Gene expression profiling of protease and non-protease genes in Trichophyton mentagrophytes isolates from dermatophytosis patients by qRT-PCR analysis

Trichophyton mentagrophytes secretes Metallocarboxypeptidase A and B of the M14 family as endoproteases and exoprotease. T. mentagrophytes produce Metalloprotease 3 and 4 which degrades the protein into the short peptides and amino acids. To understand the host fungal relationship and identification of such genes expressed during infection is utmost important. T. mentagrophytes encodes some proteins which are associated with the glyoxylate cycle. The glyoxylate cycle enzymes have been involving in virulence of dermatophytes and their up-regulation during dermatophytes growth on keratin. On comparing the expression level of virulence protease and non-protease genes, we observed, among exoprotease protease genes, Metallocarboxypeptidase B was strongly up regulated (134.6 fold high) followed by Metallocarboxypeptidase A (115.6 fold high) and Di-peptidyl-peptidases V (10.1 fold high), in dermatophytic patients as compared to ATCC strain. Furthermore, among endoprotease, Metalloprotease 4 was strongly up regulated (131.6 fold high) followed by Metalloprotease 3 (16.7 fold high), in clinical strains as compared to T. mentagrophytes ATCC strain. While among non-protease genes, Citrate Synthase was highly expressed (118 fold high), followed by Isocitrate Lyase (101.6 fold high) and Malate Synthase (52.9 fold high). All the studied virulence genes were considered the best suitable ones by geNorm, Best keeper, Norm Finder and Ref finder.

www.nature.com/scientificreports/ containing protein, utilizing it as the source of nitrogen and carbon, establishes the role of enzymes in initiating infection. The proteases in dermatophytic infection are widely recognized, and these enzymes have been identified as important virulence determinants and allergens 5 . It is estimated that proteases represent approximately 20% of the 100 most expressed secreted proteins in T. benhamiae (or Arthroderma benhamiae) during growth both in-vitro and in-vivo. Other dermatophytes like T. verrucosum including T. benhamiae were found to own 235 predicted protease-encoding genes, none unique to either species 6 . Dermatophytes release endoprotease that cleave peptide bonds within a polypeptide and exoprotease cleave peptide bonds only at the N-or the C-terminae of polypeptides 7,8 . Serine protease, leucine aminopeptidases (LAP1 and LAP2) and metalloprotease (MEP) are the predominant endoproteases, though exoproteases such as aminopeptidase, metallocarboxypeptidase (MCP) and dipeptidyl-peptidases (DPP-IV and DPP-V), have also been isolated from dermatophytes 9 . Overall there are multiples endoproteases that have been broadly categorized into two major protein families, the subtilisins, that are serine proteases (SEP), and the fungalysin or metalloprotease (MEP). Amongst the Metalloprotease Giddey et al. (2007) experimentally found high secretion levels of MEP3 and MEP4, in the culture supernatant of T. mentagrophytes grown on a soy protein medium, while T. violaceum predominantly secreted MEP2 and MEP4 [10][11][12][13] . Besides these the enzymes involved in glyoxylate cycle are also related to the pathogenicity of T. mentagrophytes, though their functions and mechanisms remain uncertain. The Isocitrate lyase (IL) and Malate synthase (MS), as the key enzymes of the glyoxylate cycle were highly induced in macrophages during S. cerevisiae infection 14. We attempted to analyze the expression patterns and dynamics of genes encoding two major families of endoproteases and exoproteases, which comprised of Metalloproteases (MEP3 and MEP4) Metallocarboxypeptidases (MCP-A and MCP-B) and key enzymes of glyoxylate cycle, Isocitrate lyase (IL), Citrate synthase (CS), Malate synthase (MS) and Dipeptidyl-peptidases (DPP-V), in T. mentagrophytes isolates from patients of chronic dermatophytosis by real-time (RT) PCR and compared the expression levels with T. mentagrophytes ATCC number 28185 strain 15,16 .
Further, to measure the expression stability of the noted differentially expressed genes which are responsible for degrading keratin into proteins and peptides, different online web based software such as geNorm 17 , BestKeeper 18 , NormFinder 19 and RefFinder 20 , have been applied to determine and validate the regulation of pathogenic virulence genes in T. mentagrophytes.

Identification of T. mentagrophytes isolates by phenotypic and genotypic methods. A total
of 40 clinical isolates of dermatophytes were identified routine phenotypic method and then T. mentagrophytes isolates were genotypically confirmed by conventional PCR shown in Fig. 1.1-3. Sequencing was performed to confirm the T. mentagrophytes isolates. The representative sequences obtained were submitted to gene bank data base with accession numbers; MH644185, MH745112 and, MH778308.
The clinical samples included 38 cases of tinea corporis and tinea cruris and 2 of onychomycosis. The mean age and disease duration in these patients was 29.8 ± 11.41 years and 9.74 ± 6.81 months, respectively. Most patients had previously received antifungal therapy but with poor clinical response and currently presented with persistent lesions. Forty clinical isolates of T. mentagrophytes were analysed against ATCC strain and the fold change was calculated. Both the groups were assessed for eight virulence genes were normalized by Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) shown in Table 1.

Expression of virulence genes of T. mentagrophytes isolates in dermatophytosis patients.
On comparing the fold change expression level we observed, the major keratinase, MCP-B of the exoprotease family was strongly up-regulated (134.6 fold higher) followed by MCP-A (115.6) and DPP-V (10.1), in T. mentagrophytes as compared to ATCC strain. Furthermore, among endoprotease, MEP4 was strongly up regulated (131.6 fold higher) followed by MEP3 (16.7 fold), in clinical isolates. While amongst non-protease genes, Citrate Synthase was highly expressed (118 fold higher), followed by Isocitrate Lyase (101.6) and Malate Synthase (52.9), in T. mentagrophytes isolates as shown in Fig. 3.
Expression stability ranking of virulence gene. The ranking of virulence gene data as per geNorm software depicting the expression stability (M) value from low to high; the NormFinder data with the stability (P) value ranging from low to high; the BestKeeper data demonstrating the coefficient of correlation (r) value from high to low while RefFinder data demonstrating the Geometric mean of ranking values ranging from low to high is shown in Table 2.
The stability analysis with geNorm showed that metallocarboxypeptidases (MCP-A and B) and metalloprotease (MEP4) genes were the more stable genes, with MCP-B ranked as the most stable gene (M value = 0.273) and serine protease DPP-V the least stable gene (M value = 0.344) amongst all virulence genes analyzed in dermatophytosis samples. RefFinder and NormFinder algorithms calculated the expression stability of 8 virulence genes demonstrated MCP-B as the most stable genes and DPP-V as the least stable one. On the contrary, BestKeeper analysis gave the DPP-V as the most stable gene in T. mentagrophytes isolates, followed by MEP4 and MEP3 as the least stable gene. Hence, BestKeeper analysis also showed MEP4 with high coefficient of Correlation (r) as most stable gene in T. mentagrophytes. Furthermore, this validation analysis using geNorm, NormFinder and genes were more stable genes followed by IL, while best keeper showed DPP-V as most stable gene along with MEP4, CS and MCP-B amongst the eight genes. Hence, MCP-B, MCP-A and MEP4 genes are the most stable and predictable virulence genes for monitoring host-fungal pathogenicity, as described by geNorm, NormFinder and RefFinder ( Table 2).

Discussion
Dermatophytes are keratinolytic fungi which preferentially survive on keratinocytes. Their anthropophillic adaptation, using a variety of host proteins, particularly keratin, as nutrient source requires secretion of several proteases to degrade skin and hair proteins. The understanding of the pathophysiological mechanisms involved in establishment of dermatophyte infection can give us critical insight for rational management strategies 21 . The level of keratinase secretion by Trichophyton species has been reportedly higher than that of Microsporum species, associating their role in pathogenesis 22 .
To establish infection, the ability of the pathogen to overcome the physical and innate host resistance, and endure the initial struggle to establish infection, while competing with normal microbial flora of skin, are all critical enabling factors. The production of various enzymes subsequently triggers a cascade of reactions, which ultimately favour the survival and growth of the pathogen on the host.
This study has attempted to analyse the change in gene expression, encoding various proteins in T. mentagrophytes, causing infection. Certain genes may be overtly engaged in the facilitating the persistence of T. mentagrophytes in the host. We observed that the patients harbouring T. mentagrophytes for several months, despite antifungal treatment, had persistent lesions. This suggested a possible biological mechanism to evade the host immune system to clear the fungus. The array of protease secreted by dermatophytes for establishing infection provides the necessary environment for the fungus to continue to proliferate and remain infective 23-25 . According to studies by Zhang et.al and Leng et al. suggested that MEP4 may be the dominant metalloprotease during the host infection stage among the dermatophytes, MEP4 and MEP5 genes have a significant contribution to T. mentagrophytes pathogenicity during the host invasion 9,26 . We demonstrated similar findings     www.nature.com/scientificreports/ Lyase were also up-regulated during infection, encoding key enzymes of the glyoxylate cycle by A. benhamiae 15 .
Little is known about the role of non protease virulence genes of glyoxylate cycle, though their involvement in the adherence process had been demonstrated in M. canis and T. mentagrophytes 15,29 . In our study we found MEP4 was strongly up-regulated (131.6 fold) as compared to MEP3 (16.7 fold) in T. mentagrophytes isolates. Several documented reports have demonstrated a uniform expression of other genes in T. mentagrophytes including Dipeptidyl peptidases (DPP-V), Metallocarboxypeptidase (MCP-A and MCP-B), and Serine Carboxypeptidases (SCP B). Secreted proteases are therefore suggested to fulfill other important functions to establish infection that are not exclusively associated with the degradation of keratin [7][8][9] . It appears that, variable expression of virulence genes are a result of several factors including growth conditions, and genomic plasticity. During keratin degradation, in dermatophytes several enzymes participate in digestion of host protein. Subtilisins and fungalysins digest proteins into large peptides, which are subsequently broken down into short chain peptides and amino acids. Once cleaved, the reduced proteins become accessible for further digestion by various other endo-and exoproteases secreted by the fungi.
The high expression of MCP-B (134.6 fold) and MCP-A (115.6 fold) by T. mentagrophytes, isolated from dermatophytosis patients, was a major highlight of our study. This finding correlates well with studies of Burmester et al. 10 , who found that only some of the typically keratin-induced proteases like MCP-B, secreted from A. benhamiae, were strongly expressed during fungus-keratinocyte interaction. We also observed Citrate Synthase was strongly up-regulated (118 fold), along with Isocitrate Lyase (101 fold) and Malate synthase (52.9 fold). The expression of various proteases in clinical isolates of T. mentagrophytes play a vital role in survival of the fungus adapting to the new challenging environment, a possible evolutionary mechanism to evade stress and persist on the host skin surface. To validate the stability and expression of various genes the geNorm software, determines the stability value, where in the smaller M-value corresponds to more stable gene. As earlier reported, a comparison on the pathogenic potential of five metalloprotease genes from T. mentagrophytes led to the proposal that MEP4 and MEP5 were most likely to affect pathogenicity, determined in a guinea pig model and a keratin degradation test, whereas expression of only MEP4 was significantly up regulated following growth in vitro on keratin, collagen, elastin or human skin sections. When T. rubrum was cultured with human keratinocytes (HaCaT), elevated expression of MEP4 and subtilisins genes was observed, whereas in a feline M. canis skin infection model there was no evidence of MEP expression during adhesion or early stages of invasion 14,15 .
Our study emphasizes the significance of various proteases though the precise role of these endoproteases, exoprotease and non-protease genes in skin and nail invasion remains to be investigated. Thus T. mentagrophytes isolated from patients of dermatophytosis despite antifungal treatment with a failed to clear the fungus indicate a complex phenomenon of survival strategy of a pathogen. The preferential expression of various proteases convey their ability to evade adverse environmental circumstances and overcome the inhibitory effects of antifungals and host defense mechanism, adapt to the hostile environment persist on lesions 1,10 .
Acquisition of drug resistance is a stepwise mechanism and resistance to Terbinafine and high MIC to azoles has been reported in T. mentagrophyte. However to achieve better survival, fungi adopt various other mechanism to emerge as dominant species amongst the rest. Hence the role of enzymes may be an important stress adaptation strategies expressed in T. mentagrophytes.

Conclusion
Superficial dermatophytosis is a common fungal infection in humans and exoproteases which are proteolytic enzymes digest hard keratinized structure. This study gives us an insight to the virulence properties of T. mentagrophytes responsible for pathogenicity in patients with dermatophytosis. Amidst various factors like rising antifungal resistance, the property of, dermatophytes to secrete several kinds of proteases during penetration, are key factors in the invasion and utilization of peptides on the stratum corneum of the host as a source of energy for their survival. Therefore, an understanding of the specific virulence factors involved in pathogenicity of dermatophytes would assist in the development of new therapeutic approaches to inhibit the proliferation and minimize the inoculum at the site of infection and facilitate the antifungal action on the dermatophytes.

Materials and methods
Study group: The present study was conducted on 40 patients clinically diagnosed with dermatophytosis, attending dermatology outpatient department of a tertiary care hospital, Delhi during a period, January 2015 to December 2018. The skin and nail samples of these patients were collected, after obtaining written informed consent and obtaining Institutional ethical clearance.
Ethical clearance was obtained from UCMS & GTB Hospital. Helsinki guidelines-the study was performed accordance with the declaration of Helsinki. It is widely regarded as a cornerstone document on human research ethics.

Screening and identification of isolates.
Each clinical specimen was suspended in a drop of 10% potassium hydroxide (KOH) for microscopic examination. A portion of the sample was cultured on Sabouraud's dextrose agar (SDA, Hi-media; India) with antibiotics Chloramphenicol (0.05 g/l), Gentamicin (20 mg/l) and Cycloheximide (0.5 g/l). All inoculated tubes were incubated at 25 °C for optimal growth up to 4 weeks. In case of growth, the etiological agent was confirmed by the characteristic morphology of the colony, microscopic appearance of the fungus on Lacto Phenol Cotton Blue (LPCB) mount. www.nature.com/scientificreports/ at 12,000 rpm for 15 min. The pellet was re-suspended in 500 µl of lysis buffer and extraction of DNA was done using standard commercially available kit protocol (Real Biotech Corporation, Taiwan). The species specific primers 30 targeting the internal transcribed spacer 2 (ITS 2) of T. mentagrophytes were used and was confirmed by a 130 bp PCR product length as shown in Table 3. Each tube contained PCR reaction mixture of 25 μl which included 2.5 μl buffer (10X), 5 μl of Q-buffer, 0.5 μl dNTPs (200 μM), MgCl 2 0.5 μl (1.5 mM), 0.15 μl Taq polymerase, 1 μl of each primer, forward and reverse (10 μM), 5 μl of the extracted DNA and nuclease free water to make up the final volume. All PCR reagents were purchased from Qiagen, USA and amplification was performed on a thermo-cycler (Eppendorf, Germany). An initial denaturation was performed at 95 °C for 10 min, followed by 35 amplification cycles of 30 s at 95 °C, 45 s at 65 °C, 30 s at 72 °C, and a final extension of 10 min at 72 °C. The amplified PCR products were analyzed by electrophoresis on 1.5% agrose gel, stained with ethidium bromide and separated at 125 V and 15 mAh current, in a 10 slot apparatus for 30 min. Molecular marker of 100 bp was used to determine the size of the amplicons. Standard ATCC control strain of T. mentagrophytes ATCC no. 28185 was used as positive control 31 .

DNA extraction and PCR for molecular confirmation.
Purification of PCR products followed by DNA sequence analysis was performed and by comparison of the nucleotides with dermatophytes reference sequences obtained from gene bank data base (http://www.ncbi. nih.gov/gene), the isolates were specified as T. mentagrophytes complex which were showing similarity of 99%.

RNA extraction and complimentary DNA synthesis.
Total RNA was extracted from 40 culture isolates of Trichophyton mentagrophytes using TRIzol Reagent (Invitrogen, USA). Briefly, 1 ml of TRIzol Reagent was added in the sample and mixed gently by micropipette to form a homogeneous cell lysate and incubated for 5 min at room temperature. 200 µl of chloroform per ml of TRIzol Reagent was added and centrifuged at 12,000 rpm for 15 min at 4 °C to obtain a colorless upper aqueous phase. RNA containing aqueous phase was taken in a fresh 1.5 ml tube and washed with 500 µl of Isopropanol. The eppendorf tube was centrifuged at 12,000 rpm for 10 min at 4 °C and supernatant was discarded. 1 ml of 75% ethanol was added to the pellet and mixed by vortex. This was followed by centrifugation at 10,000 rpm for 5 min at 4 °C. The supernatant was discarded and the pellet was resuspended in RNase free water, and incubated at 55-60 °C for 15 min. The isolated RNA was stored at − 80 °C immediately. The integrity of the RNA samples was confirmed by agarose gel electrophoresis. Concentration and purity of the samples were assessed by Nano-drop (Ependorff, USA). Extracted RNA was used as a template for c-DNA synthesis by Superscript reverse transcriptase II (Invitrogen, USA). Total RNA (2-5 µg) from each sample was reverse transcribed into c-DNA under thermal conditions, 25 °C for 10 min., 37 °C for 120 min., 85 °C for 5 min. and 4 °C for infinite time.  Tables 4 and  5. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) served as an endogenous expression control for normalization of data and the threshold cycle (Ct) values were calculated by the software. The dissociation curve analysis was performed to verify that a single product was amplified. Amplification curve and melt peaks for each virulence gene were shown in Fig. 2.1,2. The fold change of the expression of virulence genes of T. mentagrophytes in clinical isolates was calculated in compare to ATCC strain by 2 −ΔΔCt method 32 .

Expression study of virulence genes by quantitative real-time PCR (qRT-PCR
Stability index (SI) analysis. The stability index of virulence genes was calculated using the web based softwares, Best Keeper, geNorm, NormFinder and RefFinder. Best Keeper uses raw data, Ct values and Real time PCR efficiency (r) to determine the expression of pathogenic genes and combines them into an index. The software computes descriptive statistics of all derived Ct values for the eight virulence genes that is the geometric mean, arithmetic mean, minimal and maximal value, standard deviation, and coefficient of variance. The stability of virulence genes expression is based on the calculated variations as coefficient of correlation variation (r).
The geNorm program calculates genes stability measure (M value) for a putative virulence gene based on the geometric mean of all studied genes. It relies on the principle that the expression ratio of two ideal virulence genes is identical in all samples, regardless of experimental conditions or cell type. It assumes that two genes that show the best correlation in their expression levels are the most appropriate ones and an expression factor can be calculated from these genes. Gene with the lowest M value is considered the most stable in expression. In our study, raw Ct values were transformed to quantities using the comparative Ct method, where the highest relative quantities (lowest Ct) for each genes. These linear scale data were used as input data, according to    www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.