Unequal distribution of the mating type (MAT) locus idiomorphs in dermatophyte species

Fungal Genetics and Biology 118 (2018) 45–53

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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi

Unequal distribution of the mating type (MAT) locus idiomorphs in dermatophyte species

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Susanne Kosanke , Lutz Hamann, Christiane Kupsch, Sarah Moreno Garcia, Avneesh Chopra, Yvonne Gräser Institute of Microbiology and Hygiene, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Dermatophytes Mating type (MAT) locus Sexual reproduction Unisexual reproduction

The mating type (MAT) locus is the key regulator of sexual reproduction in fungi. In the dermatophytes and other Ascomycetes this genomic region exists in two distinct forms (idiomorphs) and their balanced presence is a precondition for successful mating in heterothallic fungi. But the MAT locus not only drives sexual reproduction, it has also been shown to influence pathogenicity, virulence, and/or morphological changes in pathogenic fungi of the genera Candida, Histoplasma, and Cryptococcus. In order to find out whether there are similar trends in dermatophytes, we investigated the MAT locus of 19 anthropophilic and zoophilic species via Sanger sequencing and primer walking. We identified for the first time the MAT locus idiomorphs of the dermatophyte species Microsporum audouinii (MAT1-2), M. ferrugineum (MAT1-2), Trichophyton schoenleinii (MAT1-2), T. bullosum (MAT1-1), T. quinckeanum (MAT1-1), T. concentricum (MAT1-1), T. eriotrephon (MAT1-1), and T. erinacei (MAT1-2). In addition, we determined the MAT locus sequence for dermatophyte species whose mating type idiomorphs had been described on the basis of results of classical confrontation experiments (e.g. M. canis, MAT1-2) and we confirmed recently published molecular data (e.g. T. rubrum, MAT1-2). Our results corroborate that MAT locus idiomorphs are unequally distributed in the majority of the analyzed species and the ability to mate with a partner of the opposite sex is limited to a few zoophilic species. Clonal spreads are identified that are connected to one of the idiomorphs and a higher virulence and/or a higher transmission rate to humans (T. benhamiae and T. mentagrophytes). For the imbalanced idiomorph distribution pattern we hypothesize that either: (I) one of the mating type idiomorphs may be extinct due to clonal reproduction (e.g., T. rubrum and M. canis), (II) mating partners of one species adapted to different hosts followed by speciation in the new niche (e.g., T. equinum and T. tonsurans) or (III) unisexual reproduction is the next evolutionary stage of propagation in dermatophytes which involves the extinction of one mating idiomorph.

1. Introduction The reproductive strategies in filamentous Ascomycetes are diverse. Heterothallism as well as homothallism and pseudohomothallism are observed among species of even one genus (Coppin et al., 1997). Unisexual reproduction, as a special mode of homothallism, has been described for pathogenic fungi like Cryptococcus neoformans and is expected to be present in other microbial pathogens, fungi, and eukaryotic kingdoms in general (Lin et al., 2005). Fungal sex is debated vigorously in the scientific community, because there are costs and benefits that are not easily outbalanced: on the one hand, via sexual reproduction, organisms are able to adapt to

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changing environments by outcrossing and lose deleterious alleles, but on the other hand it is an expensive process that requires two mating partners that will only transfer the half of their alleles to their offspring. In addition, recombination can also diminish or delete favorable welladapted traits (reviewed in (Ene and Bennett, 2014)). Dermatophytes belong to the Ascomycetes and the vast majority are heterothallic organisms, except for two homothallic species, Arthroderma curreyi and Arthroderma cifferii (Metin and Heitman, 2017). Dermatophytes degrade keratin in skin, hair and nails of humans (anthropophilic), animals (zoophilic) and keratinous detritus in soil (geophilic) (Weitzman and Summerbell, 1995). According to the new taxonomy, dermatophyte species are grouped into seven genera:

Corresponding author. E-mail address: [email protected] (S. Kosanke).

https://doi.org/10.1016/j.fgb.2018.07.003 Received 21 March 2018; Received in revised form 8 July 2018; Accepted 14 July 2018 1087-1845/ © 2018 Elsevier Inc. All rights reserved.

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Table 1 List of dermatophyte species used in this study and result of MAT locus analysis. (a): anthropophilic; AUT: authentic strain; ET: epitype; HT: holotype; NT: neotype; PT: paratype; T: type strain; (z): zoophilic. Reference strains were obtained from the “Westerdijk Fungal Biodiversity Institute” Utrecht, The Netherlands. Genus, Species

Trichophyton T. tonsurans (a)

Total number of strains

Reference and clinical strain number

8

Reference strains CBS 332.32 CBS 118.65 CBS 495.48 CBS 729.88

T. interdigitale (a)

29

T. mentagrophytes (z)

30

Clinical strains 4 Clinical strains 29 Reference strain CBS 642.73

Synonymized taxa, morphotypes

Geographic origin

Host

Mating type

T. tonsurans T. tonsurans sulfureum T. tonsurans crateriforme T. tonsurans sulfureum

var. epilans var.

Unknown Unknown

Unknown Human

MAT1-1 MAT1-1

var.

Unknown

Human

MAT1-1

var.

France

Human

MAT1-1

Germany and unknown

Human

MAT1-1

Germany

Human

MAT1-2

Unknown

Unknown

MAT1-1

Germany

Human, animal

24 x MAT1-2 5 x MAT11

New Zealand

Horse

MAT1-2

New Zealand

Horse

MAT1-2

New Zealand

Horse

MAT1-2

India India

Poultry Chicken

MAT1-1 MAT1-2

Unknown

Unknown

MAT1-2

The Netherlands

Human

MAT1-1

The Netherlands

Human

MAT1-1

The Netherlands and Unknown

Unknown

MAT1-1

The Netherlands

Human

MAT1-2

Unknown

Unknown

MAT1-2

Arthroderma vanbreuseghemii

Clinical strains 29

T. equinum (z)

3

Reference strains CBS 635.82 CBS 634.82 CBS 100080 T

T. simii (z)

T. quinckeanum (z)

3

5

Reference strains CBS 449.65 PT CBS 417.65 T Clinical strains 1 Reference strains CBS 106.67 CBS 318.56 NT

T. schoenleinii (a)

T. benhamiae (z)

T. concentricum (a)

16

23

13

T. equinum var. autotrophicum T. equinum var. autotrophicum T. equinum var. autotrophicum

T. mentagrophytes var. quinckeanum T. mentagrophytes var. quinckeanum

ITS type

Clinical strains 3 Reference strains CBS 433.63 Clinical strains 15 Reference strains CBS 809.72

A. benhamiae

Belgium

Dog

MAT1-1

CBS 280.83

A. benhamiae

The Netherlands

Human

MAT1-1

CBS 806.72

A. benhamiae

France

Guinea pig

MAT1-1

CBS 807.72

A. benhamiae

Spain

Human

MAT1-1

Clinical strains 10

Germany, Switzerland

MAT1-1

Yellow

1

Germany

Human and Unknown Human

MAT1-1

8

Germany, Switzerland

Cat, dog, guinea pig, rabbit

MAT1-2

White, Japanese White, European

Unknown New South Wales

Unknown Human

MAT1-1 MAT1-1

Unknown

Unknown

MAT1-1

Reference strains CBS 196.26 AUT CBS 563.83 Clinical strains 11

White, Japanese White, Japanese White, Japanese White, Japanese

(continued on next page)

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Table 1 (continued) Genus, Species

Total number of strains

Reference and clinical strain number

T. verrucosum (z)

7

Reference strains CBS 564.50 CBS 554.84 CBS 563.50

T. eriotrephon (z)

1

T. bullosum (z)

1

T. erinacei (z)

4

CBS 562.50 Clinical strains 3 Reference strains CBS 220.25 AUT Reference strains CBS 363.35 Reference strains CBS 511.73 T CBS 344.79 CBS 677.86 CBS 474.76

T. rubrum (a)

T. soudanense (a)

T. violaceum (a) Microsporum M. canis (z)

M. audouinii (a)

M. ferrugineum (a)

22

12

11

235

26

8

Reference strains CBS 118892 CBS 100084 CBS 288.86 CBS 289.86 CBS 417.52 CBS 360.53 CBS 389.58 CBS 384.64 CBS 847.73 CBS 551.86 CBS 734.88 Clinical strains 3 8 Reference strains CBS 452.61 Clinical strains 11 Clinical strains 11

Synonymized taxa, morphotypes

Geographic origin

Host

Mating type

T. verrucosum ochraceum T. verrucosum verrucosum T. verrucosum discoides T. verrucosum

var.

Unknown

Human

MAT1-2

var.

The Netherlands

Human

MAT1-2

var.

USA

Human

MAT1-2

var. album

USA

Human

MAT1-2

Unknown

Unknown

MAT1-2

Unknown

Unknown

MAT1-1

Unknown

Unknown

MAT1-1

var.

New Zealand

Hedgehog

MAT1-2

var.

The Netherlands

Human

MAT1-2

var.

Germany

Human

MAT1-2

var.

Great Britain

Human

MAT1-2

Germany Canada Canada Canada Switzerland Unknown The Netherlands The Netherlands The Netherlands Sweden Spain

Human Human Unknown Human Unknown Unknown Human Human Human Human Human

MAT1-1 MAT1-1 MAT1-1 MAT1-1 MAT1-2 MAT1-2 MAT1-2 MAT1-2 MAT1-2 MAT1-1 MAT1-2

USA Japan and Unknown

Human Unknown

MAT1-1 MAT1-1

Zaire

Human

MAT1-1

Africa

Unknown

MAT1-1

Unknown

Unknown

MAT1-1

Germany Japan Japan

Human Unknown Unknown

MAT1-1 MAT1-1 MAT1-2

Austria, Germany, Japan, Korea, Dominican Republic, Mexico, Turkey

Human and animal

MAT1-1

USA USA Canada Canada Canada

Unknown Unknown Human Human Human

MAT1-2 MAT1-2 MAT1-2 MAT1-2 MAT1-2

Great Britain, Germany, Netherlands, Canada

Human

MAT1-2

Thailand

Human

MAT1-2

T. mentagrophytes erinacei T. mentagrophytes erinacei T. mentagrophytes erinacei T. mentagrophytes erinacei

T. T. T. T. T. T. T. T. T. T.

raubitschekii fischeri kanei megninii megninii megninii megninii megninii megninii megninii

T. raubitschekii

Reference strains CBS 113480 CBS 496.86 ET CBS 495.86 ET Clinical strains 232

Reference strains CBS 215.47 CBS 344.50 CBS 108932 CBS 108933 CBS 108934 Clinical strains 21 Clinical strains 8

ITS type

Sum of all strains: 457

Hironaga et al., 1982), while this is less common in zoophilic species (Hasegawa and Usui, 1975; Weitzman and Padhye, 1978). In anthropophilic species like Trichophyton rubrum, sexual reproduction is not observed (Young, 1972). This is supported by the use of molecular markers for population genetic analysis (analysis of the reproductive

Trichophyton, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, Microsporum, and Arthroderma (de Hoog et al., 2017). Via mating experiments, sexual reproduction was observed in dermatophytes, but to varying degrees according to the environmental niches they live in: soildwelling species reproduce frequently sexually (Choi et al., 2012; 47

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found in soil isolates equally, whereas (−) mating type strains were predominantly found in clinical isolates (Kwon-Chung et al., 1974). A more recent study investigated H. capsulatum strains isolated from bats, soil and humans in Brazil and Mexico: Since only six human isolates were examined, no statement could be made regarding the possible disproportionate nature of identified MAT1 loci. In the 22 isolates of bats and from soil, neither of the two mating partners was present in significantly higher abundance, a disequilibrium was not detected (Rodríguez-Arellanes et al., 2013). The classical approach of mating type identification are crossing experiments of (+) and (−) strains of a species. This method, however, is indicative of mating type and because of the absence of the teleomorphic state in several dermatophyte species (zoophilic and anthropophilic) it cannot be applied to all dermatophyte species (Brasch, 2004). Therefore, we used the MAT locus analysis on the molecular level. The genetic structure of the MAT locus in dermatophytes is known for seven species in detail. For Trichophyton tonsurans, T. equinum, T. rubrum, Microsporum canis, and Nannizzia gypsea single strains have been analyzed by Li et al. and they revealed a genomic synteny between the species: The genes encoding the alpha-box (MAT1-1) and HMG domain (MAT1-2) proteins are flanked at the 5′-end by genes coding for a cytoskeleton assembly control protein (SLA2), cytochrome c oxidase subunit VIa (COX13), a protein similar to DNA lyase (APN2), a hypothetical protein and another hypothetical protein, all of which are closely linked to the idiomorph sequence downstream (Li et al., 2010).

mode based on recombination events) in dermatophyte species, which was unable to show sexual recombination events for T. rubrum, which is the most frequently isolated species of human skin and nail dermatophytosis. Ninety-six T. rubrum strains from four continents were analyzed and a clonal population was observed, which is very likely an adaption to a stable niche, the human host (Gräser et al., 2007; Persinoti et al., 2018). Sharma et al. examined the zoophilic Microsporum canis and revealed sexual and asexual reproduction by the identification of three genotypes within 137 strains, but with one genotype being prevalent in 74% of all human isolates, the scientists assumed a higher degree of virulence here (Sharma et al., 2007). Two opposite mating partners are essential for sexual reproduction in dermatophytes. Ribbon and Garber found a potential association between the mating partners and virulence in the 1960’s for the geophilic species Nannizzia fulva. During the infection of guinea pigs with N. fulva, the authors recognized that the two mating partners secreted proteases (e.g. elastase, alkaline phosphatase) to different degrees: the (+) mating type was able to secrete proteases and thus cause a more profound infection than the (−) mating type, which showed no enzyme production (Rippon and Garber, 1969). A link between virulence and one mating partner has been suggested for pathogenic fungi, including those of the genera Cryptococcus and Histoplasma. Infection studies with Cryptococcus neoformans in mice showed that mating type α was significantly more virulent than mating type a and an infection with α-type strains led to early death (KwonChung et al., 1992). In H. capsulatum, both (+) and (−) strains were

Fig. 1. Schematic illustration of the primer walking procedure of M. audouinii and M. ferrugineum. Primer walking was started from a sequence section in the conserved flanking regions of the MAT locus: A primer was designed at the downstream end to amplify a part of the unknown adjacent MAT locus sequence, which was then sequenced. The newly sequenced DNA strain was used to synthesize another primer that amplified the next adjacent MAT locus sequence downstream. This procedure was repeated a total of eight times and the resulting 8 sequences were aligned to obtain the complete MAT locus sequence. HMG_1 primer product (520 bp) and alpha-box primer product (380 bp) display the size and position of the sequence sections that were used to generate the phylogenetic trees (see Fig. 3). MAT1-1 and MAT1-2 are flanked by hypothetical proteins (hp). In Nannizzia gypsea the MAT locus is flanked upstream by an APN2-like gene. 48

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step of enzyme activation for 3 min at 98 °C with a subsequent cycling protocol of 40 cycles composed of denaturation at 98 °C for 5 sec, annealing at 56 °C for 5 sec, and elongation at 72 °C for 45 sec. The final elongation step proceeded at 72 °C for 1 min and PCR products cooled down to 12 °C until visualization on 1.5% (w/v) agarose gels.

In 2011, Burmester and colleagues described the MAT locus of T. benhamiae and T. verrucosum and identified an identical genetic set-up compared to the MAT locus of closely related dermatophyte species (Burmester et al., 2011). In this study, we analyzed the mating loci of several isolates from 19 dermatophyte species of the genera Trichophyton and Microsporum including their morphotypes/varieties. Hence, we present a comprehensive picture of the distribution and phylogenetic relationship of the mating loci in anthropophilic and zoophilic dermatophyte species and suggest possible correlations between MAT locus idiomorphs and virulence.

2.3. Primer walking for MAT locus identification of Microsporum audouinii and Microsporum ferrugineum Since no HMG sequences were known for closely related species of M. audouinii and M. ferrugineum, an amplification of the MAT locus of these two species was not possible. In order to design suitable PCR primers, the sequences of the MAT loci had to be determined first. To obtain these sequences a primer walking, starting from preserved flanking regions, was applied via PCR (see Section 2.2) and Sanger sequencing. The primer walking was carried out with DNA of two strains of M. audouinii (clinical isolate X74; reference strain CBS 108933) and two clinical strains of M. ferrugineum (E63 and E65). Primers pairs are given in Supplemental Table S2. The assembled sequences were aligned with the software “Geneious, version 8.1.2” (Biomatters Limited, Auckland New Zealand) and compared with previously published sequence information of the HMG domain of N. gypsea and with the alpha-box domain of Microsporum canis (Supplementary Table S1, Fig. 1).

2. Material and methods 2.1. Dermatophyte strains and DNA isolation In total, 457 strains of 19 dermatophyte species were analysed for MAT locus identification (Table 1). The species identity of these clinical and reference strains was confirmed by sequencing the internal transcribed spacer region (ITS) of the ribosomal DNA. Strains were grown on Sabouraud glucose medium for 1–2 weeks at 28 °C. DNA isolation was accomplished by using the CTAB (Hexadecyltrimethylammonium bromide) method as described by Gräser et al, 1999 (Gräser et al., 1999). 2.2. Development of PCR primers and MAT locus determination

2.4. Phylogeny of dermatophytes based on the MAT locus

Using mRNA and/or DNA multiple sequence alignments of approximately 1000 bp of the MAT loci of five dermatophyte strains (see Supplementary Table S1), primer pairs were designed in conserved regions to amplify the alpha-box and the HMG domain which are representative for MAT1-1 and MAT1-2 idiomorphs, respectively. The primer sequences are listed in Supplementary Table S2. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.fgb.2018.07.003. MAT locus sequences were obtained from the data platform “National Center for Biotechnology Information” (NCBI; https://www. ncbi.nlm.nih.gov/). The polymerase chain reaction (PCR) was run in a thermal cycler (Applied Biosystems 2720 Thermal Cycler, California USA) in a 20 µl volume reaction consisting of 10 µl 2 × Phire Plant PCR Buffer (Thermo Scientific, Massachusetts USA), 4 µl Q-Solution (Qiagen, Hilden Germany), 10 µM of each primer, Phire Hot Start II DNA Polymerase (Thermo Scientific, Massachusetts USA), and 15–100 ng/µl template DNA. The reaction was run under the following conditions: an initial

Primer pairs HMG_for_1/HMG_rev_1 (designed on the basis of the identified sequences of M. audouinii and M. ferrugineum), Tt_alpha_F/ Tt_alpha_R, and Mc_alpha_F/Mc_alpha_r (Supplementary Table S2) were used to amplify parts of the HMG domain (380 bp) and the alpha-box (520 bp) to obtain the phylogenetic relationships among dermatophyte species. Sequence information of MAT loci, available at NCBI (https:// www.ncbi.nlm.nih.gov/), were used (Supplementary Table S1). The remaining MAT locus sequences used in this study were generated by PCR and Sanger sequencing (Section 2.2, Supplementary Table S1). MAT locus sequences of either alpha-box or HMG domain were aligned in a multiple sequence alignment. The software “Geneious, version 8.1.2” (Biomatters Limited, Auckland New Zealand) was again used to set up the alignments of the MAT locus sequences of either alpha-box or HMG domain and the Neighbour Joining phylogenetic trees of the idiomorphs based on the Hasegawa-Kishino-Yano (HKY) distance matrix.

Fig. 2. Alignment of the protein sequences of HMG domains of M. audouinii and M. ferrugineum. The protein sequences have a length of 364 amino acids. Varieties of HMG domain protein sequences between the two species are underlined and in bold. 49

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3. Results

strains analyzed display both mating types with a ratio of 1 MAT1-1: 2 MAT1-2 (Table 1).

3.1. MAT locus of Microsporum audouinii and Microsporum ferrugineum 3: T. benhamiae complex Via primer walking eight overlapping sequence fragments were generated and merged into complete sequences of the MAT loci of M. audouinii and M. ferrugineum (Fig. 1). The HMG domain nucleotide sequence has a length of 1212 bp in both species, coding for a protein consisting of 364 amino acids starting with the amino acid triplet ATG coding for methionine (Fig. 2). The alignment of the HMG domain of the two sequences displays 97% identity and 26 single nucleotide polymorphisms (SNPs). Within the short HMG_1_for1/HMG_rev_1 primer product (appr. 300 bp) of M. ferrugineum, M. audouinii, and the closest relative species M. canis, for which one single strain containing the HMG domain could be identified in this study, the following similarities were observed: M. canis and M. ferrugineum 99%, M. audouinii and M. canis 98%. In comparison of the HMG domain sequences of M. audouinii and M. ferrugiuneum with HMG sequences of dermatophyte species of the genera Trichophyton and Nannizzia a sequence similarity of 72% (T. benhamiae) and 75% (N. gypsea) is present. The sequence similarity of the HMG domain of Trichophyton species and N. gypsea is higher, at approximately 82% (Table 2a). The same trend is observed in sequence alignments of the alpha-box: Trichophyton species and N. gypsea have nucleotide similarities ranging from 82% to 85% (e.g. T. benhamiae and N. gypsea 84% similarity), whereas Microsporum canis is more variable compared to species of the genera Trichophyton (e.g., T. rubrum 72% similarity) and Nannizzia (e.g., N. gypsea 75% similarity) (Table 2b).

There are two different genotypes within the species Trichophyton benhamiae (Symoens et al., 2013). Seven out of 20 T. benhamiae strains were of the yellow phenotype and all of them are MAT1-1. Strains of T. benhamiae with the white phenotype can have either the MAT1-1 or the MAT1-2 idiomorph: Among these, all 8 isolates of the white type originating from Europe are MAT1-2 whereas isolates of the white type originating from Japan (with a single SNP within the ITS sequence compared to the European isolates) are MAT1-1 (Table 1). The zoophilic T. erinacei exclusively shows MAT1-2 and in the anthropophilic T. concentricum isolates MAT1-1 was detected (Table 1). T. verrucosum isolates are all MAT1-2. One isolate each available for the species T. bullosum and T. eriotrephon is MAT1-1 (Table 1). 4: T. rubrum complex In almost all strains of the complex, including the species T. rubrum, T. soudanense, T. violaceum as well as the T. rubrum morphotypes T. kanei, T. raubitschekii and T. fischeri, the idiomorph MAT1-1 was detected. The only exception in this complex is T. megninii, another morphotype of T. rubrum, where in six of seven strains the opposite MAT1-2 was amplified (Table 1). 5: M. canis complex In total, 235 isolates of the zoophilic species M. canis were analysed. Although this species is known to reproduce sexually, 234 isolates contain the alpha-box and only a single strain contains the HMG domain (CBS 495.86). The analysed strains of M. ferrugineum (8) and M. audouinii (26) are MAT1-2.

3.2. Distribution and phylogenetic relation of MAT loci among dermatophyte species According to the new taxonomy five complexes are observed among the anthropophilic and zoophilic Trichophyton (T. interdigitale complex, T. simii complex, T. benhamiae complex, and T. rubrum complex) and Microsporum species. The phylogenetic analysis of 380 bp and 520 bp of the corresponding MAT locus sequences mirrors this situation (Fig. 3). In general, the phylogenetic trees of both MAT loci correspond to the phylogeny of a multilocus tree (de Hoog et al., 2017).

4. Discussion It has been shown that mating type and virulence may be associated in human pathogenic fungi of the genera Cryptococcus (Kwon-Chung et al., 1992) and Histoplasma (Kwon-Chung et al., 1974). In this study we wanted to reveal whether a similar link of mating type idiomorphs and virulence can be observed in dermatophyte species. The zoophilic species T. benhamiae occurs in two types, yellow and white phenotype linked with distinct ITS-sequences (Symoens et al., 2013). The natural hosts of this species are guinea pigs and rabbits frequently held as pets. Via direct contact this pathogen is easily transmitted to humans, often causing severe inflammatory infections (Brasch and Wodarg, 2015; Nenoff et al., 2014). In a recent study both types of T. benhamiae have been isolated from guinea pigs from Berlin pet shops with a ratio of 1.7 (yellow type) to 1 (white type) (Kupsch et al., 2017). However, since its first description in 2000, the yellow phenotype is considerably more often isolated from humans, being responsible for at least 80% of T. benhamiae infections in humans (Brasch and Wodarg, 2015; Nenoff et al., 2014). This suggests that strains of the

1: T. interdigitale complex The anthropophilic species T. tonsurans and T. interdigitale have a single MAT locus with the alpha-box and the HMG domain. In contrast, the zoophilic T. mentagrophytes strains show both types at a ratio of 6 MAT1-1 (alpha-box): 24 MAT1-2 (HMG domain) (Table 1). 2: T. simii complex The anthropophilic T. schoenleinii as well as the zoophilic T. quinckeanum exhibit single but opposite mating types, with 16 isolates being MAT1-2 (HMG domain) and seven strains being MAT1-1 (alphabox), respectively. T. simii is known to reproduce sexually and the three

Table 2a Nucleotide sequence similarity based on PCR products in the partial HMG-domain sequence alignment (1264 bp) of six dermatophyte species in percentage. Dermatophyte strain

T. benhamiae CBS 112273

T. benhamiae M. audouinii M. ferrugineum N. gypsea T. interdigitale T. simii

77% 73% 81% 88% 87%

M. audouinii 108933 NOMH 1279

M. ferrugineum E63

N. gypsea ATTC 48982

T. interdigitale CBS 646.73

T. simii CBS 448.65

72%

73% 97%

81% 75% 75%

88% 75% 75% 83%

87% 73% 73% 82% 92%

97% 75% 75% 73%

75% 75% 73%

50

83% 82%

92%

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Table 2b Nucleotide sequence similarity based on PCR products in the partial alpha-box sequence alignment (1218 bp) of eight dermatophyte species in percentage. Dermatophyte strain

N. gypsea M. canis T. tonsurans T. rubrum T. violaceum T. simii T. interdigitale T. benhamiae

N. gypsea ATCC 58595

74% 85% 82% 85% 85% 84% 85%

M. canis CBS 113480

T. tonsurans CBS 112818

T. rubrum CBS 118892

T. violaceum CMCC(F)T3I

T. simii CBS 417.65

T. interdigitale CBS 642.73

T. benhamiae 4-40

74%

85% 75%

82% 72% 91%

85% 75% 94% 96%

85% 76% 95% 92% 95%

84% 75% 96% 90% 93% 95%

85% 74% 94% 90% 93% 94% 93%

75% 72% 75% 76% 75% 74%

91% 94% 96% 96% 94%

96% 92% 90% 90%

95% 93% 93%

95% 94%

93%

was not available for morphological evaluation. T. megninii strains are characterized by loose white growth on BCP milk dextrose medium, the requirement for histidine and a positive urease test (Kane and Fischer, 1975). T. rubrum on the other hand shows restricted growth, a pronounced reddish pigment and a slight white aerial growth on BCP milk dextrose medium (Kane and Fischer, 1975). The ITS-sequences of T. rubrum morphotypes are almost identical and phylogenetic analysis displays a monophyletic group (Gräser et al., 2000b). Morphotype T. megninii was reported to be restricted to southern European countries like Italy and Portugal in the early 1990s (Sequeira et al., 1991), but it can be assumed that it will lose its endemic distribution via travel and emigration. In general, the isolation of this morphotype is very rare. Sexual stimulation (but not mating) of clonally reproducing dermatophytes (characterized by the formation of pseudo-cleistothecia) has been reported in the past (Young, 1968). In such experiments, T. megninii isolates reacted as the (+) mating type (Sequeira et al., 1991). This year, Persinoti and colleagues identified molecularly MAT1-2 in T. rubrum morphotype “megninii” (Persinoti et al., 2018). We support this result by showing MAT1-2 in T. rubrum morphotype “megninii” in the present study. The rare isolation of the MAT1-2 strains, however, suggests that this reproductive partner is becoming extinct. In the same way, the adaptation to humans may have led to the loss of one of the idiomorphs also in other anthropophilic dermatophyte species, like T. tonsurans (including varieties sulfureum, epilans, and crateriforme), T. interdigitale (including varieties goetzii and nodulare), T. schönleinii, and T. concentricum. While 11 strains of T. tonsurans and 12 strains of T. concentricum exhibit MAT1-1, the opposite MAT1-2 was detected in 29 strains of T. interdigitale and 16 of T. schoenleinii. For some of these dermatophyte species, the reason for the non-discovery of

yellow phenotype are more virulent to humans than white type strains. In the present study, all T. benhamiae isolates of the yellow phenotype exhibit MAT1-1 whereas eight and five strains of the white phenotype are MAT1-2 and MAT1-1, respectively. Hiruma and colleagues found a very similar MAT idiomorph distribution pattern in this species: Isolates of the yellow phenotype (group II) were all MAT1-1, white isolates were either MAT1-1 or MAT1-2 (Hiruma et al., 2015). Likewise, at the ISHAM conference in 2016, A. Čmoková reported an intriguing MAT locus distribution in 326 T. benhamiae strains (specific talk). All 238 strains of the yellow phenotype and all 22 white T. benhamiae strains causing outbreaks in Japan were MAT1-1, while the majority of white T. benhamiae strains isolated in Europe were MAT1-2. The results of our present study underline these findings: all white T. benhamiae strains with MAT1-2 are of the European ITS type whereas all strains of the Japanese genotype are MAT1-1 (Table 1). Thus, white types found in Europe have MAT1-2, while the potentially higher virulent yellow type exclusively has MAT1-1. This is an indication that there may be a connection between mating type and virulence in T. benhamiae. Kano et al. have shown that 206 strains of the anthropophilic T. rubrum from Japan exhibit the MAT1-1 locus (Kano et al., 2013). In the present study we analyzed 22 strains of T. rubrum with different morphotypes (raubitschekii, kanei, megninii, and fischeri) from the USA, Japan, Africa and Europe. In the course of last year's new phylogeny and taxonomy of the dermatophytes, Trichophyton soudanense (regarded as a morphotype of T. rubrum before the taxonomy was revised) is described as a species of its own (de Hoog et al., 2017). MAT1-1 was identified in all, with exception of 6 out of 7 T. megninii strains with MAT1-2. One single isolate of T. megninii (CBS 551.86) showing MAT11 may be a morphological misidentification. Unfortunately, this strain

Fig. 3. Neighbor joining phylogenetic trees of dermatophytes based on sequence analysis of the idiomorphs. A: MAT1-1 (alpha-box), B: MAT1-2 (HMG domain). *N. gypsea strain CBS 118893 was not analyzed in this study, but a part of the published alpha-box sequence (Accession number FJ798794.1) was included in the alignment/phylogenetic tree. **N. gypsea strain ATCC48982 was not analyzed in this study, but a part of the published HMG domain sequence (Accession number FJ798798.1) was included in the alignment/phylogenetic tree. 51

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different mammalian species, the European and the African hedgehog, which may eventually lead to clonal spreads in T. erinacei. For the first time, the MAT locus of the zoophilic dermatophyte species T. bullosum, T. eriotrephon, and T. quinckeanum was examined in this study. All isolates are MAT1-1. Strains of these species are rare and therefore only a few were analyzed (1 T. bullosum, 1 T. eriotrephon, 7 T. quinckeanum). Hence, it remains an open question whether the other reproductive partner also exists. T. equinum was known to be MAT1-2 from classical and molecular experiments (Li et al., 2010; Stockdale, 1968). All three tested strains of this study are MAT1-2, as well. The MAT locus of all T. verrucosum strains (including varieties album, discoides, and orchraceum) are MAT12. Although T. verrucosum is a zoophilic species the opposite mating partner has never been isolated (Kano et al., 2014). Partial sequences of the MAT1-1 as well as of the MAT1-2 idiomorph were used in this study to create phylogenetic trees (Fig. 3). These reflect the same dermatophyte phylogeny as the trees based on molecular markers like ITS, partial LSU, the ribosomal 60S protein, and fragments of β-tubulin (de Hoog et al., 2017). Very closely related sister-taxa like M. canis and M. audouinii show more differences with 13 SNPs in MAT12 as they show in the commonly used ITS (6 SNPs). This means that the MAT loci could be suitable molecular markers for dermatophyte species identification. However, it should be noted that in the case of dermatophyte species which may have both idiomorphs, the MAT locus must be determined beforehand.

the opposite reproductive partner could be an adaptation to a different mammalian host and its evolution into a new species in a separate ecological niche, as already briefly discussed by Summerbell and Graeser et al. (e.g. T. tonsurans (human) and the phylogenetic close relative T. equinum (horse) or T. concentricum (human) and the closely related zoophilic T. verrucosum (cattle)) (Gräser et al., 2008; Summerbell, 2000). In the Microspoum clade the anthropophilic species M. audouinii (including morphotypes langeronii and rivalieri) and M. ferrugineum have one single MAT locus idiomorph, MAT1-2, herewith shown for the first time. The zoophilic species T. mentagrophytes, T. simii, T. erinacei, T. benhamiae, and M. canis were assumed to harbor both idiomorphs and reproduce sexually. The identification of the MAT locus for the following species meets this expectation: T. mentagrophytes (6/24), T. simii (1/2), T. benhamiae (15/8). There is a striking imbalance in the idiomorph distribution in M. canis, with MAT1-2 ((+) strains) being isolated very rarely in the present study (234/1) and in previous studies (Hasegawa and Usui, 1975; Hironaga et al., 1980; Weitzman and Padhye, 1978). Among these few isolates is strain CBS 495.86 (VUT77054), which has been subjected to molecular analysis earlier: In a multilocus study with 13 microsatellite and non-microsatellite markers this strain groups within the M. canis cluster, but shows a remarkable molecular distance to the other analyzed M. canis strains (Kaszubiak et al., 2004). Within the ITS region, CBS 495.86 is even phylogenetically more closely related to M. audouinii than to M. canis (de Hoog et al., 2017; Gräser et al., 2000a). Nevertheless, this particular strain originates from a monoascospore culture obtained from a cleistothecium produced by 2 compatible M. canis strains (VUT-73015 (+) × VUT-74001 (−)) and classical mating experiments have repeatedly shown that this particular strain mates with (−) strains of M. canis (Hironaga et al., 1980). Thus, the species identity of CBS 495.86 as M. canis is confirmed. But both the molecular differences as well as the presence of MAT1-2, let us presume this might be an ancestral strain which represents an evolutionary link between the zoophilic M. canis and the human-adapted M. audouinii. The MAT locus idiomorph ratio in T. mentagrophytes is imbalanced in the isolates of our study with MAT1-2 being 3.7 times more isolated than MAT1-1. Interestingly, this species has recently been observed to cause inflammatory infections in the pubic area (tinea genitalis) of young adults, most of which did not report animal contact. Isolated strains showed a typical granulous white surface with a yellow-brown pigmentation on the reverse side (Nenoff et al., 2017). T. mentagrophytes strains with this phenotype were isolated from 37 patients at the Charité in Berlin between January 2016 and July 2017, but only two of them had an anamnesis of animal contact which is usually the rule for infections with zoophilic dermatophyte species. The molecular analysis revealed a rare but identical ITS genotype (with three SNPs to known T. mentagrophytes type IV (Heidemann et al., 2010)) and the MAT1-2 locus in all isolates (unpublished data). These data suggest that a new type or an expansion of a T. mentagrophytes clonal lineage, which carries the MAT1-2 idiomorph, is still highly virulent to humans but has already acquired the ability of transmission from human to human and may be on the way toward speciation. In the present study, the zoophilic T. erinacei shows MAT1-2 in all tested strains. By classical mating experiments, Takashio and colleagues identified strains from UK, Belgium and New Zealand as (+) mating partners (isolated from European hedgehog Erinaceus europaeus), whereas African strains from Kenya behaved as (−) strains (isolated from African hedgehog Erinaceus albiventris) (Takashio, 1979). Strain CBS 511.73 was isolated from a European hedgehog and since the other strains tested herein were isolated from humans from the Netherlands, Great Britain and Germany, it seems likely that these patients had contact with European hedgehogs, rather than African hedgehogs. Under this assumption, our results would underpin the findings of Takashio. Here again, we find an adaptation of the two idiomorphs to

5. Conclusion For several zoophilic dermatophyte species, both mating partners were detected molecularly in this study (e.g. Trichophyton benhamiae, T. simii). However, the majority of dermatophyte species seems to have reduced or lost one mating partner. Although there is a clear trend towards asexual reproduction in most of the 19 studied dermatophyte species, there is the remarkable fact that anthropophilic species like T. rubrum retain meiosis-specific genes required for sexual reproduction (Martinez et al., 2012). It seems that sexual reproduction is maintained and possibly switched on when environmental circumstances like niche alterations push for an adaptation. After the establishment of a new niche, e.g. the human host, pathogenic fungi shift from sexual to clonal reproduction. We summarize the following possible scenarios for the imbalanced presence of idiomorphs in dermatophyte species. Summerbell and Graeser discussed the first and the second hypotheses already in former publications (Gräser et al., 2008; Summerbell, 2000): 1. The extinction of one mating type idiomorph due to asexual reproduction (for example in T. rubrum and M. canis) 2. An adaptation of one mating partner to the human host with subsequent speciation may occur, which may apply e.g. for T. tonsurans, T. interdigitale, and T. concentricum, and M. audouinii. For the latter, strain CBS 495.86, which was shown to mate with M. canis but has phylogenetic similarities with M. audouinii, is possibly a relic from the time of species transition. Such an adaptation of one mating partner to humans as new niche seems to happen occasionally over time. The recently discovered T. mentagrophytes type, which is sexually transmitted from human to human (Luchsinger et al., 2015; Nenoff et al., 2017), could be a current example of this scenario. The evolution from a former sexual into an asexual organism was also suggested for the human pathogenic fungus Aspergillus fumigatus (reviewed in (Ene and Bennett, 2014)). 3. Maybe not clonality, but unisexual reproduction is the next evolutionary stage of reproductive strategies in human pathogenic fungi, which has already been hypothesized for dermatophytes (Metin and Heitman, 2017). In order to test this hypothesis, however, further experiments would be necessary, to show that there are fusions between two MAT1-1 strains or between two MAT1-2 strains. Via 52

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unisexual reproduction the risk of losing well-adapted traits could be reduced, while the progeny may enhance its fitness by the introduced limited genetic diversity (Feretzaki and Heitman, 2013).

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