Genetic Heterogeneity in the rRNA Gene Locus of Trichophyton tonsurans

Abstract

Trichophyton tonsurans is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of T. tonsurans by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated T. tonsurans from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in T. tonsurans.

FIG. 1.

PCR-RFLP genotyping of T. tonsurans variants and subvariants. Scans of agarose gels are shown in the order of performance (genotyping scheme). Assay details including fragment sizes are provided in Materials and Methods. Lanes are labeled with variant (I to V) or subvariant (a to g). For the SNP 5 panel, only subvariant f remains uncut (evidenced by the absence of the 94-bp band); the remaining subvariants demonstrate an uppermost band at either 219 or 273 bp, depending on whether the first repeat 2 region is uninterrupted or interrupted by the 54-bp motif, respectively. For the deletion-insertion panel, subvariant d indicates the band pattern seen with the 10-bp insertion, and subvariant f indicates the band pattern for isolates possessing the 14-bp deletion. M, molecular size markers; Del, deletion; Ins, insertion. -ve, negative control.

FIG. 2.

Schematic overview of the VIR region showing the positions of sequence variations within the NTS. The values in parentheses indicate the nucleotide positions of the SNPs relative to the variant I sequence. Resultant haplotype subvariants are shown in the lower panel. The arrows indicate the tandem arrangement of two repeat elements. DEL, deletion; INS, insertion.

FIG. 3.

Full-sequence data for the VIR region by variant (the repeat 1 elements are highlighted in black) and for the insertion and deletion sequences, with repeat motifs underlined.

FIG. 4.

T. tonsurans-specific duplex PCR. (a) Amplification of a 207-bp T. tonsurans-specific NTS fragment and an ∼300-bp conserved NTS fragment from a panel of dermatophytes and nondermatophyte species; (b) amplification of ITS-2 as DNA quality control for the same samples as are shown in panel a. M, molecular size markers.