Dermatophytes are the group of filamentous fungi infecting keratinized structures such as skin, hair, and nails. Knowledge about genes and molecular mechanisms responsible for pathogenicity, as well as other biological properties of Microsporum canis is still relatively poor. The qRT-PCR is a reliable technique for quantifying gene expression across various biological processes, and choosing a set of suitable reference genes to normalize the expression data is a crucial step of this technique. We investigated the suitability of nine candidate reference genes: β-act, β-tub, adp-rf, ef1-α, sdha, rpl2, mbp1, psm1, and rGTPa for gene expression analysis in the dermatophyte M. canis in response to different carbon sources, phosphate levels, and pH shifts - factors that are extremely important and necessary for growth of dermatophyte in the host tissue. The transcription stability of these genes was evaluated using NormFinder, geNorm, BestKeeper, and RefFinder software. Regarding expression stability, mbp1, β-act, and sdha were the most stable housekeeping genes which we recommend for future qRT-PCR studies on M. canis strains. To the best of our knowledge this is the first study on selection and validation of reference genes for qRT-PCR data normalization in M. canis growth in culture media which promote adhesion-inducing conditions.

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Keratin is important and needed for the growth of dermatophytes in the host tissue. In turn, the ability to invade keratinised tissues is defined as a pivotal virulence attribute of this group of medically important fungi. The host–dermatophyte interaction is accompanied by an adaptation of fungal metabolism that allows them to adhere to the host tissue as well as utilize the available nutrients necessary for their survival and growth. Dermatophyte infections pose a significant epidemiological and clinical problem. Trichophyton rubrum is the most common anthropophilic dermatophyte worldwide and its typical infection areas include skin of hands or feet and nail plate. In turn, Microsporum canis is a zoophilic pathogen, and mostly well known for ringworm in pets, it is also known to infect humans. The aim of the study was to compare the intracellular metabolite content in the T. rubrum and M. canis during keratin degradation using liquid chromatography system coupled with tandem mass spectrometer (LC-MS/MS). The metabolite “fingerprints” revealed compounds associated with amino acids metabolism, carbohydrate metabolism related to the glycolysis and the tricarboxylic acid cycle (TCA), as well as nucleotide and energy metabolism. The metabolites such as kynurenic acid, l-alanine and cysteine in case of T. rubrum as well as cysteine and riboflavin in case of M. canis were detected only during keratin degradation what may suggest that these compounds may play a key role in the interactions of T. rubrum and M. canis with the host tissue. The metabolomic results were completed by qPCR gene expression assay. Our findings suggest that metabolomic analysis of T. rubrum and M. canis growing in culture media that mimic the dermatophyte infection could allow the understanding of processes involved in the pathogenesis of dermatophytes.

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Species identification of dermatophytes by conventional mycological methods based on macro-and microscopy analysis is time-consuming and has a lot of limitations such as slow fungal growth or low specificity. Thus, there is a need for the development of molecular methods that would provide reliable and prompt identification of this group of medically important fungi. The are many reports in the literature concerning PCR identification of dermatophyte species, but still, there are not many PCR assays for the separate detection of members of the genera Microsporum, especially Microsporum canis (zoophilic species) and Microsporum audouinii (anthropophilic species). The correct distinction of these species is important to determine the source of infection to implement the appropriate action to eliminate the path of infection transmission. In this paper, we present such a PCR-based method targeting velB gene that uses a set of two primers-Mc-VelB-F (5′-CTTCCCCACCCGCAACATC-3′) and Mc-VelB-R (5′-TGTGGCTGCACCTGAGAGTGG-3′). The amplified fragment is specific due to the presence of (CAGCAC) 8 microsatellite sequence only in the velB gene of M. canis. DNA from 153 fungal samples was used in PCR assay followed by electrophoretic analysis. The specificity of the designed set of primers was also confirmed using the online BLAST-Primer tool. The positive results were observed only in the case of M. canis isolates, and no positive results were obtained neither for other dermatophytes and non-dermatophyte fungi nor for other Eukaryotes, including the human genome sequence, as well as the representatives of bacterial and viral taxa. The developed PCR assay using the proposed Mc-VelB-F and Mc-velB-R primers can be included in the algorithm of M. canis detection in animals and humans.