Abstract
The keratinolytic activities of dermatophyte species are accompanied by the secretion of enzymes, such as serine proteases, which are coded by the Subtilisin (SUB) genes. This study aimed to determine the presence of the SUB genes in the clinical and nonclinical samples of Trichophyton verrucosum and Microsporum gypseum. Isolation was carried out by direct and laboratory examination. Following that, for the determination of the presence of the SUB gene, polymerase chain reaction with specific primers was conducted. The frequencies of the SUB gene were observed in almost 66% of the isolates. Statistical analysis showed a significant relationship between the presence of the SUB gene and the samples collected from human, animals, and soil (P˂0.005). The current investigation has been the first study of the presence/absence of the SUB gene in the clinical and nonclinical isolates of T. verrucosum and M. gypseum in Iran which may be a new step to perform further studies.
Keywords: Dermatophyte, Microsporum gypseum, Pathogenicity, SUB gene, Trichophyton verrucosum
1. Introduction
The investigation of virulence genes of dermatophytes, causing diseases, has been one of the research issues in recent years. That is why the gene library of this group of microorganisms is not so rich (Jousson et al., 2004a; Tarabees et al., 2013). Dermatophytes can exclusively grow in the skin, hair, and nail of humans and animals to digest components of the cornified cell envelope (Aljabre et al., 1993; Jousson et al., 2004b; Oborilova and Rybnikar, 2005). Keratinolytic enzymes are among the most important factors which are effective in the development and spread of infection (Moallaei et al., 2009; Chinnapun et al., 2016; Robati et al., 2018). Although the role of SUB gene in the adherence and inflammation steps of infection should be more studied (Baldo et al., 2012), pathogenic fungi, belonging to the S8A SUBfamily, can produce serine proteases as their virulence factors (Monod et al., 2002).
Despite the fact that M. gypseum is endemic in different regions of the world, there has been a limited number of studies conducted on its molecular analysis (Muhsin et al., 1997; Lemsaddek et al., 2010). Some studies demonstrated low enzyme activity of Subtilisin-like protease in M. gypseum isolates (Moallaei et al., 2009). Muhsin et al. (1997) revealed the elastinolytic activity in M. gypseum and T. verrucosum. The low activities of SUB2-3 in M. gypseum and high activities of all SUB family (SUB1-7) in T. verrucosum were also reported (Lemsaddek et al., 2010).
Studies on the presence of two families, metalloproteases and Subtilisins, performed by polymerase chain reaction (PCR) using specific primers in Portugal indicated the presence of these two genomic families in the genus Trichophyton and the absence of some genomic sequences in some clinical isolates as well as a genus of microsporum (Lemsaddek et al., 2010). The investigation of the enzymatic activity and molecular characterization of Subtilisin protein in Microsporum and Trichophyton species in Iran has been performed for pathogenic and other practical purposes (Moallaei et al., 2009). Various studies have demonstrated that the secretions of proteinases (e.g., the SUB family) can not only break down proteins (e.g., creatine, elastin, and collagen) to provide fungal nutrients but also can control host defense mechanisms and cause delayed type sensitivity (Hadadi et al., 2014). The Tri r2 allergen investigation (SUB6) has been introduced as a diagnostic marker using the protein analysis (proteomics) method produced by Trichophyton in clinical and nonclinical specimens (Mehul et al., 2016).
The aim of the current study was the identification of the presence/absence of SUB gene in T. verrucosum isolated from human hair and cattle skin and in the clinical (i.e., dog and horse hair) and nonclinical (i.e., soil) isolates of M. gypseum, acting as three different Substrates for the photolytic activities of dermatophytes. The knowledge of the virulence genes can help the researchers to diagnose the infection and enable them to develop new drugs and vaccines which may be very effective treatments for dermatophytosis.
2. Material and Methods
2.1. Sample Collection
Within 22 February 2018 and 21 September 2018, 8 human isolates with dermatophytosis of T. verrucosum and M. gypseum referred to Mycology and Parasitology Laboratory, Tehran University of Medical Sciences, Tehran, Iran, 15 animal isolates with dermatophytosis of the two dermatophytes referred to the Mycology and Parasitology Laboratory, Tehran University of Medical Sciences, Tehran, Iran, and 7 soil isolates of M. gypseum were collected. The samples were kept in distilled water until examination.
2.2. Direct Examination and Culture of Dermatophytes
Direct microscopic examination was carried out by a drop of 10% potassium hydroxide solution on a clean glass slide of the samples and investigated at a magnification of 40 ×10. All the samples were cultured in Sabouraud dextrose agar with cycloheximide and chloramphenicol, enriched by thiamine and inositol added to the media under aseptic conditions, and then incubated at 37°C for 3-4 weeks. Macro- and micromorphological features, such as the texture of colonies, color, and rate of growth, were studied. For microscopic examination, a small smear of the colony was put on the glass slide, stained with lactophenol cotton blue, and covered with a cover slide; then, it was observed by an optical microscope.
2.3. Design Specific Primers for T. verrucosum and M. gypseum
Two new sets of oligonucleotide primers with the access codes (National Centre for Biotechnology Information reference sequence: XM_003025488.1 for T. verrucosum and DQ923809.1: Gene Bank for M. gypseum) were designed. The primers were synthesized at SINA CLON Company (Iran) (Table 1).
Table 1.
Information of specific primers designed for this study
| Primer name Primer sequence Length (bp) | ||
|---|---|---|
| Mg-SUBti -S1 | 5’ GCAGCA GGA CAA CGT TCC AT 3’ | 420 |
| Mg-SUBti -As1 | 5’ TGG GAG AAG GCA ACA CGA TG 3’ | 420 |
| Tv-SUBti -S1 | 5’ TGT CCA GAC CCT CGC TGA TA 3’ | 461 |
| Tv-SUBti -As1 | 5’ CAA CGA AGT TTG CAC CCC AG 3’ | 461 |
2.4. DNA Extraction
After growing the samples, the mycelium was collected and washed with distilled water and frozen at -80°C. The protocol of i-genomic Plant DNA Extraction Mini Kit (INtRON) (Lot. No. 13110450, Korea) was followed according to the manufacturer’s instructions.
2.5. PCR Amplification
Amplification was performed in a thermal cycler using a total volume of 25 μl consisting of Tag DNA polymerase, buffer, Master Mix of SINA COLON. Co., magnesium chloride, and deoxyribonucleotide triphosphates. Furthermore, primers, dermatophyte DNA, and distilled water were added to it.
For PCR reactions, the guidelines in Table 2 were used, and the PCR products were run on 2% agarose gel visualized on ultraviolet-transilluminator, and photographed by the use of gel documentation system and 1000 kb ladder as molecular weight marker. In addition, the proven clinical isolates of T. verrucosum and M. gypseum were used as positive controls.
Table 2.
Polymerase chain reactions
| Step | Time | Temperature (°C) | Cycle |
|---|---|---|---|
| Initial denaturation | 5 min | 94 | 36 |
| Denaturation | 30 sec | 94 | |
| Primer annealing | 45 sec | 63 | |
| Extension | 1 min per kb | 72 | |
| Final extension | 5 min | 72 |
2.6. Statistical Analysis
The Chi-square and student t-test were employed for analyzing the data in order to show statistical independence between the presence of the virulent gene (SUB) in the two dermatophytes (i.e., T. verrucosum and M. gypseum) and origin of the samples (i.e., human and animal isolates and soil samples).
3. Results
Based on micro- and macroscopic examination, out of 30 samples, 14 isolates were identified as T. verrucosum and 4 clinical isolates and 7 soil samples were identified as M. gypseum (3/14 human and 11/14 cattle isolates).
3.1. Molecular Identification by PCR
The positive results of PCR products on the 2% gel showed that among all the isolates, 64% (16/25) of them carried SUB gen; however, the gene could not be amplified for 36% (9/25) of the isolates (Figures 1 and 2). In addition, 44% (11/14) of the T. verrucosum clinical (i.e., human and animal) isolates were positive for the presence of SUB gene, among which the human samples completely indicated the gene activity (3/3; 100%).
Figure 1.
Polymerase chain reaction results obtained for SUB gene in T. verrucosum and M. gypseum deoxyribonucleic acid (DNA); lane M 100-1000-bp DNA molecular weight markers; C+ proved clinical isolates of T. verrucosum and M. gypseum used as positive controls; a) positive results of 400 bp fragment of SUB gene observed in T. verrucosum (11/14); lane 1: 3/11 isolates of human hair and face; lane 2: 8/11 isolates of cattle skin; b) positive results of 200 kb fragment observed in M. gypseum (5/11); lane 1: 1/5 isolate of human hair; lane 2: 2/5 dog and horse hair; lane 3: 2/5 isolates of soil
Figure 2.
Frequencies of the SUB gene in T. verrucosum and M. gypseum
In case of M. gypseum, only about 20% (5/11) of the isolates demonstrated the presence of SUB gene in which the proportion of the soil samples was 2/7; nevertheless, 3/4 clinical samples carried this gene (Table 3). Statistical analysis showed a significant relationship between the presence of the SUB gene and the samples collected from human, animals, and soil (P˂0.005).
Table 3.
Demographic characteristics
| Frequency | |||||
|---|---|---|---|---|---|
| M. gypseum | M. gypseum | T. verrucosum | T. verrucosum | Sample number | Sample type |
| Lack of genes | Presence of genes | Lack of genes | Presence of genes | ||
| 0 | 1 | 0 | 3 | 4 | Human |
| 0 | 0 | 3 | 8 | 11 | Cow |
| 0 | 1 | 0 | 0 | 1 | Horse |
| 1 | 1 | 0 | 0 | 2 | Dog |
| 5 | 2 | 0 | 0 | 7 | Soil |
| 6 | 5 | 3 | 11 | 25 | Total |
| 24 | 20 | 12 | 44 | Frequency (%) | |
4. Discussion
The production of various proteolytic secondary metabolites, such as enzymes, and lytic activity of dermatophytes have already been described (Moallaei et al., 2009; Lemsaddek et al., 2010; Achterman and White, 2012; Tarabees et al., 2013), which have proven keratinase as a considerable virulent factor, concerned with clinical dermatophytosis (Achterman and White, 2012).
Despite the high prevalence of dermatophyte infections, there has been limited information about the pathogenicity mechanisms (i.e., the production of SUB gene) of T. verrucosum and M. gypseum (Grumbt et al., 2011). This finding shows that these two dermatophytes have been poorly studied at the molecular level. In the present study, 14 clinical isolates of T. verrucosum were investigated for the determination of the SUB gene activity.
Out of the total isolates (n=14), 11 isolates showed the presence of the gene (3 human and 8 cattle isolates), and confirmed the results of the study conducted by Lemsaddek et al. (2010).
Addressing a potential function of the SUB gene in the clinical isolates of M. gypseum (Tarabees et al.,2013), it was tried to identify the presence of the gene in its clinical (i.e., human and animal) and nonclinical (i.e., soil) isolates which can act as three different SUBstrates for enzyme activity. In the present study, only three samples (human , dog , and horse ), showed the presence of the gene, which seemed to be consistent with the results of a study by Lemsaddek et al. (2010). Only, two (2/11) soil samples showed SUB gene activity that supported the investigation of the presence of the gene in the two dermatophytes in a study in Iran (Moallaei et al., 2009) and another study by Tarabees et al. (2013). Staib et al. (2010) by studying the role of unique secreted proteases in severe inflammatory skin infections in humans and rodents using microarray on the expression of at least 23 protease genes in vivo in dermatophytes. The results of their study indicated the expression of the SUB6 gene among the protease genes as an important allergen gene that was strongly increased during infection.
Giudice et al. (2012) reported no activity of SUB genes in the clinical isolates of M. gypseum. They claimed that none of the designed primers was amplified in M. gypseum and only 1 kb band was produced with ERF3R3 primers indicating 92%, 88%, 78%, and 78% similarity with Arthroderma gypseum, T. rubrum, T. tosorance, and T. verrucosum, respectively. What they reported completely differed from the results of the present study and study by Lemsaddek et al. (2010).
Due to different reports of the presence/absence of the SUB gene (Muhsin et al., 1997; Lemsaddek et al., 2010; Giudice et al., 2012), it is necessary to collect and study more samples and perform gene sequencing and gene expression.
Lemsaddek et al. (2010) manifested the relative risk of the SUB 4-7 genes in clinical and nonclinical samples with 95% confidence level. The obtained findings from their study are in line with the results of the present study that showed the role of the SUB genes family in the prevalence of the two dermatophytes infection
At least two SUB-lysine family gene sequences were reported in the clinical and nonclinical collected samples; nevertheless, none of the seven genomic sequences in four clinical and nonclinical Trichophyton isolates were detected in a similar study (Lemsaddek et al., 2010).
Since the present study has been the first report of SUB gene activity in the clinical and nonclinical isolates of T. verrucosum and M. gypseum in Iran, it can be used as a platform for performing further studies in this regard. The obtained results also showed that there was a significant difference in the presence of SUB gene in the clinical and nonclinical samples.
Authors' Contribution
Study concept and design: F. N.
Acquisition of data: S. J. H. and F. N.
Analysis and interpretation of data: S. J. H. and M. B.
Drafting of the manuscript: F. N. and S. J. H.
Critical revision of the manuscript for important intellectual content: J. H. and S. R.
Statistical analysis: S. J. H. and F. N.
Administrative, technical, and material support: S. J. H. and F. N.
Ethics
We hereby declare all ethical standards have been respected in preparation of the submitted article.
Conflict of Interest
The authors declare that they have no conflict of interest.
Grant Support
This study a self-funded study.
Acknowledgement
The authors would like to express their gratitude to the Department of Medical Mycology at Tehran University of Medical Sciences, Tehran, Iran, as well as ATIYESALAMAT Alborz Laboratory, Karaj, Iran.
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