Visualising virulence factors: Trichophyton benhamiaes subtilisins demonstrated in a guinea pig skin ex vivo model

Dermatophytoses rank among the most frequent communicable diseases in humans, and the zoonotic transmission is increasing. The zoophilic dermatophyte Trichophyton (T.) benhamiae is nowadays one of the main causes of tinea faciei et corporis in children. However, scientific data on molecular pathomechanisms and specific virulence factors enabling this ubiquitous occurrence are scarce.


| INTRODUC TI ON
Fungal infections of skin, nail and hair caused by dermatophytes are common worldwide and represent a growing health burden in animals and man. 1 The high prevalence of dermatophytoses is connected to the dermatophytes' extraordinary ability to degrade keratins. These insoluble fibrous proteins represent major constituents of the aforementioned host structures and are degraded into short peptides and free amino acids to be used as nutrients.
The thereto necessary proteolytic activity is exerted through synergistically acting endo-and exoproteases secreted by dermatophytes. 2,3 However, an 'ecological switching', that is a differential expression of these secreted proteases depending on the host species or experimental system, was noticed. [4][5][6][7][8] For Trichophyton (T.) spp., major endoproteases expressed during in vitro growth were subtilisins (Sub) 3 and 4 and fungalysins Mep 3 and 4; major exoproteases were aminopeptidases Lap1 and 2, dipeptidyl-peptidases DppIV and DppV and metallocarboxypeptidase A (MCPA). 3 In contrast, the most abundant protease found during experimental animal infection was Sub6. 5,8 It was also found in numerous patient samples of dermatophyte nail infections but not with trauma or non-dermatophyte infections. 5,9 Hence, Sub6 is considered a robust marker of in vivo trichophytosis and onychomycosis caused by Trichophyton spp.
The aforementioned findings suggest distinct functions for different proteases of the same protein family during in vitro growth and in vivo infection and demand for further research integrating results from different experimental approaches. 5,8,10 Recently, we developed an ex vivo infection model based on guinea pig skin explants (GPSE) for the investigation of dermatophytoses induced by T benhamiae. 11 Ex vivo models comprise the physiological 3D microenvironment with crucial features, for example hair follicles, which is an important advantage compared with other in vitro or (2D) cell culture systems.
The zoonotic dermatophyte T. benhamiae was only recently categorised as an emerging pathogen due to frequent misdiagnosis and increased incidences. [12][13][14] Its main reservoir and the most common source of human infection are guinea pigs (GP), which are often held as household pets. 12,15 Asymptomatic carriage (mainly GP) but also a wide range of symptoms (erythematous eczema, itch, scales, crusts, alopecia and scars), immunologic responses and localisations of tinea are described for both companion animals and human patients. [15][16][17] Since the aforementioned experimental GPSE set-up was proven a suitable model for the early stages of T. benhamiae skin infection, it was now employed to analyse the expression of important virulence factors on protein level. Therefore, Trichophyton isolates derived from two distinct groups of hosts (human patients vs. infected GP) were applied, immunofluorescence (IF) stainings were carried out, and the obtained expression patterns were semiquantitatively evaluated. Additionally, the general mode of fungal invasion was examined and compared with skin biopsies of naturally infected GP.

| Fungal isolates and infection experiments
Isolates of T. benhamiae were recovered from human patients (n = 10) and infected GP (n = 10). Species identity was analysed by sequencing of the internal transcribed spacer (ITS) region of the fungal rDNA from cultures grown on Sabouraud-Dextrose Agar (4%; Sifin Diagnostics GmbH; 14 days, 28°C). Total DNA was extracted according to the manufacturer´s instructions of the QIAamp® DNA Mini Kit (Qiagen) with an additional overnight Proteinase K digestion at 56°C and 600 rpm agitation. The ITS region was amplified using the universal primers LSU266 and V9D (for primer sequences and thermal profile see ref. 18 ) and the Red HS Taq Master Mix (Biozym). 18  Preparation of conidia suspensions for inoculation and GPSE, and infection experiments were carried out as previously described. 11 Briefly, skin explants were prepared from the flank region of disinfected and clipped GP after euthanasia with state approval and in accordance with the ethical policies of the journal. Explants were transferred to cell culture inserts, supplied with growth medium and directly inoculated with 1 × 10 3 CFU in 2 µL PBS of one of the above-mentioned T. benhamiae isolates. A T. mentagrophytes isolate was prepared and employed equally; 1 × 10 3 cells of Geotrichum candidum being a yeast-like fungus were counted and directly applied. 20 Inoculated GPSE and controls were incubated for 10 days at 30°C, 5% CO 2 and 95% relative humidity. Fixation in paraformaldehyde (4%) and paraffin-embedding of samples according to standard protocols ensued at days 3, 5, 7 and 10.

| Histological and IF stainings
Histological analyses of infected GPSE were conducted using 1-µmthin skin sections subjected to the PAS reaction (standard protocols) and an upright Olympus BX 51 microscope (Olympus Deutschland GmbH). Skin biopsies of naturally infected GP were obtained from feed animals from the Zoo Leipzig after euthanasia; skin sections thereof were prepared and evaluated identically.
To visualise Trichophyton isolates in selected samples, a DyLight 594-coupled anti-Trichophyton antibody was produced and applied as previously described (note: different fluorophore). 11 To assess hyphal invasion of GPSE, double IF stainings of the desmosomal cadherin desmoglein-1 (Dsg1) and fungal elements were carried out in selected samples. Therefore, dewaxed and rehydrated GPSE sections were subjected to heat-induced epitope retrieval in Citrate Buffer (pH 6, 10 x Antigen Retriever

| Statistics
Data were analysed using SigmaStat 2.03 and SigmaPlot 7.0 software (Systat Software GmbH, Erkrath, Germany). The Mann-Whitney U test was employed to detect statistically significant differences between the two groups of isolates and time points, respectively. Pvalues of <.05 (*; **0.05 < P ≤ .02) were considered significant, and results were presented as mean ± SD.

| RE SULTS
All of the 20 recovered dermatophytes were identified as T. benhamiae (identical to GenBank accession nos. KU257463.1 (white colony phenotype (w), n = 1) and MF614429.1 (yellow colony phenotype (y), n = 19)). The phylogenetic analysis included all isolates used during this study and closely related Trichophyton spp. (in total 29 sequences). The evaluated species form distinct and very robust clusters (bootstrap values all >77%). All T. benhamiae isolates were found in one clade, which was subdivided according to their colony phenotypes; the different origin of isolation was not reflected. Initially, fungal elements of all Trichophyton isolates were mainly found in the epidermis ( Figure 1A). By no later than d7, hyphae were found in the dermis and all skin structures were destroyed. Digestion of hair remnants ( Figure 1B) and sporulation ( Figure 1C) were frequently observed by the end of culture. A preference for intra-or intercellular invasion of the skin could not be determined since fungal elements were found inside cells and also in-between cells, that is co-localised to Dsg1-stained structures ( Figure 1D). An isolate of the non-dermatophyte Geotrichum candidum served as non-invasive control ( Figure 1E): this geophilic fungus proliferated well in culture but did not invade the tissue. The observed morphological alterations of GPSE (acantholysis, pyknotic nuclei) were common changes as expected during long-term tissue culture. 11 The applied T. mentagrophytes isolate formed a massive mycelium on top of and around GPSE ( Figure 1F); invasion and destruction were accelerated but basically similar to that caused by the T. benhamiae isolates.
Skin biopsies of naturally infected GP (n = 18) served as references for experimentally infected GPSE. Fungal presence in sections of those biopsies was verified histologically using the PAS reaction (Figure 2A,B) and IF stainings specific for Trichophyton spp ( Figure 2C). Obviously, the causative agent was isolated and subjected to routine mycological culture and diagnostics. Species identity as T. benhamiae was confirmed through ITS sequencing or MALDI-TOF-MS (data not shown). Interestingly, isolates of both colony phenotypes were found in the animal facility, sometimes even isolated from the same animal.
In skin sections of naturally infected GP, hyphae and numerous conidia were found concentrated in and around hair follicles; the infection seemed to spread from these postulated portals of entry. 24 The expression of the in vivo trichophytosis marker Sub6 was confirmed using the described customised anti-peptide antibody ( Figure 2D; Sub3 and MCPA were also demonstrated; data not shown).
Sections of infected GPSE were analysed immunohistochemically for the abundance of the virulence factors Sub3, Sub6 and MCPA (Figure 3; non-infected GPSE were stained as negative controls, no signal was observed; data not shown). IF signals for Sub6 were found dispersed throughout the whole mycelium grown on and in GPSE (distinct progression proximal to distal from point of inoculation; Figure 3A). Strongest signals were seen mainly around keratinised structures (stratum corneum, hair follicles) and in conidia and hyphae, respectively ( Figure 3B,C). Sub3 and MCPA were found at cell peripheries in hyphae and conidia ( Figure 3D-F). Furthermore, Sub3 was most abundant in conidia F I G U R E 1 Representative images of the infected ex vivo skin model illustrate different localisations of fungal elements, the interaction of hyphae and skin cells and different growth patterns of dermatophytes and non-dermatophytes. A-C, Illustrations of different localisations of Trichophyton (T.) benhamiae isolates applied to guinea pig skin explants (GPSE): A, hyphae are found in the epidermal stratum corneum (SC) and in deeper layers with progressing culture time. B, All tested isolates were also found in hair remnants and follicles (* hair isthmus); most of them showed tricholysis. C, Often, conidia (black arrows) were deposited in the dermis towards the end of culture. D, The interaction between T. benhamiae and GPSE is visualised using immunofluorescence stainings: desmoglein-1 as an integral part of (corneo)desmosomes is depicted in green, the T. benhamiae isolate is shown in red. Fungal elements are found in and in-between cells, that is co-localised with Dsg1 (anti-Trichophyton-DyLight 594, arrows; nuclear counterstain with bisbenzimide 33342 (Hoechst) is depicted in blue; dashed line highlights basement membrane). E, The yeast-like non-dermatophyte Geotrichum candidum served as a non-invasive control: it proliferated well during the 10-d culture, and colonised GPSE, tissue invasion and destruction were rarely seen. The observed morphological alterations of GPSE (acantholysis, pyknotic nuclei) are expected changes during tissue culture (d5). Selected isolates with white and yellow tally-all being confirmed members of the yellow growth type 12,13 -are depicted in Figure S1, and a phylogenetic tree comprising all applied isolates and outgroups is shown in Figure S2.

| D ISCUSS I ON
We used an ex vivo skin infection model that previously proved suitable to study early stages of dermatophytoses 11 and analysed its ability to support the expression of important virulence factors.
Therefore, isolates of the zoonotic dermatophyte T. benhamiae derived from two different groups of hosts were applied to the skin model and the infection was compared with skin biopsies of naturally infected GP.
To ensure species identity of all applied isolates, their ITS region was sequenced revealing that only one belongs genetically to the white colony phenotype group although more white and brown tally were observed (see Figure S1). This emphasises the need for species identification through molecular biological methods such as PCR-ELISA, (real time) PCR, sequencing and/or MALDI-TOF-MS. 12,15,25 Moreover, even using these sophisticated methods, caution must be exercised when deducing identity from deposited database sequences/spectra since a universal consensus in species description and classification is still not reached. 26 critically evaluate sequences/spectra and given information to conclude secured results.
The phylogenetic analysis of the T. benhamiae isolates disclosed distinct clusters according to their colony phenotype (y vs w) as it was seen by others as well. 28 The different origin of isolation was not reflected, which is also documented for T. verrucosum isolates F I G U R E 2 Trichophytosis and expression of the virulence factor subtilisin 6 in skin biopsies of naturally infected guinea pigs were confirmed using specific customised antibodies in immunofluorescence stainings. The colonisation of hair follicles by a T. benhamiae isolate is illustrated using the PAS reaction (arrows in A and B; A: bar ≙ 100 µm, magn. 20×; B: bar ≙ 50µm, magn. 40×) and immunofluorescence stainings with a customised anti-Trichophyton-DyLight 594 antibody (depicted in red in C; bar ≙ 100µm, magn. 10×). The expression of the important virulence factor subtilisin 6 (depicted in green) is demonstrated in a hair follicle from the skin biopsy of a naturally infected guinea pig (D, hair follicle is isolated and devoid of hair due to handling; bar ≙ 25µm, magn. 40×). The nuclear counterstain with Bisbenzimide 33342 (Hoechst) in C and D is depicted in blue derived from humans and cattle. 29 The authors ascribe this observation to the fact that human infection mostly originates from contact with infected or asymptomatic animals. Such a zoonotic transmission is also hypothesised for humans with T. benhamiae infections especially when contact with GP is reported. 12,16,28,30 The experimental GPSE infection was compared with skin biopsies obtained from GP naturally infected with T. benhamiae and is described simultaneously intra-and intercellularly most likely through concerted mechanical and enzymatic forces. [35][36][37][38] Proteases are considered the most important dermatophyte virulence factors especially during the establishment of the infection. 39 Consequently, we confirmed Sub3, Sub6 and MCPA immunohistochemically in skin biopsies of naturally infected GP. To the best of our knowledge, this is the first report of a visualisation of virulence factor expression in host tissue. Additionally, this finding proved our customised antibodies valuable tools for further research on, for example, the chronology and secretion levels of these virulence factors during natural infection. Such data are currently not available but urgently needed. 39 We focused on the most important advantage of IF stainings, that is the opportunity to localise targets. Using our model, all examined proteases were mainly found associated with the cell walls of fungal elements. This phenomenon is also described, for example, for a Cryptococcus neoformans laccase and might be explained with the thus possible direct interaction with extracellular substances and host immune cell products facilitating the survival of the respective pathogen. 40 We observed most Sub3 signals in conidia, which is in accordance with other groups who found Sub3-mRNA upregulated in   44 However, many proteases are produced as (pre) proproteins inside the cell and further processed until they reach their final destination and full functionality.
Dermatophytoses are an ever-growing global health burden, but the knowledge on specific pathomechanisms remains scarce.
The expression of subtilisins (Sub) 3 and 6 by Trichophyton (T.) benhamiae isolates derived from infected humans (hum) and guinea pigs (GP) cultured in an ex vivo infection model was semiquantitatively analysed and compared. Equally acquired immunofluorescence images of infected GPSE were scored, statistically analysed (Mann-Whitney U test) and compared; P-values are given in the tables below the graphs, significant differences are indicated with asterisks (*/**) in the graphs and with black boxes in the tables, respectively. T. benhamiae isolates derived from human patients showed a significantly higher expression of Sub3 on d7 of culture compared with GP-derived isolates (**, left). Also, hum-derived isolates showed a significantly higher expression of Sub6 on d3 of culture (*, right). Generally, the staining signals for both virulence factors increased towards the end of culture for both groups of isolates (partly significant differences) important prerequisites to gain more insight into virulence factor production and functionality during natural and experimentally induced dermatophyte infection.

CO N FLI C T O F I NTE R E S T
All authors have nothing to disclose.