Title: PRP8 intein applied to identification and phylogeny of dermatophytes.
Authors: Hans Garcia Garces, Raquel Theodoro Cordeiro, Eduardo Bagagli. 


Inteins are self-splicing protein elements found in some housekeeping genes important for cell survival. They can be found as a full-length intein when they have a splicing and a homing endonuclease (HE) domains or as a mini-intein when only the splicing domain is present. The PRP8 intein is inserted into the PRP8 protein, a core protein of the spliceosome complex responsible for removing introns during the splicing process. It was observed that variations in the nucleotide sequences of the PRP8 intein reflects the phylogenetic relationships of fungal species and its longitude polymorphism enables a proper molecular identification by electrophoresis techniques. Since a PRP8 intein was by us detected in dermatophytes we aimed to determine the phylogenetic relationship based in PRP8 intein and to identify clinically important Microsporum and Nannizzia species by amplification and visualization of some polymorphic regions (HE region) with no need of sequencing. Forty strains of 11 dermatophytes species were previously molecularly identified (by Internal Transcribed Spacers (ITS) and D1/D2 DNA ribosomal regions) and then, sequenced for PRP8 intein nucleotide region. Nucleotide PRP8 sequences were analyzed and employed for phylogenetic studies by using the software MEGA v 6.0. For electrophoretic molecular identification two sets of primers that amplified a size variable polymorphic region of the HE domain were designed and amplification trough PCR and visualization in agarose gel were performed.   The phylogenetic trees obtained by using the PRP8 nucleotide and amino acid sequences were concordant with other phylogenetic constructions that use other genetic regions (ITS and D1/D2). Arthroderma uncinatum was grouped within the Microsporum Clade (M. audouinii and M. canis). E. floccosum clustered within the Nannizzia genus clade, that encompassed the N. incurvata, N. gypsea, N. persicolor and N. fulva species. All Trichophyton species clustered as a separated clade, a proper resolution was achieved within the T. rubrum and T. interdigitale strains. A molecular identification by using amplification by PCR and visualization by agarose gel electrophoresis enabled the identification of herein studied Microsporum and Nannizzia species. Primers HE1F and HE2R were used for identifying Microsporum species (M. audouinii and M. canis), that possessed the biggest amplicon with 938 bp. Trichophyton species (T. rubrum and T. interdigitale) and Nannizia species were separated from Microsporum species with amplicons ranging from 620 to 680 bp. The specific primers HE1F and HE3R made possible to distinguish species within the Nannizzia genus. N. fulva e N. persicolor presented 350 bp, followed by N. gypsea with 327 bp and N. incurvata with 300 bp. We concluded that the nuclear region of the PRP8 intein is a suitable genetic marker for classifying and identifying dermatophytes.

PRP8 intein applied to phylogeny and species identification of dermatophytes