Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum

The electrophoretic karyotype of the dermatophyte Trichophyton rubrum was established using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Five chromosomal bands of approximately 3.0 to 5.8 megabase pairs (Mbp) each were observed and together indicated that 22.05 Mbp of the total genome are organized as chromosomal macromolecules. In addition to establishing the number and size of T. rubrum chromosomes, these results open perspectives for the construction of chromosome-specific libraries and for the physical mapping of genes of interest, thus permitting future gene linkage studies in this pathogen. A detailed understanding of the karyotype and genomic organization of T. rubrum should contribute to further genetic, taxonomic and epidemiological studies of this dermatophyte.


Introduction
Trichophyton rubrum is a cosmopolitan filamentous fungus that can infect human keratinized tissue (skin, nails and, rarely, hair), and is one of the most frequently encountered dermatophytes (Costa et al., 2002;Jennings et al., 2002;Monod et al., 2002).This pathogen, which normally causes well-characterized superficial infections, also produces skin infections in unusual parts of the body in immunodepressed patients (Squeo et al., 1998;Sentamilselvi et al., 1998;Smith et al., 2001).Despite the incidence and medical importance of this dermatophyte, little is known about its genetic and biological characteristics.Classic genetic analysis is not possible with T. rubrum because no sexual phase is known.In addition, the number of chromosomes in this species has not been determined by light microscopy since mitotic material is generally unsuitable for the study of fungal chromosomes (Zolan, 1995).
Pulse-field gel electrophoresis (PFGE) is an effective method for separating chromosome-sized DNA, and is an important tool for basic genetic studies, especially in lower eukaryotes such as fungi and yeasts.Furthermore, summa-tion of the sizes of the different molecules separated by this technique provides an estimate of the genome size.Pulse-field gel electrophoresis and a refinement known as contour-clamped homogeneous electric field (CHEF) gel electrophoresis, has been used to determine the molecular karyotype of many fungi, including Aspergillus nidulans (Brody and Carbon, 1989), Aspergillus niger (Verdoes et al., 1994), Neurospora crassa (Orbach et al., 1988), Paracoccidioides brasiliense (Montoya et al., 1999), Aspergillus fumigatus (Tobin et al., 1997), and pathogenic yeasts such as Candida albicans and Candida glabrata (Fodor et al., 2002).
Since the karyotype of T. rubrum is unknown we have standardized the conditions for obtaining protoplasts in order to determine the electrophoretic karyotype of this species using CHEF gel electrophoresis and to estimate its genome size.

Strain
A clinical isolate of T. rubrum (H6) obtained from a patient admitted to the University Hospital of Ribeirão Preto, SP, Brazil, and identified by standard methods (McGinnis, 1980;Fachin et al., 1996) was used in this study.

Preparation of intact chromosomal DNA
After incubation of isolate H6 in Sabouraud glucose agar for 20 days at 28 °C, conidia and mycelium from one petri dish were carefully harvested and incubated with 100 mL of liquid Sabouraud glucose medium at 28 °C and 250 rpm for 18 h.The resulting mycelium was harvested by vacuum filtration through Whatman paper (Whatman International, Maidstone, UK) and washed with sterile water.Protoplasts were obtained by incubating the mycelium in 30 mL of lytic solution (600 mg of Glucanex (Novo Nordisk, Swiss), 50 mM KH 2 PO 4 , pH 7.5, and 0.7 M KCl) for 3-4 h at 28 °C.The suspension of protoplasts was filtered through glass wool and the protoplasts were collected by centrifugation (1575 g, 5 min).The pellet was resuspended and washed twice in STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 ).The final pellet was resuspended in protoplasting buffer (0.9 M sorbitol, 0.125 M EDTA, pH 7.5) to a protoplast concentration of 1.0 x 10 8 -1.5 x 10 8 cells per mL.The suspension was heated to 42 °C and an equal volume of molten 1.4% (w/v) InCert agarose (Seaken Gold agarose, FMC Bioproducts) in protoplasting buffer precooled to 42 °C was added.The agarose/protoplast mixture was then poured into a plug mold and solidified at room temperature for 30 min.Protoplast lysis was induced by adding proteinase K (2 mg/mL) in digestion buffer (0.5 M EDTA, pH 8.0, 10 mM Tris-HCl, 1% Na-N-laurylsarcosinate) at 50 °C for 72 h.Finally, the plug was washed three times in 50 mM EDTA, pH 8.0, at 50 °C and once at room temperature, and then stored at 4 °C in 50 mM EDTA.

Results
Intact chromosomes of strain H6 of T. rubrum were identified by CHEF gel electrophoresis (Figure 1).The ini-tial electrophoretic conditions did not adequately separate the smallest chromosomes (Figure 1A), but a second CHEF gel with conditions optimized for this size range showed a more efficient separation of these chromosomes (Figure 1B).Under the conditions used here, at least five chromosome bands were detected.Assuming a linear separation, comparison with the S. pombe and H. wingei molecular weight standards showed that these bands were 3.0, 3.05, 4.6, 5.6 and 5.8 Mbp in size.The chromosomes shown in Figure 1 have been numbered according to size, starting with the largest.This numbering is arbitrary and does not take into consideration the presence of possible co-migrating chromosomes.

Discussion
CHEF gel electrophoresis showed that the molecular karyotype of the clinical isolate H6 of T. rubrum consists of five chromosomal bands which range in size from 3.0 Mbp to 5.8 Mbp, and correspond to a total genome size of 22.05 Mbp.Thus, at least 22.05 Mbp of the genome are organized as chromosomal macromolecules.Table 1 shows the chromosome and genome sizes of other fungi compared to T. rubrum.
Chromosome length polymorphisms are common in fungi (Skinner et al., 1991) and variations in the molecular karyotype of T. rubrum may exist among different isolates.Such variability has been observed in yeast and fungal species such as Candida albicans (Pittet et al., 1991) and Septoria nodorum (Cooley and Caten, 1991), and may reflect chromosomal rearrangements, such as deletions, duplications and translocations, as well as different degrees of pathogenicity in the same species.CHEF analysis is useful in fungal epidemiological studies, and has revealed interand intraspecies variations in Candida infections (Pittet et al., 1991).The genetic variability of T. rubrum isolates has been demonstrated by sequence analysis of the 18S rDNA gene combined with morphological and enzymatic analysis (Nascimento and Martinez-Rossi, 2001).
The ability to separate intact chromosomes has led to the assignment of cloned genes to chromosomal locations without the need for crosses or progeny tests.Any cloned DNA segment may be assigned to a chromosome by Southern hybridization experiments (Skinner et al., 1991;Scherer and Magee, 1990).This strategy has been used to locate the MDR-like genes AfuMDR1 and AfuMDR2 of A. fumigatus (Tobin et al., 1997).
In addition to establishing the number and size of T. rubrum chromosomes, the present results open perspectives for constructing chromosome-specific libraries, for determining the presence of translocations, duplications and deletions, and for identifying the chromosomal location of genes of interest.This information should facilitate genetic linkage studies in this pathogen.The construction of a genetic map for T. rubrum will be useful for cloning genes of interest by "chromosome walking" to cognate genes (Agnan et al., 1997).The information obtained will contribute to our understanding of the genomic organization of this dermatophyte.Finally, determination of the T. rubrum genome size is important in order to determine the genome coverage of any genomic library of this fungus to be used in genome projects.The sequencing of the T. rubrum genome, together with that of 14 other fungi, has recently been proposed as part of the Fungal Genome Initiative (FGI) (http://www-genome.wi.mit.edu/seq/fgi).This project will allow the application of genomic technologies to the study of fungal pathogenicity, and is particularly important given the limited usefulness of classic genetic approaches for studying this species.

Figure 1 -
Figure 1 -CHEF electrophoretic karyotype of strain H6 of T. rubrum (1).(A) Separation of intact chromosomal DNAs (I to III) by CHEF in a 0.7% (w/v) agarose gel.M is the S. pombe standard.(B) Optimized CHEF separation of chromosomes IV and V in a 0.8% (w/v) agarose gel.M is the H. wingei standard.The approximate lengths of the chromosomal bands are indicated.