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Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis
Fungal Genetics and Biology 39 (2003) 60–69 www.elsevier.com/locate/yfgbi
Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis Luciano dos S. Feitosa,a,1 Patrıcia S. Cisalpino,b,1 M arcia R. Machado dos Santos,a a a Renato A. Mortara, T^ ania F. Barros, Fl avia V. Morais,a Rosana Puccia,a Jose Franco da Silveira,a and Zoilo P. de Camargoa,* b
a Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP/EPM – Universidade Federal de S~ ao Paulo, S~ ao Paulo, SP, Brazil Departamento de Microbiologia, Instituto de Ci^ encias Biol ogicas, UFMG – Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Received 2 August 2002; accepted 24 January 2003
Abstract Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: P. brasiliensis; Clinical and environmental isolates; Gene mapping; Genome size
1. Introduction Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America (Lacaz, 1994). The disease presents two progressive clinical forms. The chronic form affects rural workers, mainly male adults, evolves gradually in the lungs, presenting or not disseminated lesions depending on the integrity of cell immunity. Acute (or subacute) PCM affects mainly children and young adults of both sexes, progresses rapidly and disseminates through the lymphatic system, with lymph node hypertrophy, intense hepatosplenomegaly, and involvement of other organs. If not treated, the severe acute form (juvenile
*
Corresponding author. Fax: +55-11-5571-5877. E-mail address: [email protected] (Z.P. de Camargo). 1 These authors contributed equally to this work.
type) frequently culminates with the patientÕs death (Franco, 1987; Franco et al., 1987). The genetic composition of P. brasiliensis is poorly known. A teleomorphic, sexual stage has not been determined, greatly impairing classical genetic analysis. There are few reports on the isolation and characterization of mutants. The multinucleated nature of the fungus, even of the unicellular, parasitic yeast form, has been pointed out as the reason for the phenotypic instability and high number of revertants of the in vitrogenerated mutants (Hallak et al., 1982; McEwen et al., 1987; Queiroz-Telles, 1994). The development of pulsed-field gel electrophoresis (PFGE) has allowed the genomic characterization, gene mapping, and molecular epidemiological biotyping of strains from microorganisms refractory to genetic analysis (Altboum, 1994; Pan and Cole, 1992; Perfect et al., 1989; Steele et al., 1989). Montoya et al. (1997) and Cano et al. (1998), respectively, studying eight and two clinical isolates, described conditions for the
1087-1845/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00003-3
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separation of P. brasiliensis chromosome-sized DNA molecules by PFGE. Recently, Montoya et al. (1999) by karyotyping five environmental isolates reported a discrete chromosomal polymorphism, i.e., clinical isolates showing five identical chromosomal DNA bands, with the environmental isolates closely resembling the clinical isolates, distinguished only by a slight difference in mobility of two bands. In contrast, in our previous work, we found that the karyotype profiles of two isolates were highly polymorphic. Two probes were mapped to chromoblots and we concluded that it would be very difficult to correlate chromosome-sized DNA molecules and karyotype profiles from different P. brasiliensis isolates without probing them with a panel of specific genes or sequences (Cano et al., 1998). In the present report, we describe intraspecific variation observed by karyotyping 12 P. brasiliensis isolates. In an attempt to assess strain relatedness we mapped nine homologous genes to chromosomes of isolates from different patients with different clinical forms (chronic and acute) and of different geographical origins. This represents the first comparative mapping study among different isolates. Gene mapping allowed the identification of synteny groups, and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. We also addressed the question of the fungus ploidy by comparing data on genome sizes generated by PFGE and microfluorometry for all 12 isolates.
2. Materials and methods 2.1. Microorganisms The 12 clinical and environmental isolates of P. brasiliensis selected for this study are listed in Table 1.
Table 1 Characteristics of the P. brasiliensis isolates analyzed in this study Isolate name or number
Origin
Source
B-339 (ATCC-32069) 113 SS (ATCC-200272) Bt01 4947 18 265 (ATCC-200443) 18749 Ibi a I9 EPM 45 Tatu
Brazil Brazil Brazil Brazil Argentina Brazil Brazil Peru Brazil Colombia Argentina Brazil
Chronic PCM Chronic PCM Acute PCM Chronic PCM Chronic PCM Chronic PCM Chronic PCM Acute PCM Soil Chronic PCM Chronic PCM Armadillo
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Saccharomyces cerevisiae haploid strain 167-C and diploid strain BCV-59 (Castilho-Valavicius et al., 1992) were provided by Dr. Beatriz Castilho (Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP/EPM, S~ao Paulo, SP, Brazil). Fungal isolates were maintained by periodic subculturing in slanted tubes of PYG medium (yeast extract 5 g, bacto peptone 10 g, dextrose 15 g, and Agar 15 g l1 ) at 35 )37 °C. 2.2. Separation of chromosome-sized DNA molecules of P. brasiliensis by PFGE The procedures were carried out essentially as described by Cano et al. (1998), with minor modifications. P. brasiliensis yeast cells were subcultured three times in PYG medium at 5-day intervals in order to obtain cells in the late exponential phase (San-Blas and Cova, 1975; San-Blas and San-Blas, 1994). Erlenmeyer flasks containing 50 ml of PYG broth were inoculated with the entire growth of two culture slants, placed in a reciprocating shaker at 120 rpm and grown for 5–7 days at 35 °C. Cells were washed three times in 50 mM EDTA, resuspended in the same solution and sonicated (Branson Ultrasonic 450) for 5 s at low intensity (20 kHz) to disperse cellular aggregates. Cell concentration was adjusted to 1 109 cells ml1 and the preparation was mixed with an equal volume of low melting agarose (2% w/v in water); approximately 5 107 cells were immobilized in each gel plug (100 ll). Spheroplasts were obtained by digestion of cell walls with Novozym 234, 2.5 mg ml1 in PBS, pH 7.5, for 1 h at 37 °C. The blocks were dialyzed three times against 250 mM EDTA, pH 8.0, at 37 °C to inactivate the enzyme. Spheroplasts were disrupted for 24 h at 56 °C in a solution containing 500 mM EDTA, pH 8.0, 10 mM Tris–Cl, pH 8.0, 1% (w/v) sarkosyl, and 1 mg proteinase K per ml. The blocks were washed with 500 mM EDTA and stored at 4 °C in the same solution. Electrophoretic separation was performed under PFGE conditions in a Gene Navigator System (Pharmacia Biotech) with a hexagonal electrode array. The separations were carried out in 0.6% (w/v) agarose gel in 1.0 TAE (40 mM Tris acetate, pH 7.5, 2 mM EDTA, pH 8.0) kept at constant temperature (10 °C). Original running conditions (Cano et al., 1998) were slightly modified to achieve better resolution of chromosomal bands ranging from 4.7 to 10 Mb: we used homogeneous pulses (N/S, E/W) with interpolation for 120 h at 42 V: phase 1, pulse time 900 s (run time 12 h); phase 2, pulse time 1800 s (run time 12 h); phase 3, pulse time 2700 s (run time 36 h); phase 4, pulse time 3600 s (run time 36 h); and phase 5, pulse time 4500 s (run time 24 h). After electrophoresis, the gels were stained with ethidium bromide 0.5 lg/ml1 and photographed. Chromosome-sized DNA molecules were
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subjected to acid depurination in the presence of 0.25 M HCl for 5 min and transferred to nylon membranes (Hybond N, Amersham) using 0.5 M Tris, pH 7.0, and 0.4 N NaOH plus 1.5 M NaCl as neutralization and transfer solutions, respectively (Birren and Lai, 1993). The CHEF DNA size marker Schizosaccharomyces pombe (Bio-Rad) was used as the chromosome-sized DNA standard. The apparent molecular size of each chromosomal band was plotted as a function of the distance of migration on the gel, considering the distance migrated by S. pombe known standard markers (Cox et al., 1990; Pan and Cole, 1992). The densitometric analysis of chromosome-sized DNA bands resolved by PFGE was performed using Kodak Digital Science 1D Image Analysis Software, version 3.0. 2.3. DNA probes P. brasiliensis cloned genes were used as probes: a 630 bp BamHI/HindIII fragment which contains 70% of the coding region of the Gp43 antigen gene was isolated from plasmid pUCGPb16A (GenBank U26160) (Cisalpino et al., 1996). Two fragments of about 600 bp corresponding to the homologous chitin synthase genes CHS1 and CHS2 were amplified from total DNA of the B-339 isolate by the method of Bowen et al. (1992), cloned into pUC18 (Sureclone system, Pharmacia), and sequenced (GenBank AF416865 and AF416866, respectively). A 28S rRNA gene fragment was amplified from the B-339 isolate by the method of Peterson and Sigler (1998), cloned into pUC18 and sequenced (GenBank AF416745). In an effort to determine the degree of gene synteny among the P. brasiliensis isolates, we chose several genes that are linked in isolate B-339: PbLON, an ATP-dependent proteinase gene (GenBank AF239178) and its neighbor PbMDJ1-homologue (GenBank AF334811) were identified in a 6.5 kb genomic clone isolated from a P. brasiliensis genomic library (Barros and Puccia, 2001); a Ran binding protein homologue gene (RanBP, unpublished data) was found flanking the Gp43 gene in a 3.8 kb EcoRI fragment cloned from isolate B-339 (Cisalpino et al., 1996). A fragment of the gene coding for a putative N-acetyl-glycosaminidase (NAG) (GenBank AF395815) and a putative mannosyltransferase gene (named clone 11) (GenBank AF374353) from P. brasiliensis were provided by Dr. Celia Maria de Almeida Soares (Departamento de Ci^encias Fisiol ogicas, ICB, Universidade Federal de Goi as, Goi^ ania, Goias, Brazil). Chromosomal bands of 9.5 and 3.5 Mb of isolate 113 were separated by PFGE and excised from the agarose gels. Gel slices were digested with bagarase (Maule, 1993) and the DNA recovered from each whole band was radiolabeled and used as a probe.
2.4. Radiolabeling of probes and Southern blot hybridization The DNA fragments cited above were radiolabeled (Rad primer labeling kit, Gibco-BRL) and used as probes in Southern blots. Hybridizations were carried out overnight at 42 °C in 50% (v/v) formamide, 5 SSC (1 sodium saline citrate ¼ 0.15 M NaCl, 0.015 M sodium citrate), 5 DenhardtÕs solution, 50 lg yeast tRNA, 100 lg sonicated herring sperm DNA per ml, and 0.1% (w/v) SDS. The filters were washed twice in 0.1 SSC/0.1% (w/v) SDS at 56 °C. 2.5. Preparation of DAPI-stained cells, confocal microscopy, and estimation of genome size P. brasiliensis cells were stained with DAPI and prepared for confocal microscopy as described by Cano et al. (1998). Images were observed with a Bio-Rad 1024UV confocal system attached to a Zeiss Axiovert 100 microscope using a 40 N.A. 1.2 Plan-Apochromatic (DIC) water immersion objective. All images were collected by Kalman averaging of at least 10 frames (512 512 pixels) using an aperture (pinhole) of 1.5 mm, with the zoom set at 3.5 and the photomultiplier gain kept at 1200 during all image acquisitions. DAPIstained nuclei in different fields that could be clearly distinguished were then subjected to serial optical sectioning (0.36 lm steps) and the fluorescence intensity of the volume of each nucleus was estimated using the processing software Lasersharp 3.2TC (Bio-Rad). Fluorescence and DIC prints were generated by dye sublimation on a Codonics NP1600 printer. The genome size of P. brasiliensis in megabases (GS) was calculated as follows: ðSc nÞ ðPb DFIÞ ; Sc n DFI where (Sc n) is the genome size (12.1 Mb) of an haploid strain of S. cerevisiae; (Pb DFI) and (Sc n DFI) are the values of DNA fluorescence intensity of DAPI-stained nuclei of P. brasiliensis and of a haploid S. cerevisiae strain, respectively. Note that the same calculation can be done using the genome size of the diploid strain of S. cerevisiae and its corresponding DFI value.
GS ¼
3. Results and discussion 3.1. Molecular karyotype and chromosomal polymorphism of P. brasiliensis In this paper, we refer to a DNA band visible on pulsed field gel after staining with ethidium bromide as a ‘‘chromosomal band,’’ which could contain one, two or more, not necessarily homologous, co-migrating
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Fig. 1. Karyotype profiles of 12 P. brasiliensis isolates. We present ethidium bromide-stained chromosomal bands separated by PFGE (arrowheads indicate the origin of the gels) and the graphic results generated by densitometry (Kodak Digital Science 1D) of the karyotypes of each isolate (A–G). The numbers on the left and on the top row of each graph indicate the sizes of the chromosomal bands in megabases (Mb) (smaller arrows point to the corresponding peaks). P. brasiliensis isolates are indicated at the top of each panel (A–G). The chromosomal bands of S. pombe (5.7, 4.6, and 3.5 Mb) were used as chromosome-size DNA standard.
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gration properties of circular DNA seem to be generally proportional to the total running time. For instance, the mtDNA of A. chrysogenum was estimated to be 27 kb in size, but it migrated in PFG like 1.8– 1.9 Mb linear DNA molecules (Skatrud and Queener, 1989). A relative stoichiometry staining of the chromosomal bands was observed in the majority of isolates, but some striking differences could be found: the 9.5 band was heavily stained in isolates 113 and SS when compared to other isolates (Fig. 1A). The largest chromosome-sized molecules resolved in this study were approximately 9 Mb (or 10 Mb) in size, close to the upper limits for pulsed field gel electrophoresis and we cannot rule out co-migration of two bands in the compression zone. The fluorescence intensity of bands of isolates 18749, 4947, and 18 was always comparatively weaker than that of isolates Bt01, EPM 45, and Tatu (Fig. 1A). Although all possible care was taken to standardize the number of cells ð5 107 Þ in the agarose plugs, variations in fluorescence intensity of chromosomal-sized DNA molecules among the isolates indicated that there still was DNA content variation among blocks of different isolates. Interfering factors could be the number of nuclei per yeast cell, considering the multinucleated nature of these cells, and the fungus ploidy (see Table 2), which will be discussed later.
chromosomes. The karyotype profiles of 12 P. brasiliensis isolates shown in Figs. 1A–G were confirmed by densitometric analysis of staining bands (Figs. 1B–G) and hybridization with 32 P-labeled P. brasiliensis total DNA (data not shown). The analysis of karyotype profiles revealed the presence of distinct chromosomal migration patterns showing either four bands (isolates B339, 113, SS, Bt01, 4947, 18 and 265; Figs. 1B–D) or five bands (isolates 18749, Ibi a, I9, EPM45, Tatu; Figs. 1E–G), in the 2.5–9.5 Mb range. The chromosomes are comparable in size and number to those of fungi belonging to the ‘‘Schizosacharomyces pombe-like’’ group, whose electrophoretic separation requires long pulse times over a period of 5–8 days (Neurospora crassa, Orbach et al., 1988; Histoplasma capsulatum, Steele et al., 1989; Aspergillus niger, Debets et al., 1990; Coccidioides immitis, Pan and Cole, 1992; Aspergillus fumigatus, Tobin et al., 1997; Trichoderma harzianum, G omez et al., 1997). A band of 9.5 Mb was observed in all isolates, and bands of 4.7 (4.5–5.0) and 2.5 Mb were detected in most isolates. On the other hand, several bands were found in a few isolates: a 3.5 Mb band was only detected in isolate 113; 8.3 and 8.4 Mb bands were present in isolates 113 and SS; and band of 7.7 and 7.8 Mb were found in isolates B-339 and I9. A fuzzy band of about 1.0 Mb stained with ethidium bromide was observed in all isolates. Its nature is not known, but the band could be the result of some degradation of large chromosomal bands during spheroplast preparation. It should also be considered that mitochondrial DNA (mtDNA) is present in the agarose plugs and, although it does not enter the gel, the mi-
3.2. Mapping of P. brasiliensis genes to the chromosomesized DNA molecules Nine P. brasiliensis gene probes were hybridized to Southern blots containing the chromosomal bands of 12
Table 2 Estimate of the genome size of P. brasiliensis isolates by microfluorometry of DAPI-stained nuclei: comparison with values obtained by summation of PFGE bands P Ploidy ratio Microorganisms Number of DFI/SDb Genome sized of PFGE a (Mb) nuclei bandsc S. c. BCV-59 S. c. 167-3C P. b. B-339 P. b. 113 P. b. SS P. b. Bt01 P. b. 4947 P. b. 18 P. b. 265 P. b. 18749 P. b. Ibi a P. b. I9 P. b. EPM45 P. b. Tatu a
1 1 5.7 2.0 4.6 3.8 2.6 4.3 3.7 2.5 5.4 5.0 6.0 4.3
3.19E + 04/0.49E + 04 1.33E + 04/0.37E + 04 6.33E + 04/1.06E + 04 3.09E + 04/0.86E + 04 3.80E + 04/0.81E + 04 3.39E + 04/1.23E + 04 5.73E + 04/1.70E + 04 6.86E + 04/1.49E + 04 5.22E + 04/1.28E + 04 8.83E + 04/1.60E + 04 8.32E + 04/1.46E + 04 8.01E + 04/1.60E + 04 3.42E + 04/1.10E + 04 7.99E + 04/0.80E + 04
—
– 28.9 23.8 24.9 24.0 23.7 23.3 23.4 29.6 30.1 31.2 30.5 30.5
24.2e 3.7 12.1e 3.4 53:0 8:8 25:8 7:2 31:7 7:0 28:3 10:3 47:8 14:1 57:2 2:4 43:5 10:5 73:6 13:3 69:4 12:2 66:8 13:3 28:5 9:6 66:6 6:7
Average number of nuclei per yeast cell determined by confocal fluorescence microscopy. DFI ¼ DNA mean (SD) fluorescence intensity measured in DAPI-stained nuclei by confocal microscopy. cP bands, weighted sum of P. brasiliensis chromosomal bands separated by PFGE. d Genome size of P. brasiliensis estimated according the equation GS ¼ Sc n Pb DFI=Sc DFI (see text). e Genome size of S. cerevisiae obtained from GenBank. b
2 (1.7–2.3) 1 (0.7–1.3) 1.8 (1.5–2.1) 1.1 (0.8–1.4) 1.3 (1.0–1.6) 1.2 (0.7–1.6) 2.0 (1.4–2.6) 2.4 (2.3–2.5) 1.8 (1.4–2.3) 2.5 (2.0–2.9) 2.3 (1.9–2.7) 2.1 (1.7–2.6) 1.0 (0.6–1.2) 2.2 (1.9–2.4)
2n n 2n n n n 2n 2n 2n 2n 2n 2n n 2n
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Fig. 2. Mapping of P. brasiliensis genes to the chromosome-sized DNA molecules. Autoradiograms resulting from Southern hybridization of 12 P. brasiliensis isolates with nine cloned genes (A–D) and a schematic representation of gene mapping (E). (A) Synteny group I composed by clone 11, PbLON, and PbMDJ1 homologue genes; (B) synteny group II composed by GP43, Ran BP homologue, and NAG homologue genes; (C) synteny group III composed by 28S rDNA and CHS1 genes; (D) CHS2 gene hybridization pattern. The numbers on the left indicate the sizes of the chromosomal bands in megabases (Mb). Arrows indicate the origin of the gel (slot). P. brasiliensis isolates are indicated on the top row of each autoradiogram. (E) Schematic representation of the karyotype and syntenic distribution of nine genes among the chromosomal bands of 12 isolates of P. brasiliensis. The small vertical colored rectangles represent the nine different probes that were mapped to the chromosomal bands of P. brasiliensis isolates (see color legend). The numbers on the left indicate the sizes of chromosomal bands in megabases (Mb). P. brasiliensis isolates are P indicated at the top of the figure. The number of chromosome-sized DNA bands and the summation of chromosomal band sizes ( ) for each isolate is indicated at the bottom of the figure. Arrows indicate the origin of the gel (slot).
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isolates. Fig. 2 shows the autoradiograms of chromoblot hybridization (Figs. 2A–D) and a schematic representation of the results (Fig. 2E). The PbMDJ1 probe hybridized to the same bands carrying the PbLON gene, confirming that they are linked in all isolates (Fig. 2A), and the clone 11 probe (a putative mannosyltransferase) also showed the same hybridization profile (Fig. 2A). This suggests that a synteny group could be used as a marker for a specific chromosome since it was found in the same band in all isolates: in 10 isolates it mapped to a chromosomal band of 9.5 Mb, but to an 8.3 Mb band in isolate 113, and to a 6.7 Mb band in isolate B-339. It is possible that isolate 113 originally had two distinct chromosomal bands of 9.5 Mb, and one of them originated the 8.3 Mb band by deletion of a large fragment (1.2 Mb). The GP43 and Ran BP gene probes displayed exactly the same hybridization patterns, confirming that they are also linked in all isolates studied here, a fact that would define a second chromosome (Fig. 2B). The NAG gene probe was located in the same chromosomal bands recognized by the GP43 and Ran BP probes in 10 of the isolates studied (Fig. 2B). There was a clear dissociation of these two genes from the NAG gene in isolates SS and B-339, i.e., in the former the GP43 and Ran BP genes mapped to a 9.5 Mb band while the NAG gene remained in the 4.7 Mb chromosomal band and in the latter they appeared in the 5.0 Mb band while the NAG gene mapped to a 9.5 Mb band. These results suggest the occurrence of chromosomal rearrangements and, in this group, the NAG gene would be more distant from genes GP43 and Ran BP. Hybridization of chromoblots with the rRNA probe revealed discrete polymorphic rRNA genes containing chromosomes among the different isolates (Figs. 2C and E). The rRNA and CHS1 probes mapped to the same chromosomal bands in 11 isolates (Figs. 2C and E): to a single 2.5 Mb band in eight isolates (Bt01, 4947, 18, 265, 18749, Ibi a, I9, and Tatu), and to two chromosomal bands in three other isolates (113, SS, and EPM 45), one of which was the 2.5 Mb, a band that can be quite conserved, and to an additional band of 3.5, 4.5 or 4.8 Mb, respectively. But the most divergent mapping pattern was that of isolate B339: while the rRNA probe mapped to a 6.7 Mb band, CHS1 mapped to the 7.7 Mb band, suggesting once again the occurrence of sequence rearrangements. The chitin synthase genes (CHS1 and CHS2) were not linked and mapped to different chromosome-sized DNA bands in all isolates. The hybridization pattern for the CHS2 gene probe was not similar to that of any of the above-mentioned groups (Fig. 2D) and could define a fourth P. brasiliensis chromosome. The results are indicative of the existence of intraspecific genetic variation. It appeared to be more variability among the three P. brasiliensis largest chromosomes,
bands of substantially different mobility, from which all nine probes hybridized. In natural isolates, the occurrence of important variations in chromosome sizes between different strains is especially frequent in fungi lacking a sexual cycle (Fierro and Martin, 1999). Candida albicans has a highly variable karyotype and many efforts have been made to understand the mechanism by which diversity is generated and chromosomal rearrangement has been proposed as a source of genetic variation (Magee, 1993; Rutschenko-Bulgac et al., 1990). Therefore, even though P. brasiliensis is asexual and does not go through a meiotic cycle, we suggest that the chromosomal rearrangements could provide a means for genetic variation in this organism From the results of hybridization of probes to P. brasiliensis chromosomal bands we could define three synteny groups containing two or three genes (group I: PbMDJ1, PbLON, and clone 11; group II: GP43, Ran BP, and NAG; group III: rDNA and CHS1) that are much conserved among 12 P. brasiliensis isolates, suggesting that, in spite of gross differences, there is an underlying similarity in the genome organization of different isolates. In Candida albicans it was observed that the overall structure of the genome is remarkably constant among isolates despite the chromosomal size variations and the occurrence of translocations (Magee, 1998). It is interesting to mention that the synteny group II was mapped to the 9.5 Mb band of isolate 113 where probe CHS2 did also map. Two probes from this group (GP43 and RanBP) mapped to the 9.5 band of isolate SS, where group I genes (MDJ1, LON, and clone 11) did also map. If these groups were located in two different chromosomes, and if CHS2 mapped to a third distinct chromosome, it would seem possible that each 9.5 Mb band of isolates 113 and SS is composed of two co-migrating heterologous chromosomes. This hypothesis is coherent with a previous observation of the high fluorescence intensity of the 9.5 Mb bands of isolates 113 and SS in ethidium bromide-stained gels (Fig. 1A). In order to establish the correlation among the 9.5 Mb bands, observed in all isolates, a band of that size from isolate 113 was removed from the gel, radiolabeled, and hybridized to chromoblots. The same procedure was carried out with a 3.5 Mb band which was detected only in isolate 113. Fig. 3 shows the autoradiograms and the resulting hybridization patterns. The 9.5 Mb labeled band hybridized exclusively with itself in isolate 113, suggesting that its genes are not distributed in the other chromosomal bands of this isolate. It strongly hybridized with the 9.5 Mb band of isolates SS, EPM45, Tatu, and I9 and weakly with the 9.5 Mb band of isolates B-339, 4947, 18, and 265. Hybridization signals were also seen in other sized bands of all isolates, except 113 and SS. If the large 9.5 band is a doublet, as suggested by the fluorescence intensity of agarose gels
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Having large chromosome segments that span these loci cloned in cosmids, BAC or YACs, will help to identify genes involved in the parasiteÕs survival, pathogenicity, and diagnosis. 3.3. Correlation between karyotype profiles and epidemiological characteristics of the fungal isolates
Fig. 3. Hybridization of different P. brasiliensis isolates using the whole 9.5 and 3.5 Mb chromosomal bands from isolate 113 as probes. The chromosomal bands of 9.5 and 3.5 Mb from isolate 113 were separated by PFGE, excised from the gel, radiolabeled, and hybridized to Southern blot with bands of 10 P. brasiliensis isolates. Panel A and B show the hybridization patterns, respectively, obtained with the whole 9.5 and 3.5 Mb chromosomal bands from isolate 113 used as probes. The numbers on the left indicate the sizes of the chromosomal bands in megabases (Mb). Arrows indicate the origin of the gel (slot). P. brasiliensis isolates are indicated at the top of each autoradiogram.
and by the hybridization patterns with specific genes, it should in fact probe to other bands in the other strains (Fig. 3). These data open the possibility of the existence of two 9.5 Mb heterologous chromosomes in most P. brasiliensis isolates which may have been fortuitously visualized in isolates SS and 113. Results of hybridization with the 3.5 Mb band probe showed homogenous signals distributed over all chromosomal bands in all isolates. The patterns showing cross-hybridization of the 9.5 and 3.5 Mb bands are strongly suggestive of the existence of repetitive sequences in the genome of P. brasiliensis. Virtually nothing is known about repetitive elements of the P. brasiliensis genome. Chibana et al. (2000) showed that a middle repeated DNA sequence of Candida is involved in chromosomal length polymorphism and serves as a site for ectopic pairing for recombination leading to translocations between nonhomologous chromosomes, a mechanism of karyotypic rearrangement that may serve as a model for chromosomal rearrangement in a variety of fungi and other eukaryotes. The establishment of the molecular karyotype of P. brasiliensis is an essential step in the construction of detailed physical and genetic maps of the fungus genome as a resource for large-scale DNA sequencing projects. Mapping of many other genes and probes will be necessary to construct relevant physical linkage groups for the P. brasiliensis genome and to identify genetic markers of interest which may be linked to particular phenotypic traits in this organism. Genetic and physical maps of the P. brasiliensis genome would also facilitate the identification and characterization of genetic loci.
The fungal samples analyzed here were isolated from different sources (human patients, soil, and an armadillo), were representative of both clinical forms, the acute juvenile form and the chronic PCM, and were originating from different geographical regions (Brazil, Argentina, Peru, and Colombia) (Table 1). No correlation could be established between the karyotype profile and the clinical–epidemiological characteristics of the isolates. We found similar, though not identical, karyotype profiles for samples isolated from clinical and environmental sources, for isolates from patients with chronic and acute PCM, and for isolates from different geographic areas (Fig. 2 and Table 1). The karyotype profile of isolate 18749 from a Peruvian patient with acute PCM was similar to the profile of a Brazilian isolate (Ibia) collected from soil. The karyotype profile of the armadilloÕs isolate (Tatu) was similar to that of two clinical isolates, both corresponding to chronic PCM, one originating from Colombia (I9) and the other from Argentina (EPM45). Isolates Bt01 and 4947 from patients with chronic PCM and from distinct geographical regions, i.e., Brazil and Argentina, respectively (Fig. 2, Table 1), showed similar profiles. 3.4. P. brasiliensis genome size and ploidy It became apparent from the results shown above that the DNA content varied considerably among isolates. Since P. brasiliensis yeast cells are multinucleated, variations in the number of nuclei/cell would provide an explanation for the variability in DNA content. Indeed, the number of nuclei per cell of each isolate was found to range from 2 (isolate 113) to 6 nuclei per cell (isolate EPM 45); four nuclei per cell was the average number found in five out of 12 isolates (Bt01, 265, 18, SS, and Tatu) (Table 2). The results of genome sizing for 12 isolates using the same method as that employed by Cano et al. (1998) are shown in Table 2. Using this approach, the size of the nuclear genome of the fungus was shown to be about half of that estimated by fluorescence intensity measurements (45.7–60.9 Mb), suggesting that the nuclei of P. brasiliensis yeast forms were diploid. We found similar results for eight isolates (B-339, 4947, 18, 265, 18749, Ibia, I9, and Tatu) out of 12 studied (Table 2). However, we found indexes with a ratio close to 1.0 for four isolates (namely 113, SS, Bt01 and EPM45), suggesting that these isolates could be haploid (Table 2, see
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P column of bands). We used S. cerevisiae in order to check the validity of the correlation of fluorescence intensity with total nuclear DNA content and ploidy, since it was necessary to demonstrate a 1:2 ratio for the haploid and diploid strains, respectively. Optical and photometric methods (flow microfluorometry, fluorescent microscopy, photometry, and cytofluorometry) have been employed to estimate the DNA content and ploidy of many fungi (Lauer et al., 1977; Olaiya and Sogin, 1979; Pan and Cole, 1992; Riggsby et al., 1982; Suzuki et al., 1994; Tanaka et al., 1993; Torres-Guerrero, 1999; Wyder et al., 1998). The P. brasiliensis genome sizes calculated by the equation proposed in the present study ranged from 25.8 to 73.6 Mb. Comparison of the genome sizes estimated by this equation with those estimated by the addition of sizes of chromosomal bands provided a new insight into the ploidy of P. brasiliensis. Our data suggest that diploid and haploid isolates exist and that the existence of aneuploids should also be considered. Data on ploidy, although interesting, could not be considered as definitive, mainly because both methods presented limitations such as the impossibility to state that chromosomal bands are monosomic or disomic, and the difficulty of inferring cell ploidy on the basis of nuclear DNA content, which can vary during the cell cycle (cells could have been arrested either at the G1 or G2 phases of the cell cycle). We believe that the question of P. brasiliensis ploidy deserves further studies to clarify the proposition of the existence of haploid, diploid or aneuploid isolates of the fungus, as is the case for other pathogenic fungal species (Carr and Shearer, 1998; Lengeler et al., 2001; Sia et al., 2000; Torres-Guerrero, 1999).
Acknowledgments We thank Dr. Celia Maria de Almeida Soares (Universidade Federal de Goi as, Goi^ ania, Goi as, Brazil) for providing the gene fragments used as probes and Dr. Beatriz Castilho (UNIFESP/EPM, S~ ao Paulo, SP, Brazil) for the S. cerevisiae strains. This work was supported by grants from FAPESP (ZPC and JFSF), CNPq, CAPES, FAPEMIG (PSC), and PRONEX-CNPq (PSC).
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L.S. Feitosa et al. / Fungal Genetics and Biology 39 (2003) 60–69 Montoya, A.E., Alvarez, A.L., Moreno, M.N., Restrepo, A., McEwen, J.G., 1999. Electrophoretic karyotype of environmental isolates of Paracoccidioides brasiliensis. Med. Mycol. 37, 219–222. Olaiya, A.F., Sogin, S.J., 1979. Ploidy determination of Candida albicans. J. Bacteriol. 140, 1043–1049. Orbach, M.J., Vollrath, D., Davis, R.W., Yanofsky, C., 1988. An electrophoretic karyotype of Neurospora crassa. Mol. Cell. Biol. 8, 1469–1473. Pan, S., Cole, G.T., 1992. Electrophoretic karyotype of clinical isolates of Coccidioides immitis. Infect. Immun. 60, 4872–4880. Perfect, J.R., Magee, B.B., Magee, P.T., 1989. Separation of chromosomes of Cryptococcus neoformans by pulsed field gel electrophoresis. Infect. Immun. 57, 2624–2627. Peterson, S.W., Sigler, L., 1998. Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens. J. Clin. Microbiol. 36, 2918–2925. Queiroz-Telles, F., 1994. Paracoccidioides brasiliensis ultrastructural findings. In: Franco, M.F., Lacaz, C.S., Restrepo-Moreno, A., Del Negro, G. (Eds.), Paracoccidioidomycosis, second ed. CRC Press, Boca Raton, FL, pp. 27–47. Riggsby, W.S., Torres-Bauza, L.J., Wills, J.W., Townes, T.M., 1982. DNA content, kinetic complexity, and the question of ploidy in Candida albicans. Mol. Cell. Biol. 2, 853–862. Rutschenko-Bulgac, E.P., Sherman, F., Hicks, J.B., 1990. Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans. J. Bacteriol. 172, 1276–1283. San-Blas, F., Cova, L.J., 1975. Growth curves of the yeast-like form of Paracoccidioides brasiliensis. Sabouraudia 13, 22–29.
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San-Blas, F., San-Blas, G., 1994. Protoplast production from the yeast phase of Paracoccidioides brasiliensis. In: Maresca, B., Kobayashi, G.S. (Eds.), Molecular Biology of Pathogenic Fungi, second ed. Telos Press, New York, NY, pp. 469–478. Sia, R.A., Lengeler, K.B., Heitman, J., 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet. Biol. 29, 153–163. Skatrud, P.L., Queener, S.W., 1989. An electrophoretic molecular karyotype of an industrial strain of Cephalosporium acremonium. Gene 78, 331–338. Steele, P.E., Carle, G.F., Kobayashi, G.S., Medoff, G., 1989. Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA. Mol. Cell. Biol. 9, 983–987. Suzuki, T., Hitomi, A., Magee, P.T., Sakaguchi, S., 1994. Correlation between polyploidy and auxotrophic segregation in the imperfect yeast Candida albicans. J. Bacteriol. 176, 3345– 3353. Tanaka, T., Tanaka, R., Taguchi, H., Nishimura, K., 1993. Analysis of ploidy and sexual characteristics of natural isolates of Cryptococcus neoformans. Can. J. Microbiol. 39, 958–963. Tobin, M.B., Peery, R.B., Skatrud, P.L., 1997. An electrophoretic molecular karyotype of a clinical isolate of Aspergillus fumigatus and localization of the MDR-like genes Afu MRD1 and Afu MRD2. Diagn. Microbiol. Infect. Dis. 29, 67–71. Torres-Guerrero, H., 1999. Ploidy in Sporothrix schenckii. Fungal Genet. Biol. 27, 49–54. Wyder, M.A., Rasch, E.M., Kaneshiro, E.S., 1998. Quantitation of absolute Pneumocystis carinii nuclear DNA content. Trophic and cystic forms isolated from infected rat lungs are haploid organisms. J. Euk. Microbiol. 45, 233–239.
Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis Luciano dos S. Feitosa,a,1 Patrıcia S. Cisalpino,b,1 M arcia R. Machado dos Santos,a a a Renato A. Mortara, T^ ania F. Barros, Fl avia V. Morais,a Rosana Puccia,a Jose Franco da Silveira,a and Zoilo P. de Camargoa,* b
a Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP/EPM – Universidade Federal de S~ ao Paulo, S~ ao Paulo, SP, Brazil Departamento de Microbiologia, Instituto de Ci^ encias Biol ogicas, UFMG – Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Received 2 August 2002; accepted 24 January 2003
Abstract Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: P. brasiliensis; Clinical and environmental isolates; Gene mapping; Genome size
1. Introduction Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America (Lacaz, 1994). The disease presents two progressive clinical forms. The chronic form affects rural workers, mainly male adults, evolves gradually in the lungs, presenting or not disseminated lesions depending on the integrity of cell immunity. Acute (or subacute) PCM affects mainly children and young adults of both sexes, progresses rapidly and disseminates through the lymphatic system, with lymph node hypertrophy, intense hepatosplenomegaly, and involvement of other organs. If not treated, the severe acute form (juvenile
*
Corresponding author. Fax: +55-11-5571-5877. E-mail address: [email protected] (Z.P. de Camargo). 1 These authors contributed equally to this work.
type) frequently culminates with the patientÕs death (Franco, 1987; Franco et al., 1987). The genetic composition of P. brasiliensis is poorly known. A teleomorphic, sexual stage has not been determined, greatly impairing classical genetic analysis. There are few reports on the isolation and characterization of mutants. The multinucleated nature of the fungus, even of the unicellular, parasitic yeast form, has been pointed out as the reason for the phenotypic instability and high number of revertants of the in vitrogenerated mutants (Hallak et al., 1982; McEwen et al., 1987; Queiroz-Telles, 1994). The development of pulsed-field gel electrophoresis (PFGE) has allowed the genomic characterization, gene mapping, and molecular epidemiological biotyping of strains from microorganisms refractory to genetic analysis (Altboum, 1994; Pan and Cole, 1992; Perfect et al., 1989; Steele et al., 1989). Montoya et al. (1997) and Cano et al. (1998), respectively, studying eight and two clinical isolates, described conditions for the
1087-1845/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00003-3
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separation of P. brasiliensis chromosome-sized DNA molecules by PFGE. Recently, Montoya et al. (1999) by karyotyping five environmental isolates reported a discrete chromosomal polymorphism, i.e., clinical isolates showing five identical chromosomal DNA bands, with the environmental isolates closely resembling the clinical isolates, distinguished only by a slight difference in mobility of two bands. In contrast, in our previous work, we found that the karyotype profiles of two isolates were highly polymorphic. Two probes were mapped to chromoblots and we concluded that it would be very difficult to correlate chromosome-sized DNA molecules and karyotype profiles from different P. brasiliensis isolates without probing them with a panel of specific genes or sequences (Cano et al., 1998). In the present report, we describe intraspecific variation observed by karyotyping 12 P. brasiliensis isolates. In an attempt to assess strain relatedness we mapped nine homologous genes to chromosomes of isolates from different patients with different clinical forms (chronic and acute) and of different geographical origins. This represents the first comparative mapping study among different isolates. Gene mapping allowed the identification of synteny groups, and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. We also addressed the question of the fungus ploidy by comparing data on genome sizes generated by PFGE and microfluorometry for all 12 isolates.
2. Materials and methods 2.1. Microorganisms The 12 clinical and environmental isolates of P. brasiliensis selected for this study are listed in Table 1.
Table 1 Characteristics of the P. brasiliensis isolates analyzed in this study Isolate name or number
Origin
Source
B-339 (ATCC-32069) 113 SS (ATCC-200272) Bt01 4947 18 265 (ATCC-200443) 18749 Ibi a I9 EPM 45 Tatu
Brazil Brazil Brazil Brazil Argentina Brazil Brazil Peru Brazil Colombia Argentina Brazil
Chronic PCM Chronic PCM Acute PCM Chronic PCM Chronic PCM Chronic PCM Chronic PCM Acute PCM Soil Chronic PCM Chronic PCM Armadillo
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Saccharomyces cerevisiae haploid strain 167-C and diploid strain BCV-59 (Castilho-Valavicius et al., 1992) were provided by Dr. Beatriz Castilho (Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP/EPM, S~ao Paulo, SP, Brazil). Fungal isolates were maintained by periodic subculturing in slanted tubes of PYG medium (yeast extract 5 g, bacto peptone 10 g, dextrose 15 g, and Agar 15 g l1 ) at 35 )37 °C. 2.2. Separation of chromosome-sized DNA molecules of P. brasiliensis by PFGE The procedures were carried out essentially as described by Cano et al. (1998), with minor modifications. P. brasiliensis yeast cells were subcultured three times in PYG medium at 5-day intervals in order to obtain cells in the late exponential phase (San-Blas and Cova, 1975; San-Blas and San-Blas, 1994). Erlenmeyer flasks containing 50 ml of PYG broth were inoculated with the entire growth of two culture slants, placed in a reciprocating shaker at 120 rpm and grown for 5–7 days at 35 °C. Cells were washed three times in 50 mM EDTA, resuspended in the same solution and sonicated (Branson Ultrasonic 450) for 5 s at low intensity (20 kHz) to disperse cellular aggregates. Cell concentration was adjusted to 1 109 cells ml1 and the preparation was mixed with an equal volume of low melting agarose (2% w/v in water); approximately 5 107 cells were immobilized in each gel plug (100 ll). Spheroplasts were obtained by digestion of cell walls with Novozym 234, 2.5 mg ml1 in PBS, pH 7.5, for 1 h at 37 °C. The blocks were dialyzed three times against 250 mM EDTA, pH 8.0, at 37 °C to inactivate the enzyme. Spheroplasts were disrupted for 24 h at 56 °C in a solution containing 500 mM EDTA, pH 8.0, 10 mM Tris–Cl, pH 8.0, 1% (w/v) sarkosyl, and 1 mg proteinase K per ml. The blocks were washed with 500 mM EDTA and stored at 4 °C in the same solution. Electrophoretic separation was performed under PFGE conditions in a Gene Navigator System (Pharmacia Biotech) with a hexagonal electrode array. The separations were carried out in 0.6% (w/v) agarose gel in 1.0 TAE (40 mM Tris acetate, pH 7.5, 2 mM EDTA, pH 8.0) kept at constant temperature (10 °C). Original running conditions (Cano et al., 1998) were slightly modified to achieve better resolution of chromosomal bands ranging from 4.7 to 10 Mb: we used homogeneous pulses (N/S, E/W) with interpolation for 120 h at 42 V: phase 1, pulse time 900 s (run time 12 h); phase 2, pulse time 1800 s (run time 12 h); phase 3, pulse time 2700 s (run time 36 h); phase 4, pulse time 3600 s (run time 36 h); and phase 5, pulse time 4500 s (run time 24 h). After electrophoresis, the gels were stained with ethidium bromide 0.5 lg/ml1 and photographed. Chromosome-sized DNA molecules were
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subjected to acid depurination in the presence of 0.25 M HCl for 5 min and transferred to nylon membranes (Hybond N, Amersham) using 0.5 M Tris, pH 7.0, and 0.4 N NaOH plus 1.5 M NaCl as neutralization and transfer solutions, respectively (Birren and Lai, 1993). The CHEF DNA size marker Schizosaccharomyces pombe (Bio-Rad) was used as the chromosome-sized DNA standard. The apparent molecular size of each chromosomal band was plotted as a function of the distance of migration on the gel, considering the distance migrated by S. pombe known standard markers (Cox et al., 1990; Pan and Cole, 1992). The densitometric analysis of chromosome-sized DNA bands resolved by PFGE was performed using Kodak Digital Science 1D Image Analysis Software, version 3.0. 2.3. DNA probes P. brasiliensis cloned genes were used as probes: a 630 bp BamHI/HindIII fragment which contains 70% of the coding region of the Gp43 antigen gene was isolated from plasmid pUCGPb16A (GenBank U26160) (Cisalpino et al., 1996). Two fragments of about 600 bp corresponding to the homologous chitin synthase genes CHS1 and CHS2 were amplified from total DNA of the B-339 isolate by the method of Bowen et al. (1992), cloned into pUC18 (Sureclone system, Pharmacia), and sequenced (GenBank AF416865 and AF416866, respectively). A 28S rRNA gene fragment was amplified from the B-339 isolate by the method of Peterson and Sigler (1998), cloned into pUC18 and sequenced (GenBank AF416745). In an effort to determine the degree of gene synteny among the P. brasiliensis isolates, we chose several genes that are linked in isolate B-339: PbLON, an ATP-dependent proteinase gene (GenBank AF239178) and its neighbor PbMDJ1-homologue (GenBank AF334811) were identified in a 6.5 kb genomic clone isolated from a P. brasiliensis genomic library (Barros and Puccia, 2001); a Ran binding protein homologue gene (RanBP, unpublished data) was found flanking the Gp43 gene in a 3.8 kb EcoRI fragment cloned from isolate B-339 (Cisalpino et al., 1996). A fragment of the gene coding for a putative N-acetyl-glycosaminidase (NAG) (GenBank AF395815) and a putative mannosyltransferase gene (named clone 11) (GenBank AF374353) from P. brasiliensis were provided by Dr. Celia Maria de Almeida Soares (Departamento de Ci^encias Fisiol ogicas, ICB, Universidade Federal de Goi as, Goi^ ania, Goias, Brazil). Chromosomal bands of 9.5 and 3.5 Mb of isolate 113 were separated by PFGE and excised from the agarose gels. Gel slices were digested with bagarase (Maule, 1993) and the DNA recovered from each whole band was radiolabeled and used as a probe.
2.4. Radiolabeling of probes and Southern blot hybridization The DNA fragments cited above were radiolabeled (Rad primer labeling kit, Gibco-BRL) and used as probes in Southern blots. Hybridizations were carried out overnight at 42 °C in 50% (v/v) formamide, 5 SSC (1 sodium saline citrate ¼ 0.15 M NaCl, 0.015 M sodium citrate), 5 DenhardtÕs solution, 50 lg yeast tRNA, 100 lg sonicated herring sperm DNA per ml, and 0.1% (w/v) SDS. The filters were washed twice in 0.1 SSC/0.1% (w/v) SDS at 56 °C. 2.5. Preparation of DAPI-stained cells, confocal microscopy, and estimation of genome size P. brasiliensis cells were stained with DAPI and prepared for confocal microscopy as described by Cano et al. (1998). Images were observed with a Bio-Rad 1024UV confocal system attached to a Zeiss Axiovert 100 microscope using a 40 N.A. 1.2 Plan-Apochromatic (DIC) water immersion objective. All images were collected by Kalman averaging of at least 10 frames (512 512 pixels) using an aperture (pinhole) of 1.5 mm, with the zoom set at 3.5 and the photomultiplier gain kept at 1200 during all image acquisitions. DAPIstained nuclei in different fields that could be clearly distinguished were then subjected to serial optical sectioning (0.36 lm steps) and the fluorescence intensity of the volume of each nucleus was estimated using the processing software Lasersharp 3.2TC (Bio-Rad). Fluorescence and DIC prints were generated by dye sublimation on a Codonics NP1600 printer. The genome size of P. brasiliensis in megabases (GS) was calculated as follows: ðSc nÞ ðPb DFIÞ ; Sc n DFI where (Sc n) is the genome size (12.1 Mb) of an haploid strain of S. cerevisiae; (Pb DFI) and (Sc n DFI) are the values of DNA fluorescence intensity of DAPI-stained nuclei of P. brasiliensis and of a haploid S. cerevisiae strain, respectively. Note that the same calculation can be done using the genome size of the diploid strain of S. cerevisiae and its corresponding DFI value.
GS ¼
3. Results and discussion 3.1. Molecular karyotype and chromosomal polymorphism of P. brasiliensis In this paper, we refer to a DNA band visible on pulsed field gel after staining with ethidium bromide as a ‘‘chromosomal band,’’ which could contain one, two or more, not necessarily homologous, co-migrating
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Fig. 1. Karyotype profiles of 12 P. brasiliensis isolates. We present ethidium bromide-stained chromosomal bands separated by PFGE (arrowheads indicate the origin of the gels) and the graphic results generated by densitometry (Kodak Digital Science 1D) of the karyotypes of each isolate (A–G). The numbers on the left and on the top row of each graph indicate the sizes of the chromosomal bands in megabases (Mb) (smaller arrows point to the corresponding peaks). P. brasiliensis isolates are indicated at the top of each panel (A–G). The chromosomal bands of S. pombe (5.7, 4.6, and 3.5 Mb) were used as chromosome-size DNA standard.
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gration properties of circular DNA seem to be generally proportional to the total running time. For instance, the mtDNA of A. chrysogenum was estimated to be 27 kb in size, but it migrated in PFG like 1.8– 1.9 Mb linear DNA molecules (Skatrud and Queener, 1989). A relative stoichiometry staining of the chromosomal bands was observed in the majority of isolates, but some striking differences could be found: the 9.5 band was heavily stained in isolates 113 and SS when compared to other isolates (Fig. 1A). The largest chromosome-sized molecules resolved in this study were approximately 9 Mb (or 10 Mb) in size, close to the upper limits for pulsed field gel electrophoresis and we cannot rule out co-migration of two bands in the compression zone. The fluorescence intensity of bands of isolates 18749, 4947, and 18 was always comparatively weaker than that of isolates Bt01, EPM 45, and Tatu (Fig. 1A). Although all possible care was taken to standardize the number of cells ð5 107 Þ in the agarose plugs, variations in fluorescence intensity of chromosomal-sized DNA molecules among the isolates indicated that there still was DNA content variation among blocks of different isolates. Interfering factors could be the number of nuclei per yeast cell, considering the multinucleated nature of these cells, and the fungus ploidy (see Table 2), which will be discussed later.
chromosomes. The karyotype profiles of 12 P. brasiliensis isolates shown in Figs. 1A–G were confirmed by densitometric analysis of staining bands (Figs. 1B–G) and hybridization with 32 P-labeled P. brasiliensis total DNA (data not shown). The analysis of karyotype profiles revealed the presence of distinct chromosomal migration patterns showing either four bands (isolates B339, 113, SS, Bt01, 4947, 18 and 265; Figs. 1B–D) or five bands (isolates 18749, Ibi a, I9, EPM45, Tatu; Figs. 1E–G), in the 2.5–9.5 Mb range. The chromosomes are comparable in size and number to those of fungi belonging to the ‘‘Schizosacharomyces pombe-like’’ group, whose electrophoretic separation requires long pulse times over a period of 5–8 days (Neurospora crassa, Orbach et al., 1988; Histoplasma capsulatum, Steele et al., 1989; Aspergillus niger, Debets et al., 1990; Coccidioides immitis, Pan and Cole, 1992; Aspergillus fumigatus, Tobin et al., 1997; Trichoderma harzianum, G omez et al., 1997). A band of 9.5 Mb was observed in all isolates, and bands of 4.7 (4.5–5.0) and 2.5 Mb were detected in most isolates. On the other hand, several bands were found in a few isolates: a 3.5 Mb band was only detected in isolate 113; 8.3 and 8.4 Mb bands were present in isolates 113 and SS; and band of 7.7 and 7.8 Mb were found in isolates B-339 and I9. A fuzzy band of about 1.0 Mb stained with ethidium bromide was observed in all isolates. Its nature is not known, but the band could be the result of some degradation of large chromosomal bands during spheroplast preparation. It should also be considered that mitochondrial DNA (mtDNA) is present in the agarose plugs and, although it does not enter the gel, the mi-
3.2. Mapping of P. brasiliensis genes to the chromosomesized DNA molecules Nine P. brasiliensis gene probes were hybridized to Southern blots containing the chromosomal bands of 12
Table 2 Estimate of the genome size of P. brasiliensis isolates by microfluorometry of DAPI-stained nuclei: comparison with values obtained by summation of PFGE bands P Ploidy ratio Microorganisms Number of DFI/SDb Genome sized of PFGE a (Mb) nuclei bandsc S. c. BCV-59 S. c. 167-3C P. b. B-339 P. b. 113 P. b. SS P. b. Bt01 P. b. 4947 P. b. 18 P. b. 265 P. b. 18749 P. b. Ibi a P. b. I9 P. b. EPM45 P. b. Tatu a
1 1 5.7 2.0 4.6 3.8 2.6 4.3 3.7 2.5 5.4 5.0 6.0 4.3
3.19E + 04/0.49E + 04 1.33E + 04/0.37E + 04 6.33E + 04/1.06E + 04 3.09E + 04/0.86E + 04 3.80E + 04/0.81E + 04 3.39E + 04/1.23E + 04 5.73E + 04/1.70E + 04 6.86E + 04/1.49E + 04 5.22E + 04/1.28E + 04 8.83E + 04/1.60E + 04 8.32E + 04/1.46E + 04 8.01E + 04/1.60E + 04 3.42E + 04/1.10E + 04 7.99E + 04/0.80E + 04
—
– 28.9 23.8 24.9 24.0 23.7 23.3 23.4 29.6 30.1 31.2 30.5 30.5
24.2e 3.7 12.1e 3.4 53:0 8:8 25:8 7:2 31:7 7:0 28:3 10:3 47:8 14:1 57:2 2:4 43:5 10:5 73:6 13:3 69:4 12:2 66:8 13:3 28:5 9:6 66:6 6:7
Average number of nuclei per yeast cell determined by confocal fluorescence microscopy. DFI ¼ DNA mean (SD) fluorescence intensity measured in DAPI-stained nuclei by confocal microscopy. cP bands, weighted sum of P. brasiliensis chromosomal bands separated by PFGE. d Genome size of P. brasiliensis estimated according the equation GS ¼ Sc n Pb DFI=Sc DFI (see text). e Genome size of S. cerevisiae obtained from GenBank. b
2 (1.7–2.3) 1 (0.7–1.3) 1.8 (1.5–2.1) 1.1 (0.8–1.4) 1.3 (1.0–1.6) 1.2 (0.7–1.6) 2.0 (1.4–2.6) 2.4 (2.3–2.5) 1.8 (1.4–2.3) 2.5 (2.0–2.9) 2.3 (1.9–2.7) 2.1 (1.7–2.6) 1.0 (0.6–1.2) 2.2 (1.9–2.4)
2n n 2n n n n 2n 2n 2n 2n 2n 2n n 2n
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Fig. 2. Mapping of P. brasiliensis genes to the chromosome-sized DNA molecules. Autoradiograms resulting from Southern hybridization of 12 P. brasiliensis isolates with nine cloned genes (A–D) and a schematic representation of gene mapping (E). (A) Synteny group I composed by clone 11, PbLON, and PbMDJ1 homologue genes; (B) synteny group II composed by GP43, Ran BP homologue, and NAG homologue genes; (C) synteny group III composed by 28S rDNA and CHS1 genes; (D) CHS2 gene hybridization pattern. The numbers on the left indicate the sizes of the chromosomal bands in megabases (Mb). Arrows indicate the origin of the gel (slot). P. brasiliensis isolates are indicated on the top row of each autoradiogram. (E) Schematic representation of the karyotype and syntenic distribution of nine genes among the chromosomal bands of 12 isolates of P. brasiliensis. The small vertical colored rectangles represent the nine different probes that were mapped to the chromosomal bands of P. brasiliensis isolates (see color legend). The numbers on the left indicate the sizes of chromosomal bands in megabases (Mb). P. brasiliensis isolates are P indicated at the top of the figure. The number of chromosome-sized DNA bands and the summation of chromosomal band sizes ( ) for each isolate is indicated at the bottom of the figure. Arrows indicate the origin of the gel (slot).
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isolates. Fig. 2 shows the autoradiograms of chromoblot hybridization (Figs. 2A–D) and a schematic representation of the results (Fig. 2E). The PbMDJ1 probe hybridized to the same bands carrying the PbLON gene, confirming that they are linked in all isolates (Fig. 2A), and the clone 11 probe (a putative mannosyltransferase) also showed the same hybridization profile (Fig. 2A). This suggests that a synteny group could be used as a marker for a specific chromosome since it was found in the same band in all isolates: in 10 isolates it mapped to a chromosomal band of 9.5 Mb, but to an 8.3 Mb band in isolate 113, and to a 6.7 Mb band in isolate B-339. It is possible that isolate 113 originally had two distinct chromosomal bands of 9.5 Mb, and one of them originated the 8.3 Mb band by deletion of a large fragment (1.2 Mb). The GP43 and Ran BP gene probes displayed exactly the same hybridization patterns, confirming that they are also linked in all isolates studied here, a fact that would define a second chromosome (Fig. 2B). The NAG gene probe was located in the same chromosomal bands recognized by the GP43 and Ran BP probes in 10 of the isolates studied (Fig. 2B). There was a clear dissociation of these two genes from the NAG gene in isolates SS and B-339, i.e., in the former the GP43 and Ran BP genes mapped to a 9.5 Mb band while the NAG gene remained in the 4.7 Mb chromosomal band and in the latter they appeared in the 5.0 Mb band while the NAG gene mapped to a 9.5 Mb band. These results suggest the occurrence of chromosomal rearrangements and, in this group, the NAG gene would be more distant from genes GP43 and Ran BP. Hybridization of chromoblots with the rRNA probe revealed discrete polymorphic rRNA genes containing chromosomes among the different isolates (Figs. 2C and E). The rRNA and CHS1 probes mapped to the same chromosomal bands in 11 isolates (Figs. 2C and E): to a single 2.5 Mb band in eight isolates (Bt01, 4947, 18, 265, 18749, Ibi a, I9, and Tatu), and to two chromosomal bands in three other isolates (113, SS, and EPM 45), one of which was the 2.5 Mb, a band that can be quite conserved, and to an additional band of 3.5, 4.5 or 4.8 Mb, respectively. But the most divergent mapping pattern was that of isolate B339: while the rRNA probe mapped to a 6.7 Mb band, CHS1 mapped to the 7.7 Mb band, suggesting once again the occurrence of sequence rearrangements. The chitin synthase genes (CHS1 and CHS2) were not linked and mapped to different chromosome-sized DNA bands in all isolates. The hybridization pattern for the CHS2 gene probe was not similar to that of any of the above-mentioned groups (Fig. 2D) and could define a fourth P. brasiliensis chromosome. The results are indicative of the existence of intraspecific genetic variation. It appeared to be more variability among the three P. brasiliensis largest chromosomes,
bands of substantially different mobility, from which all nine probes hybridized. In natural isolates, the occurrence of important variations in chromosome sizes between different strains is especially frequent in fungi lacking a sexual cycle (Fierro and Martin, 1999). Candida albicans has a highly variable karyotype and many efforts have been made to understand the mechanism by which diversity is generated and chromosomal rearrangement has been proposed as a source of genetic variation (Magee, 1993; Rutschenko-Bulgac et al., 1990). Therefore, even though P. brasiliensis is asexual and does not go through a meiotic cycle, we suggest that the chromosomal rearrangements could provide a means for genetic variation in this organism From the results of hybridization of probes to P. brasiliensis chromosomal bands we could define three synteny groups containing two or three genes (group I: PbMDJ1, PbLON, and clone 11; group II: GP43, Ran BP, and NAG; group III: rDNA and CHS1) that are much conserved among 12 P. brasiliensis isolates, suggesting that, in spite of gross differences, there is an underlying similarity in the genome organization of different isolates. In Candida albicans it was observed that the overall structure of the genome is remarkably constant among isolates despite the chromosomal size variations and the occurrence of translocations (Magee, 1998). It is interesting to mention that the synteny group II was mapped to the 9.5 Mb band of isolate 113 where probe CHS2 did also map. Two probes from this group (GP43 and RanBP) mapped to the 9.5 band of isolate SS, where group I genes (MDJ1, LON, and clone 11) did also map. If these groups were located in two different chromosomes, and if CHS2 mapped to a third distinct chromosome, it would seem possible that each 9.5 Mb band of isolates 113 and SS is composed of two co-migrating heterologous chromosomes. This hypothesis is coherent with a previous observation of the high fluorescence intensity of the 9.5 Mb bands of isolates 113 and SS in ethidium bromide-stained gels (Fig. 1A). In order to establish the correlation among the 9.5 Mb bands, observed in all isolates, a band of that size from isolate 113 was removed from the gel, radiolabeled, and hybridized to chromoblots. The same procedure was carried out with a 3.5 Mb band which was detected only in isolate 113. Fig. 3 shows the autoradiograms and the resulting hybridization patterns. The 9.5 Mb labeled band hybridized exclusively with itself in isolate 113, suggesting that its genes are not distributed in the other chromosomal bands of this isolate. It strongly hybridized with the 9.5 Mb band of isolates SS, EPM45, Tatu, and I9 and weakly with the 9.5 Mb band of isolates B-339, 4947, 18, and 265. Hybridization signals were also seen in other sized bands of all isolates, except 113 and SS. If the large 9.5 band is a doublet, as suggested by the fluorescence intensity of agarose gels
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Having large chromosome segments that span these loci cloned in cosmids, BAC or YACs, will help to identify genes involved in the parasiteÕs survival, pathogenicity, and diagnosis. 3.3. Correlation between karyotype profiles and epidemiological characteristics of the fungal isolates
Fig. 3. Hybridization of different P. brasiliensis isolates using the whole 9.5 and 3.5 Mb chromosomal bands from isolate 113 as probes. The chromosomal bands of 9.5 and 3.5 Mb from isolate 113 were separated by PFGE, excised from the gel, radiolabeled, and hybridized to Southern blot with bands of 10 P. brasiliensis isolates. Panel A and B show the hybridization patterns, respectively, obtained with the whole 9.5 and 3.5 Mb chromosomal bands from isolate 113 used as probes. The numbers on the left indicate the sizes of the chromosomal bands in megabases (Mb). Arrows indicate the origin of the gel (slot). P. brasiliensis isolates are indicated at the top of each autoradiogram.
and by the hybridization patterns with specific genes, it should in fact probe to other bands in the other strains (Fig. 3). These data open the possibility of the existence of two 9.5 Mb heterologous chromosomes in most P. brasiliensis isolates which may have been fortuitously visualized in isolates SS and 113. Results of hybridization with the 3.5 Mb band probe showed homogenous signals distributed over all chromosomal bands in all isolates. The patterns showing cross-hybridization of the 9.5 and 3.5 Mb bands are strongly suggestive of the existence of repetitive sequences in the genome of P. brasiliensis. Virtually nothing is known about repetitive elements of the P. brasiliensis genome. Chibana et al. (2000) showed that a middle repeated DNA sequence of Candida is involved in chromosomal length polymorphism and serves as a site for ectopic pairing for recombination leading to translocations between nonhomologous chromosomes, a mechanism of karyotypic rearrangement that may serve as a model for chromosomal rearrangement in a variety of fungi and other eukaryotes. The establishment of the molecular karyotype of P. brasiliensis is an essential step in the construction of detailed physical and genetic maps of the fungus genome as a resource for large-scale DNA sequencing projects. Mapping of many other genes and probes will be necessary to construct relevant physical linkage groups for the P. brasiliensis genome and to identify genetic markers of interest which may be linked to particular phenotypic traits in this organism. Genetic and physical maps of the P. brasiliensis genome would also facilitate the identification and characterization of genetic loci.
The fungal samples analyzed here were isolated from different sources (human patients, soil, and an armadillo), were representative of both clinical forms, the acute juvenile form and the chronic PCM, and were originating from different geographical regions (Brazil, Argentina, Peru, and Colombia) (Table 1). No correlation could be established between the karyotype profile and the clinical–epidemiological characteristics of the isolates. We found similar, though not identical, karyotype profiles for samples isolated from clinical and environmental sources, for isolates from patients with chronic and acute PCM, and for isolates from different geographic areas (Fig. 2 and Table 1). The karyotype profile of isolate 18749 from a Peruvian patient with acute PCM was similar to the profile of a Brazilian isolate (Ibia) collected from soil. The karyotype profile of the armadilloÕs isolate (Tatu) was similar to that of two clinical isolates, both corresponding to chronic PCM, one originating from Colombia (I9) and the other from Argentina (EPM45). Isolates Bt01 and 4947 from patients with chronic PCM and from distinct geographical regions, i.e., Brazil and Argentina, respectively (Fig. 2, Table 1), showed similar profiles. 3.4. P. brasiliensis genome size and ploidy It became apparent from the results shown above that the DNA content varied considerably among isolates. Since P. brasiliensis yeast cells are multinucleated, variations in the number of nuclei/cell would provide an explanation for the variability in DNA content. Indeed, the number of nuclei per cell of each isolate was found to range from 2 (isolate 113) to 6 nuclei per cell (isolate EPM 45); four nuclei per cell was the average number found in five out of 12 isolates (Bt01, 265, 18, SS, and Tatu) (Table 2). The results of genome sizing for 12 isolates using the same method as that employed by Cano et al. (1998) are shown in Table 2. Using this approach, the size of the nuclear genome of the fungus was shown to be about half of that estimated by fluorescence intensity measurements (45.7–60.9 Mb), suggesting that the nuclei of P. brasiliensis yeast forms were diploid. We found similar results for eight isolates (B-339, 4947, 18, 265, 18749, Ibia, I9, and Tatu) out of 12 studied (Table 2). However, we found indexes with a ratio close to 1.0 for four isolates (namely 113, SS, Bt01 and EPM45), suggesting that these isolates could be haploid (Table 2, see
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P column of bands). We used S. cerevisiae in order to check the validity of the correlation of fluorescence intensity with total nuclear DNA content and ploidy, since it was necessary to demonstrate a 1:2 ratio for the haploid and diploid strains, respectively. Optical and photometric methods (flow microfluorometry, fluorescent microscopy, photometry, and cytofluorometry) have been employed to estimate the DNA content and ploidy of many fungi (Lauer et al., 1977; Olaiya and Sogin, 1979; Pan and Cole, 1992; Riggsby et al., 1982; Suzuki et al., 1994; Tanaka et al., 1993; Torres-Guerrero, 1999; Wyder et al., 1998). The P. brasiliensis genome sizes calculated by the equation proposed in the present study ranged from 25.8 to 73.6 Mb. Comparison of the genome sizes estimated by this equation with those estimated by the addition of sizes of chromosomal bands provided a new insight into the ploidy of P. brasiliensis. Our data suggest that diploid and haploid isolates exist and that the existence of aneuploids should also be considered. Data on ploidy, although interesting, could not be considered as definitive, mainly because both methods presented limitations such as the impossibility to state that chromosomal bands are monosomic or disomic, and the difficulty of inferring cell ploidy on the basis of nuclear DNA content, which can vary during the cell cycle (cells could have been arrested either at the G1 or G2 phases of the cell cycle). We believe that the question of P. brasiliensis ploidy deserves further studies to clarify the proposition of the existence of haploid, diploid or aneuploid isolates of the fungus, as is the case for other pathogenic fungal species (Carr and Shearer, 1998; Lengeler et al., 2001; Sia et al., 2000; Torres-Guerrero, 1999).
Acknowledgments We thank Dr. Celia Maria de Almeida Soares (Universidade Federal de Goi as, Goi^ ania, Goi as, Brazil) for providing the gene fragments used as probes and Dr. Beatriz Castilho (UNIFESP/EPM, S~ ao Paulo, SP, Brazil) for the S. cerevisiae strains. This work was supported by grants from FAPESP (ZPC and JFSF), CNPq, CAPES, FAPEMIG (PSC), and PRONEX-CNPq (PSC).
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