Phylogeny of dermatophytes with genomic character evaluation of clinically distinct Trichophyton rubrum and T. violaceum

Abstract

Trichophyton rubrum and T. violaceum are prevalent agents of human dermatophyte infections, the former being found on glabrous skin and nail, while the latter is confined to the scalp. The two species are phenotypically different but are highly similar phylogenetically. The taxonomy of dermatophytes is currently being reconsidered on the basis of molecular phylogeny. Molecular species definitions do not always coincide with existing concepts which are guided by ecological and clinical principles. In this article, we aim to bring phylogenetic and ecological data together in an attempt to develop new species concepts for anthropophilic dermatophytes. Focus is on the T. rubrum complex with analysis of rDNA ITS supplemented with LSU, TUB2, TEF3 and ribosomal protein L10 gene sequences. In order to explore genomic differences between T. rubrum and T. violaceum, one representative for both species was whole genome sequenced. Draft sequences were compared with currently available dermatophyte genomes. Potential virulence factors of adhesins and secreted proteases were predicted and compared phylogenetically. General phylogeny showed clear gaps between geophilic species of Arthroderma, but multilocus distances between species were often very small in the derived anthropophilic and zoophilic genus Trichophyton. Significant genome conservation between T. rubrum and T. violaceum was observed, with a high similarity at the nucleic acid level of 99.38 % identity. Trichophyton violaceum contains more paralogs than T. rubrum. About 30 adhesion genes were predicted among dermatophytes. Seventeen adhesins were common between T. rubrum and T. violaceum, while four were specific for the former and eight for the latter. Phylogenetic analysis of secreted proteases reveals considerable expansion and conservation among the analyzed species. Multilocus phylogeny and genome comparison of T. rubrum and T. violaceum underlined their close affinity. The possibility that they represent a single species exhibiting different phenotypes due to different localizations on the human body is discussed.

Keywords: Adhesion; Arthrodermataceae; Character analysis; Dermatophytes; Genome; Phylogeny; Protease; Trichophyton rubrum; Trichophyton violaceum.

Fig. 1

Phenotypes of two anthropophilic dermatophytes. A–C.Trichophyton violaceum, CBS 141829. A. colony on SGA, 3 wk, 27 °C, obverse and reverse. B. non-sporulating hyphae. C. clinical image of the isolate (tinea capitis). D–G.Trichophyton rubrum, CBS 139224. D. colony on SGA, 3 wk, 27 °C, obverse and reverse. E. microconidia. F. macroconidia. G. clinical image of the isolate (onychomycosis). Scale bar = 10 μm.

Fig. 2

Maximum likelihood phylogenetic tree of rDNA ITS of 264 dermatophyte strains, using RaxML v. 8.0.0 under gtrcat model and 1000 bootstrap replications. Bootstrap support above 80 % is shown above branches. Species complexes are indicated when ITS distinction of taxa was not unambiguous (marked with ∼). Guarromyces ceretanicus CBS 269.89 was used as outgroup. Abbreviations used: A = authentic, ET = epitype, NT = neotype, T = type; MT = mating type. Numbers in bold are authentic or reference for described taxa.

Fig. 2

Maximum likelihood phylogenetic tree of rDNA ITS of 264 dermatophyte strains, using RaxML v. 8.0.0 under gtrcat model and 1000 bootstrap replications. Bootstrap support above 80 % is shown above branches. Species complexes are indicated when ITS distinction of taxa was not unambiguous (marked with ∼). Guarromyces ceretanicus CBS 269.89 was used as outgroup. Abbreviations used: A = authentic, ET = epitype, NT = neotype, T = type; MT = mating type. Numbers in bold are authentic or reference for described taxa.

Fig. 3

Comparison of five gene-trees based on maximum datasets of strains analyzed (ITS n = 238, LSU n = 219, TUB2 n = 198, TEF3 n = 211, RP-60S L1 n = 222), compared with a set of strains for which all genes were sequenced (n = 147). Phylogenetic analysis was done with RaxML, MrBayes, using Guarromyces ceretanicus or Ctenomyces serratus as outgroup. Bootstrap values > 80 % are shown with the branches.

Fig. 4

Mitochondrial genomes of Trichophyton rubrum CBS 139224 and T. violaceum CBS 141829. Green blocks: tRNA coding genes, blue arrows: genes, yellow arrows: protein coding sequences, red arrows: rDNA coding sequence. ORFs are shown with blue arrows without corresponding yellow arrows.

Fig. 5

A, B. Approximate MAT1-1 locus of dermatophytes. A. Locus as present in T. rubrum CBS 118892, T. tonsurans CBS 112818, T. verrucosum HKT0517, T. benhamiae CBS 112371, N. gypsea CBS 118893, and M. canis CBS 113480. B.MAT1-2 locus present in T. equinum CBS 127.97. C, D.MAT1-1 locus with numbers of amino acids. C.T. rubrum CBS 139224. D.T. violaceum CBS 141829.

Fig. 6

Non-rooted Maximum likelihood trees of dermatophyte deuterolysins (M35 family) and fungalysins (metalloproteinases, M36 family) constructed with Mega v. 6.0 with 500 bootstrap replications. *M35 family members denominated herewith.

Fig. 7

Non-rooted Maximum likelihood trees of dermatophyte secreted proteases (S8A families) constructed with Mega v. 6.0 with 500 bootstrap replications.