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1 Institute of Biological Sciences, University of Wales, Aberystwyth, Wales, UK
2 Department of Dermatology, Kanazawa Medical University, Uchinada, Japan
3 Mycology Reference Centre, Department of Microbiology, General Infirmary, and University of Leeds, Leeds, UK
Correspondence
Colin J. Jackson
coj{at}aber.ac.uk
Received 20 April 2006
Accepted 11 July 2006
Abbreviations: LSU, large subunit; NTS, nontranscribed spacer; SRE, subrepeat element; SSU, small subunit; VIR, variable internal repeat.
The GenBank/EMBL/DDBJ accession number for the NTS region of T. mentagrophytes var. interdigitale, containing the coordinates of the three subrepeat loci, is DQ486866.
Supplementary data are available with the online version of this paper.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The two most common agents of tinea pedis are the anthropophilic dermatophytes Trichophyton mentagrophytes var. interdigitale and Trichophyton rubrum (Zaias & Rebell, 2003). Both species cause very similar clinical conditions, but are quite well separated in the phylogeny of the genus. T. mentagrophytes var. interdigitale has been placed in a separate species, T. interdigitale, by some authors (Gräser et al., 1999a), but this group of dermatophytes, isolated almost exclusively from the skin and nails of human feet, is distinctive regardless of name. Isolates of T. mentagrophytes var. interdigitale can show substantial variation in colony morphology and appearance, but these characters can alter greatly on subculture, and are therefore inappropriate for use as phenotypic strain markers.
Molecular subtyping offers an alternative way of identifying individual fungal isolates for epidemiological purposes. We have previously developed a genotyping procedure based upon variable tandem subrepeat elements (SREs) in the rDNA gene cluster of T. rubrum (Jackson et al., 2000). Recently, T. mentagrophytes var. interdigitale has also been shown to possess genetic polymorphisms that map to the rDNA (Mochizuki et al., 2003). Here, we report the molecular characterization of the nontranscribed spacer (NTS) of the rDNA of T. mentagrophytes var. interdigitale, and identify three variable loci within it that consist of tandemly repetitive subelements. PCR amplification of each of these polymorphic NTS loci generated simple, discriminatory patterns, which when combined produced a robust molecular subtyping scheme for this dermatophyte.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. All were randomly selected isolates cultured from clinical samples submitted from patients with dermatophytoses, though the exact specimen type was not available in some cases. Twenty samples were obtained from the Mycology Reference Centre at Leeds University Medical School, Leeds, UK, and 14 from the Department of Dermatology at St Thomas's Hospital, London. Strains from Japan (six), India (two) and Belgium (one) were provided by the Department of Dermatology, Kanazawa Medical University, Japan. The clinical isolates were identified on the basis of microscopy and colony morphology from the primary cultures, and by restriction profiling of ITS regions 1 and 2 (Jackson et al., 1999; Mochizuki et al., 2003).
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PCR amplification and cloning of the NTS region.
The NTS region was successfully amplified from three T. mentagrophytes var. interdigitale isolates (2111, EP0914 and KMU.Aik) using a modification of the procedure described previously (Jackson et al., 2000). The PCR reaction contained 1x Expand Long Buffer 3, 2.75 mM magnesium chloride, 500 µM each deoxynucleoside triphosphate (dATP, dCTP, dTTP, dGTP), 300 nM of each conserved primer [forward 25SCON2, reverse NS1-R (White et al., 1990; Table 2)], 3.75 units of Expand Long high fidelity Taq DNA polymerase (Expand Long Template PCR System, Roche) and 1 µl genomic DNA template (
20 ng), in a total volume of 50 µl. The thermal profile was altered to optimize the yield, with an initial denaturation of 95 °C for 2.5 min, followed by ten cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 35 s and extension at 68 °C for 75 s. This was followed by 20 cycles using exactly the same thermal profile, but with an additional 20 s of extension time on each repeated cycle. A terminal extension of 4 min at 68 °C completed the reaction.
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PCR amplification of the subrepeat regions S0, S1 and S2.
A single generic PCR reaction mixture and thermal profile was developed to allow amplification of all three polymorphic loci using the same conditions. PCR primers were designed for the specific amplification of each of the three loci containing repeats TmiS0, TmiS1 and TmiS2 (Table 2
). The PCR mixture contained 1x reaction buffer (Promega), 1.5 mM magnesium chloride, 20 pmol each primer, 0.2 mM each dNTP and 1.25 units Taq polymerase (Promega), in a total volume of 49 µl. One microlitre of genomic DNA template was added per reaction containing >104 copies of the target sequence (20 ng per reaction). Thermal cycling conditions were an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 90 s. A terminal extension of 5 min at 72 °C completed the reaction. Polymorphisms between PCR products from each locus were identified by comparative gel electrophoresis
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Identification of SREs
Dot-matrix self-comparison of the NTS sequence from isolate 2111 identified three separate regions containing repetitive elements. Each individual repeat unit was identified using co-ordinates from the dot matrix, and also by comparison with known SREs from T. rubrum (Jackson et al., 2000) and T. tonsurans (Gaedigk et al., 2003). The repeats in each of the three regions varied markedly in number, length and sequence homology. The first region, named TmiS0, contained eight copies of a short, highly degenerate repeat that varied in length from 32 to 43 residues, with an interruption of 20 bp in the fourth repeat (Fig. 1). There were no counterparts of the TmiS0 elements in either T. rubrum or T. tonsurans.
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). Homologues of these partial elements have been categorized as discrete repetitive units in T. tonsurans (Gaedigk et al., 2003), but sequence alignments suggest that in T. mentagrophytes var. interdigitale, they are specifically truncated forms of entire TmiS1 repeats. A limited number of VIRs from T. tonsurans have been found to contain an ‘interruption’ sequence (Gaedigk et al., 2003). When multiple TmiS1 elements were sequenced from different strains of T. mentagrophytes var. interdigitale, this ‘interruption’ was present in every copy (n=12), and thus formed an integral part of the TmiS1 repeat structure in this species.
The third region, named TmiS2, contained four full-length repeats of between 85 and 89 bp, and two partial copies of 70 and 66 bp, respectively. The TmiS2 repeats showed substantial positional and primary sequence conservation with the TrS2 repeat region of T. rubrum and TvS2 of T. violaceum, in contrast to T. tonsurans, which contains no repetitive elements at this location. A comparison of the S2 repeats in these species is available in Supplementary Fig. S2 in JMM Online.
Strain identification by PCR fingerprinting of the three Tmi subrepeat loci
Amplification products from each of the three loci showed different types of strain-specific polymorphisms (Fig. 2a–c). The least polymorphic locus was TmiS2, for which 37/42 isolates (88 %) had a single-banded PCR product of 684 bp. This was designated pattern type II. Two isolates (EP0843 and EK1471) produced a smaller fragment of approximately 600 bp (type I), which was also amplified from the teleomophic species Arthroderma vanbreuseghemii. Two further isolates (EF2608 and EP1174) produced progressively larger fragments of approximately 920 and 1000 bp, respectively, designated types III and IV. The TmiS2 pattern types I–IV are illustrated in Fig. 2(c). The size of the TmiS2 elements was approximately 85 bp, and hence the type I pattern may represent five repeats; type II, six repeats; type III, ten repeats and type VI, 11 repeats.
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), with ten different PCR types identified from the 42 isolates. However, a majority of strains (27/42; 64 %) again showed one common pattern type (type D), represented by a single 800 bp PCR fragment. The nine other variable types, designated A–C and E–J, were identified either from low numbers of isolates or in single strains only (Table 1). The first six types (A–F) had a single band of variable size, but four isolates (types G–J) had complex patterns containing multiple bands (Fig. 2a). Two isolates from India (SM8774 and SM8779) failed to amplify with these primers. A characteristic low-molecular-mass band of approximately 450 bp was amplified from the teleomorph A. vanbreuseghemii (lane B, Fig. 2a). Preliminary sequence data for some of the PCR fragments from types A–E indicate that a combination of repeat element multiplications and insertion/deletion events is responsible for the polymorphisms observed at this locus.
The TmiS1 region showed the greatest variation, but as with the other loci, a single PCR pattern predominated. This pattern, designated type 2, was represented by a 1175 bp PCR fragment containing two copies of the TmiS1 SRE, and was present in 23/42 isolates (55 %). Five simple pattern types (types 1–5) were observed with incremental size variations corresponding to full copies of the TmiS1 element. Five multiple-banded, complex PCR profiles (types 6–10) were identified for eleven isolates (lanes 6–10, Fig. 2b). Two isolates from India (SM8774 and SM8779) again failed to amplify with these primers. A unique, complex pattern (type 0; Fig. 2b, lane 0) was amplified from the TmiS1 locus of A. vanbreuseghemii.
Pattern variability at each individual repeat locus appeared to occur independently of the other two. By combining the PCR profiles, a total of 19 PCR types were recognized from the 42 isolates (Table 3), with a further unique type for A. vanbreuseghemii (pattern 20, type B 0 1; Table 3). Reproducibility was high, with DNA extracted from the same strain by different investigators producing identical PCR profiles. Two isolates from the Indian subcontinent produced fragments with the TmiS2 primers, but could not be typed from the S0 and S1 regions. Phenotypically, these isolates were T. mentagrophytes var. interdigitale, and showed ITS restriction profiles characteristic of this species. However, isolates previously identified as T. mentagrophytes var. mentagrophytes (Mochizuki et al., 2003) which were from non-foot sites with patients reporting animal contact as a likely causal factor, also produced an S2 product alone when amplified with the three sets of fingerprinting primers (data not shown). We are therefore further investigating the molecular identity of the Indian strains by sequence analysis of the ITS and NTS regions.
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In contrast to the divergence of the TmiS1 and TrS1 repeats, the TrS2 elements in T. rubrum have close sequence homology to the corresponding TmiS2 repeats in T. mentagrophytes var. interdigitale, and these conserved repetitive regions, proximal to the SSU gene, may have a role in the regulation of rDNA transcription.
Pattern distributions among clinical sites/countries
There are remarkable parallels between the distribution of PCR types in T. mentagrophytes var. interdigitale and of those in T. tonsurans and T. rubrum. In all three species, a majority of isolates belong to only one PCR type, and this type is characterized by having either one or two copies of the main repeat element. In T. rubrum and T. mentagrophytes var. interdigitale, the repeat locus closest to the SSU rRNA gene (TmiS2, TrS2) shows much less interstrain polymorphism than the second, larger locus (TmiS1, TrS1). The predominance of a single PCR type, and its occurrence in different geographic locations, imply clonality in all these species. In the current study, no clear associations existed between the PCR type and the geographic origin or clinical site of isolates, but a much larger and better-documented sample base is required in order to identify any such links.
Conclusion
Molecular studies have demonstrated that, as with T. rubrum (Gräser et al., 1999b), there is a lack of differentiation between strains of T. mentagrophytes var. interdigitale at the genetic level (Mochizuki et al., 1996). Adapted strains may have spread throughout the human population very rapidly (Charif & Elewski, 1997), and this recent emergence or ‘epidemic’ spread of a limited number of strain types has generated the pattern distributions we observe here. It is possible that this explosive increase in dermatophyte population size has influenced the accumulation of polymorphisms in specific genetic markers, such as the rRNA subrepeat elements reported here, since the copy number of such tandemly repetitive regions is regulated by events occurring at mitosis, such as unequal crossover (Szostak & Wu, 1980). These markers, and other mini- and microsatellites, currently represent the most effective, and often the only practical means, for the molecular subtyping of anthropophilic dermatophytes (Jackson et al., 1999, 2000; Gaedigk et al., 2003; Mochizuki et al., 2003; Ohst et al., 2004). Our report of a rapid, simple and discriminatory method for subtyping T. mentagrophytes var. interdigitale will provide a valuable resource for future investigations into the epidemiology and pathogenesis of this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  J.-C. Chang, M. M.-L. Hsu, R. C. Barton, and C. J. Jackson High-Frequency Intragenomic Heterogeneity of the Ribosomal DNA Intergenic Spacer Region in Trichophyton violaceum Eukaryot. Cell, April 1, 2008; 7(4): 721 - 726. [Abstract] [Full Text] [PDF]
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