INCIDENCE OF FUNGALYSINS VIRULENCE GENES (MEP1-5) IN DERMATOPHYTES ISOLATED FORM INFECTED CASES IN EGYPT

INCIDENCE OF FUNGALYSINS VIRULENCE GENES (MEP1-5) IN DERMATOPHYTES ISOLATED FORM INFECTED CASES IN EGYPT

TARABEES, R.; SABRY, M. and ABDEEN, E.

Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Sadat City, Minufyia University

Corresponding Author: Tarabees, R

Email: redahealth@yahoo.com

Tel: 00201092608732

INTRODUCTION

Dermatophytes are specialised filamentous fungi that considered as the most common cause of superficial mycoses in humans and animals (Weitzman and Summerbell 1995). Although Dermatophyte infection is mainly superficial, however, immunocompormised patients can experience severe, disseminated disease (Rodwell     et al. 2008). Despite the fact that dermatophyte infection is treatable, however, there is a high rate of reinfection either because of relapse or new infection (Gupta and Cooper 2008). Dermatophytes induced superficial mycoses resulting in treatment costs of close to half a billion dollars annually in USA (Smith et al., 1998). The scarcity of information concerning the dermatophytes virulence characteristics might be because the fact that these fungi have not to date been intensively studied at the molecular level comparing with other fungal pathogens such as Candida albicans and Aspergillus Fumigatus. Recently, the dermatophyte full genome sequence has only become available, and introduction of new molecular tools like PCR and microarray have been used as an accurate reliable method. The recent advances in the molecular diagnosis of dermatophytes have been reviewed in details in Achterman and white (Achterman and White 2012). The better understanding of this mechanism of virulence is the rational basis for the development of effective treatment and prophylactic strategies.

The host-fungus relationship in dermatophytes infection is complex and still required further investigation. There are many studied have carried out to characterise the secreted dermatophytic proteases at the molecular level. The newly introduced genetic tools such as PCR and microarray that allow rapid, effective, and accurate functional investigation of dermatophytes genes and identification of the most important virulence factors involved in dermatophytes pathogenesis. The keratin, collage, elastin and other skin proteins may be substrate for the endo-and exoprotease produced by dermatophytes (Monod et al., 2005; Monod 2008), and the invasion of dermatophytes into the tissue requires the elongation of germ tube and secretion of various proteolytic and lipolytic exoenzymes(Duek  et al., 2004). Dermatophytes are true pathogenic fungi infecting healthy animals and the production of metalloproteases during in vivo infection is seen to have a role in dermatophytes virulence (Brouta et al., 2002).

Several proteases have already been isolated from different species of dermatophytes and showed keratinolytic, elastinolytic and/or collagenolytic activities (Abdel-Rahman 2001; Moallaei. H et al., 2006). Brouta and co-workers described a gene family [MEP1, MEP2 and MEP3] coding for three endometalloproteases in M.canis canis and reported the production of MEP2 and MEP3 proteases during in vivo experiences in guinea pigs. MEP3 was characterized and shows collagenolytic, elastinolytic and keratinolytic activities (Brouta et al., 2001). Other authors recognized two new genes, MEP4 and MEP5, in M. canis as a part of a five member gene family (named MEP1-5) encoding for secreted endometalloproteases (fungalysins), also identified in Trichophyton rubrum and T. mentagrophytes (Jousson et al., 2004).

So far, there is no data about the incidence of these fungalysins genes (MEP1-5) is Trichophyton verrucosum isolated samples form Egypt. Consequently, the main aim of this was to screen the incidence of five fungalysins virulence genes (MEP1-5) associated dermatophytes infection by using PCR. The data obtained from this study showed that, the incidence of MEP1-4 was only 10% and 20% for MEP5.

MATERIALS and METHODS

1- Animals and Samples collection: A total of 100 hair and skin scrapings were collected from suspected ringworm lesions of infected horses and cattle from different localities in Minufyia and Shargia Governorates. The skin lesions were cleaned and disinfected with 70 % ethyl alcohol to remove surface contaminants. The scales were scrapped from the edges of the lesions by using blunt scalpel blade until blood was drawn. The basal root portion of stubby or damaged looking hair which contains the most useful diagnostic materials was collected by plucking the hair with sterile forceps. Samples were then collected in a sterile labelled closed envelop and transferred as soon as possible to laboratory for mycological examination (Sinski et al., 1979).

2- Laboratory isolation and identification of Dermatophytes:

Samples were cultivated on Sabouraud^,s dextrose agar medium with cyclohexamide and antibiotics (Kwon-Chung and Bennett 1992) Sabouraud^,s dextrose agar medium enriched with thiamine and inositol (Wawrzkiewicz and Wawrzkiewicz 1192). The antibiotics, thiamine, and inositol were added to the sterile media under full aseptic conditions.

A- Direct microscopical examination:

The skin scrapings and the broken hairs were place on a clean glass slide, then a drop of 20% KOH was added and covered with a cover slide, and gently heated and left for 1 hour. The slides were than examined for fugal elements (hyphea and spores around (ectothrix) or within the hairs (endothrix)) by using low and high power.

B- Isolation of dermatophytes:

The collected specimens from different animals were cultivated Sabouraud^,s dextrose agar supplemented with antibiotics, actidion, thiamine, and inositol, the inoculated media were then incubated at 25⁰C and 37⁰C for 3-4 weeks for rapid isolation of Trichophyton verrucosum.

C- Identification of the isolated dermatophytes (Rebell and Taplin 1970):

The isolated fungi were identified according to macroscopical and microscopical morphology of the isolates.

A-     Macroscopical examination of the cultures:

The examination involves, rate of growth, colour, texture of the colony or consistency (Cottony, fluffy, suede-like, and wiry), its surface topography (flat, folded, plicate, rugose) and reverse side of colony (pigmentation of the medium), margins, elevation and detachability from the agar surface.

B- Microscopical examination of the cultures:

B1-wet mount preparation (Collee et al., 1996)

A small part of the colony was gently teased out on the slide with a drop stain [lactophenol cotton blue] using a flamed bent inoculating needle or straight wire. A cover slip was applied with gentle pressure then examined by low and high power.

B2-Slide culture technique (Kwon-Chung and Bennett 1992)

The test was carried out to examine the presence of macro and micro conidia. a sterile glass slide was placed over a bent glass rod in bottom of sterile Petri dish, block of SDA. 1cm × 1cm was inoculated with the examined fungus. Few millilitres of sterile distilled water were added in the bottom of the Petri dish to keep its humidity. The plates were then incubated at 25 ^ₒC for 2-3 weeks and examined daily for identification of micro and macro conidia of T. verrucosum.

3- Selection of T. verrucosum used for DNA isolation preparation:

The selection of T. verrucosum samples was based on the conventional isolation and identification methods (macroscopically and microscopically, urease production…etc) The selected stains should be have the following criteria, strain should be a local strains, highly virulent strains isolated from badly infected animals, rapid grower in vitro with intensive spore forming (microcondia) (Brandebusemeyer 1990).

4- DNA isolation and PCR technique

The PCR technique was carried on isolated samples to verify verrucosum. DNA was extracted from the positive samples (10 positive samples were verified macroscopically and microscopically)

A- Identification of Trichophyton verrucosum

The Identification of verrucosum was primarily based on the detection of Simple repetitive Oligonucleotide (GACA) 4 as a single primer for identification of species of dermatophytes. DNA was extracted from the culture samples according to method previously described by Liu et al. (Liu et al., 2000) [20] and from the direct hair and skin scraping using Genejet Plant Genomic DNA extraction kits [Sigma Scientific services Co, Egypt] (Engy 2012).

1-  Amplification reaction using Simple repetitive Oligonucleotide [GACA]4 (FAGGI et al., 2001)

The dermatophyte genomic DNA samples (2 µl) were amplified by PCR in a reaction mixture (20 µl) containing 10 µl of Maximo Taq Pol. 2 X pre-mix, 1 µl of each primer, and fill up to 20 µl PCR grade water. Samples were amplified as follows: initial denaturation at 95°C for 5 min, followed by 39 cycles of I min at 93°C, annealing for 1 min at 50°C and extension for 1 min at 72°C. This was followed by a final extension step of 7 min at 72°C The PCR products (10 µl /sample) were electrophoresed through 1% agarose gel and then stained with ethidium bromide and were visualized under UV light.

2- Amplification reaction using MEP1-5 primers (Lemsaddek. L et al., 2010).

The dermatophyte genomic DNA samples (2 µl) were amplified by PCR in a reaction mixture (20 µl) containing 10 µl of Maximo Taq Pol. 2 X pre-mix, 1 µl of each primer; and fill up to 20 µl PCR grade waterPCR cycling conditions consisted of: 94°C for 3 min followed by 35 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C and a final extension step of 10 min at 72°C. The PCR products (10 µl) were resolved by agarose gel electrophoresis (1%), and then stained with ethidium bromide and were visualized under UV light. The following table shows the primers sequences [23]. These primers were designed mainly for Trichophyton rubrum based on the gene sequence of MEP1-5 available in genebank database.

RESULTS

1- Isolation and identification of T. verrucosum strains:

In this study we identified T. verrucosum depend on macroscopic appearance. Where it was very slow growing with heaped up, button like with folded white colony and non pigmented reverse side as shown in photo (1) and on microscopic appearance, the fungus gave characteristic chlamydospores arranged in chains as shown in photo (2A), and using slide culture technique for demonstration of macro and micro conidia, where it give Clavate to pyriform microconidia as shown in photo (2B).

2- Molecular identification of T. verrucosum by using Simple repetitive Oligonucleotide (GACA) 4 primer

Hair and skin scraping samples from affected horses and cattle that give positive result by conventional methods were tested by using Simple repetitive Oligonucleotide (GACA) 4 primer. The primer was able to amplify two DNA fragments of about 200 and 600 bp that specific to T. verrucosum from all tested samples as shown in photo (Koop et al.).

3- Amplification of MEP1-5 genes using specific primers.

In the present study, the incidence of metalloproteases enzymes encoding genes was screened using PCR. The test was carried out on 10 positive samples that were verified using simple repetitive Oligonucleotide (GACA)₄. The amplification of MEP1-4 was successful in 10% while MEP5 was 20% of the screened samples (photo. 4A-C).

DISCUSSION

Dermatophytosis is one of the most frequent skin diseases of pets and other livestock including cattle and horses. Dermatophytosis spread among animal by direct and indirect contact and also its zoonotic importance has been revealed. Although, the contagiousness among animal communities, high cost of treatment, difficulties of control measures, and public health consequences of animal ringworm explain their great importance. However, the molecular basis of the Pathogenicity of theses fungi is largely unclear. Thus, the better understanding of this basis is considered the rational element in the developing of the appropriate therapeutic intervention.

In the present study we identified T. verrucosum depending up on macroscopic and microscopic appearance based in the identification key of the veterinary mycology laboratory manual (Hungerford et al. 1998) and the laboratory handbook of dermatophytes (Kane et al., 1997). T. verrucosum characterized by slow growing with heaped up, button like with folded white colony and non pigmented reverse side as shown in photo (1):, and microscopically, it gave characteristic chlamydospores arranged in chains as shown in photo [2]  this agree with koop et al. (Koop et al., 2008) and Clavate to pyriform microcondia, as shown in photo (2): and this agree with (Hassan 1998) and with (Calina et al., 2010) who depend on culturing on selective medium (Sabouraud,s dextrose agar medium and dermatophyte test medium) morphological and culture characterization for isolation  and identification of Trichophyton strains. In addition, the positive samples [conventional isolation and identification methods] were verified as T. verrucosum using simple repetitive Oligonucleotide [GACA]₄ [photo, 3]. The usage of simple repetitive Oligonucleotide [GACA]₄ as fingerprint for identification of Trichophyton was reviewed in (FAGGI et al., 2001).

Dermatophytes are known to infect keratinised tissue like hair, skin, and nails and such ability is considered as a major virulence attribute of dermatophytes infection (Achterman and White, 2012). The secretion of proteases by dermatophytes in vivo, which are thought to be responsible for the colonisation and degradation of keratinised tissue during the infection. Screens have been used mainly to identify gene products likely to play a role in virulence, and dermatophytes have been shown to secrete more than 20 proteases in vitro when grown in media containing protein as a sole source for nitrogen (Burmester et al., 2011). Although, the molecular techniques such as PCR and microarray have elucidated many genes involved in the dermatophytosis infection like fungalysins, however, there a scarcity of data regarding the screening of these genes in Trichophyton verrucosum. In addition, there is no data regarding the incidence of these virulence genes in dermatophytes isolated from local strains in Egypt.

In the present study, the amplification of five genes encoding proteases protein secreted from dermatophytes and played indispensable role in its pathogenesis was screened by PCR. PCR provides a rapid and sensible tool for identification of dermatophytes isolates, that is independent of morphological and biochemical characteristics and thus enhances laboratory diagnosis of dermatophytosis (Liu et al., 2000).The data collected from the study revealed that, the amplification of MEP1-4 was only successful in 10% of the screened samples and 20% for MEP5. The data are consistent with the findings of Burmester et al. (2011), who found that only some of the keratin-induced proteases were strongly expressed during fungus-keratinocyte interaction. On the contrary, these findings are contradictory with that of Staib et al. (Staib et al., 2010), who reported none of the genes encoding the in vitro keratin-specific metalloproteases MEP1 and MEP3 was up-regulated during infection. Among the fungalysins family genes, MEP3 has already been characterised and showed elastinolytic, keratinolytic and collagenolytic activities (Brouta et al., 2002). Despite the fact that, this gene was successfully amplified only in 10% of the screened samples, knowing the proteolytic activity of MEP3, its effective absence has to be proven by analysis of more isolates. In case of confirmation, it can be emphasized its replacement by another gene coding for a protein with similar activities. These finding is consistent with that of Lemsaddek et al. (2010), who reported the presence of MEP1-3 in the screened samples of dermatophytes. Of note, among the screened samples by Lemsaddek      et al. (2010) none of them was Trichophyton verrucosum.

The presence of MEP4 was only detected in 10% of screened samples. The successful amplification of MEP4 in the Trichophyton verrucosum isolated from infected cases in Egypt is considered as a new finding. The successful amplification of MEP4 was detected in many studies. The presence of MEP4 was detected in more than half of the analyzed isolates. The protein encoded by MEP4, was the most secreted by M. canis, T. mentagrophytes and T. rubrum on soy medium (Jousson et al., 2004). Using the same medium, (Giddey et al., 2007) detected these proteases on the supernatant of A. vanbreuseghemii, T. equinum, Ttonsurans, T. rubrum, T. soudanense and T. violaceum. mRNAs corresponding to MEP4 was also detected on keratin medium inoculated with T. rubrum(Maranhão FCA et al., 2007).

Regarding the amplification of MEP5, MEP5 was successfully amplified in 20% of screened dermatophytes samples. The presence of MEP5 in our samples is consistent with the findings of Lemsaddek et al.,, who reported the successful amplification of MEP5 only in eight species. Although, the high incidence of MEP5 gene among our collection of isolates seems to indicate an important role of its coding protein in the infection process. However, a future study including an additional number of isolates, representing all species, will help to unravel the putative role of MEP5 protein during the infectious process. Of note, the high incidence of MEP1-5 was observed in the clinical samples isolated from infected horses, and mainly that obtained directly from hair samples. This observation indicates that, the pattern of virulence gene expression is varies from in vivo and in vitro conditions. In addition, this emphasises the paramount role of these genes in dermatophytes infection. Thus, further investigations are still urgently required to elucidate the crucial role of these genes, and verify the most important conditions that alter the putative expression of these genes either in vivo or in vitro.

CONCLUSION

The data presented in this study for our knowledge is considered the first screening data regarding the incidence of fungalysins (MEP1-5) in culture samples and hair of infected cases in Egypt. The high incidence was observed in horse samples (DNA obtained directly from hair samples) emphasises the important role of these genes in dermatophytes infection in horses. Moreover, more investigations are urgently required to clarify the variation in virulence between cattle and horses.

Conflict of interest:

The authors would like to declare there is no conflict of interest.

REFERENCES

Abdel-Rahman, S.M. (2001): "Polymorphic exocellular protease expression in clinical isolates of Trichophyton tonsurans. Mycopathologia. 150: 117-120.".

Achterman, R.R. and White, T.C. (2012):                     "Dermatophyte virulence factors: identifying and analyzing genes that may contribute to chronic or acute skin infections. Int J. Microbiol 2012, 358305.".

Brandebusemeyer, E. (1990): "Untersuchungen der virulenz, Vertraglichkeit und Wirksamkeit einer Vakzine gegen die Trichophytie des Rindes an Rind und Meerschweinchen. Diss. Tierarztl. Hochsch. Hannover, 1990.".

Brouta, F.; Descamps, F.; Fett, T.; Losson, B. and B. Gerday C.M. (2001): "Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis. Med Mycol.39: 269-275.".

Brouta, F.; Descamps, F.; Monod, M.; Vermout, S.; Losson, B. and Mignon, B. (2002). "Secreted metalloprotease gene family of Microsporum canis. Infect Immun 70, 5676-5683.".

Burmester, A.; Shelest, E.; Glockner, G.; Heddergott, C.; Schindler, S.; Staib, P.; Heidel, A.; Felder, M.; Petzold, A.; Szafranski, K.; Feuermann, M.; Pedruzzi, I.; Priebe, S.; Groth, M.; Winkler, R.; Li, W.; Kniemeyer, O. Schroeckh, V.; Hertweck, C.; Hube, B.; White, T.C.; Platzer, M.; Guthke, R.; Heitman, J.; Wostemeyer, J.; Zipfel, P.F.; Monod, M. and Brakhage, A.A. (2011): "Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi. Genome Biol 12, R7.".

Calina, D.; G. Rapuntean, N.N.; Fit, G.  and Olariu, R. (2010): "Aspects regarding immune prophylaxis enzootic bovine ringworm. Cercetari Agronomice Moldova, Vol. XLIII, No. 1[141]: 73-78.".

Collee, J.G.D.; J.P.; A.G. Fraser, B.P. Marmion and Simmons, A. (1996): "Mackie and McCartney Practical Medical Microbiology. 14thEd. The English Language Book Society and Churchill Living Stone Edinburgh and New York.".

Duek, L.; Kaufman, G.; Ulman, Y. and Berdicevsky, I. (2004): "The pathogenesis of dermatophyte infections in human skin sections. J. Infect. 2004; 48:[175-180].".

Engy, E.E.F. (2012): "Application of molecular techniques for rapid diagnosis of dermatophytes infection in horses. M. V. Sc. Thesis [Microbiology] Fac. Vet. Med. Cairo Univ.".

Faggi, E.; Pini, G.; Campisi, E.; Bertellini, C.; Difonzo, E. and Mancianti, F. (2001): "Application of PCR to Distinguish Common Species of Dermatophytes. JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2001, p. 3382–3385".

Giddey, K.; Monod, M.; Barblan, J.; Potts, A.; Waridel, P. and M. Zaugg, C.Q. (2007): " Comprehensive analysis of proteins secreted byTrichophyton rubrum and Trichophyton violaceum under in vitro conditions. J. Proteome Res. 2007; 6: 3081-3092.".

Gupta, A.K. and Cooper, E.A. (2008): "Update in antifungal therapy of dermatophytosis. Mycopathologia 166, 353-367.".

Hassan, W.H. (1998): "Microbiological studies on dermatomycosis in farm animals. M.V.Sc. Thesis, [Bacteriol. Mycol. Immunol.] Fac. Vet. Med., Cairo Univ., [Beni-Suef].".

Hungerford, L.L.; Campbell, C.L. and Smith, A.R. (1998): "Veterinary Mycology Laboratory Manual. Iowa State University, Press, Ames, USA.".

Jousson, O.; Lechenne, B.; Bontems, O.; Capoccia, S.; Mignon, B.; Barblan, J. and A.M.M. Quadroni M. (2004): "Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum. Microbiology 2004 [150];    301-310."

Jousson, O.; Lechenne, B.; Bontems, O.; Capoccia, S.; Mignon, B.; Barblan, J. and M. Quadroni, M.M. (2004): "Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum. Microbiology 2004 [150]; 301-310.".

Kane, J.; Summerbell, R.; Sigler, L.K.S. and Land, G. (1997): "Laboratory Handbook of Dermatophytes. Star Publishing Company, Korea.".

Koop, B.F.; Von Schalburg, K.R.; Leong, J.; Walker, N.; Lieph, R.; Cooper, G.A.; Robb, A.; Beetz-Sargent, M.; Holt, R.A.; Moore, R.; Brahmbhatt, S.; Rosner, J.; Rexroad, 3rd, C.E.; McGowan, C.R. and Davidson, W.S. (2008). "A salmonid EST genomic study: genes, duplications, phylogeny and microarrays." BMC Genomics 9: 545.

Koop, B.F.; Von Schalburg, K.R.; Leong, J.; Walker, N.;  Lieph, R.; Cooper, G.A.; Robb, A.; Beetz-Sargent, M.; R.A. Holt, Moore, R.; S. Brahmbhatt, Rosner, J.; C.E. Rexroad, M. 3rd, C.R.  and Davidson, W.S. (2008): "A salmonid EST genomic study: genes, duplications, phylogeny and microarrays. BMC Genomics 9, 545.".

Kwon-Chung, K.J. and Bennett, J.E. (1992): "Medical Mycology. Lea and Febiger, Malvern, Pennsylvania, USA.".

Lemsaddek, L.; Chambel, L. and Tenreiro, R. (2010): "Incidence of fungalysin and subtilisin virulence genes in dermatophytes. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, A. Mendez. Vials [Ed].".

Liu, D.C.S.; Baird, R. and Pedersen, J. (2000): "Application of PCR to the identification of dermatophyte fungi. J. Med. Microbial, 49: 493-497.".

Maranhão, FCA.; N. Paião FG. and M.-R. (2007): "Isolation of transcripts over[expressed in human pathogen Trichophyton rubrum during growth in keratin. Microb Pathogenesis. 2007; 43[4]: 166-72.".

Moallaei, H.; Zaini, F.; Larcher, G. and J. Beuche. B.B. (2006): "Partial purification and characterization of a 37 kDa extracellular proteinase from Trichophyton vanbreuseghemii. Mycopathologia. 161:3    69–75.".

Monod, M. (2008): "Secreted proteases from dermatophytes. Mycopathologia. 2008;166: [285-294].".

Monod, M.; Lechenne, B.; Jousson, O.; Grand, D.; Zaugg, C.; Stocklin, R. and Grouzmann, E. (2005): "Aminopeptidases and dipeptidyl [peptidases secreted by the dermatophyte Trichophyton rubrum. Microbiology. 2005; 151: 145–155.".

Rebell, G. and Taplin, D. (1970): "Dermatophytes, their recognition and identification. Revised Edition.".

Rodwell, G.E.J.; Bayles, C.L.; Towersey, L. and Aly, R. (2008): "The prevelance of dermatophyte infection in patients infected with human immunodeficiency virus. international journal of Dermatology 47, 339-343.".

Sinski, J.T.; Wallis, B.M. and Kelly, L.M. (1979): "Effect of storage temperature on viability of Trichophyton mentagrophytes in infected guinea pig skin scales. Clin. Microbiol., 10[6]: 841-842.".

Smith, E.S.; Feldman, S.R.; Fleicher, A.B.; Leshin, B. and McMicheal, A. (1998): "Characteristics of office-based visits for skin cancer: dermatologists have more experience than other physicans in managing malignant and premalignant skin conditions. Dermatologic surgery 24, 981-985.".

Staib, P.; C. Zaugg, M.B.; Weber, J.; Grumbt, M.; Pradervand, S.; Harshman, K. and Monod, M. (2010): "Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection. Microbiology 156, 884-895.".

Wawrzkiewicz, K. and Wawrzkiewicz, J. (1192): "An inactivated vaccine against ringworm. Comp. Immunol. Microbiol. Infect. Dis., 15: 31-40.".

Weitzman, I. and Summerbell, R.C. (1995): "The dermatophytes. Clin Microbiol Rev 8, 240-259.".