomalbands, the number previously attributed to the Downsstrain [6]. If total DNA mass were to be considered, se-ven isolates had estimated genome sizes equal or largerthan the 32 Mbp previously estimated for Downs strain[7] (see Table 1). Possessing the same genome size ornumber of chromosomal bands, however, is not suffi-cient evidence to infer genomic similarity. The abilityof PFGE of identifying Downs-like genotypes is yet tobe defined, as well as the biological relevance of suchfinding.

The cophenetic correlation is a measure of the agree-ment between the similarity represented in the dendro-gram and the actual degree of similarity expressed bythe Dice coefficient. A minimum 70% correlation indi-cates that the dendrogram does not greatly distort the ori-ginal structure in the input data [16]. As indicated on eachnode in Fig. 1, all the cophenetic correlations obtained inour study were above this value, thus reassuring therobustness of the clustering obtained (72–100%).

Thirteen distinct electrokaryotypes were observed,supporting inter-strain chromosomal polymorphism, asit has been described for several pathogenic fungi,including other dimorphic species [3,4,12,17–19]. Profiledifferences were more marked in chromosomal DNA

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band sizes than in their numbers and no variation wasfound between isolates obtained from different bodilysites in a single patient (Fig. 1). We observed that certainisolates from Argentina with the same number of chro-mosomal bands clustered distantly, as shown in Fig. 1,karyotypes I and X. This polymorphism was only dueto differences in band sizes. Provided that these subtledifferences are consistent and meaningful, PFGE couldbecome a useful tool to discriminate between strains ofH. capsulatum sharing a geographical niche.

In spite of the apparently high chromosome-lengthpolymorphism, three clusters of identical patterns wereidentified. Karyotype I included isolates from fourArgentinean patients. Karyotype II was composed oftwo isolates obtained from different bodily sites in a sin-gle patient. In karyotype V segregated two isolates fromtwo different Mexican patients with no record of epide-miological link with each other.

In this study, the electrophoretic profile of the refer-ence strain G186B was almost 50% different from theprofiles of the clinical isolates. The divergence of itschromosomal banding pattern from those of wild strainssuggests that this laboratory strain may have undergonegenomic rearrangements throughout its long-term his-tory of in vitro passages. In addition, its small genomesize points towards a loss rather than a gain in genomicmaterial, compared with natural isolates.

It is interesting to note that all H. capsulatum isolatesfrom Mexico showed substantial polymorphism amongeach other whereas a number of isolates from Argentinaformed two closer groups. Our results regarding the ge-netic homogeneity of strains isolated in Argentina sup-port previous findings by RAPD-PCR [20]. Morerecently, in a phylogeographical study involvingDNA se-quence variation of protein-coding genes, Kasuga et al.[21] found clonality among strains from Argentina anda wider genetic diversity among strains from Mexico.

In sum, the present PFGE study indicates that thegenome of H. capsulatum varies widely. This researchalso shows that the chromosomal band number andthe estimated genome sizes of clinical isolates are inrange with those previously reported for highly domesti-cated laboratory strains. No clear relationship wasfound between electrokaryotypes and bodily site or geo-graphical source, either due to the small number of iso-lates analyzed or to unsuitability of the method foranswering epidemiological or phylogenetic questions.Further studies are required to establish whether thekaryotypes here described can be assigned to genuine ge-netic linkage groups.

Acknowledgment

The study was partially supported by CONACyTRef. No. 34443.

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