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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 1  |  Page : 87-92
Is moderation of protease production an adaptation of well-defined anthropization in dermatophytes?


1 Faculty of Medicine, AIMST University, Malaysia
2 Department of Advanced Zoology and Biotechnology, Sri Paramakalyani College, Malaysia
3 School of Medicine, AIMST University, Malaysia
4 Sri Paramakalyani Centre for Environmental Sciences, M S University, Alwarkurichi, India
5 R & D Center, Mikasa Cosmetics Limited, Chennai, India
6 Annamalai University, Chidambaram, India

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Date of Web Publication19-Jan-2010
 

   Abstract 

The protease activity of different isolates of dermatophytes representing different ecological groups namely geophilic, zoopahilic and anthropophilic, in their vegetative and sporulation growth phases were compared. Unlike their geophilic and zoophilic counterparts, all the isolates of anthropophilic dermatophytes viz. Trichophyton rubrum, T. mentagrophytes, T. tonsurans, T. violaceum and Epidermophyton floccosum recorded reduced protease activity during artificially induced sporulation phase in comparison to their vegetative growth phase. Even among the anthropophilic group, a classical moderation of protease activity was recorded in Trichyphyton rubrum which also correlates to its clinical manifestation. This enzyme moderation could also be an evolutionary adaptation of the anthropization of these species

Keywords: Dermatophytes, anthropization, protease, sporulation, enzyme moderation

How to cite this article:
Gokulshankar S, Ranjitsingh A, Venkatesan G, Ranjith M S, Vijayalakshmi G S, Prabhamanju M, Subashini S. Is moderation of protease production an adaptation of well-defined anthropization in dermatophytes?. Indian J Pathol Microbiol 2010;53:87-92

How to cite this URL:
Gokulshankar S, Ranjitsingh A, Venkatesan G, Ranjith M S, Vijayalakshmi G S, Prabhamanju M, Subashini S. Is moderation of protease production an adaptation of well-defined anthropization in dermatophytes?. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 14];53:87-92. Available from: http://www.ijpmonline.org/text.asp?2010/53/1/87/59191



   Introduction Top


Protease elaboration of various species of dermatophytes and their role in pathogenesis had been reported by several workers. [1],[2],[3] Most of the previous workers have studied the extra cellular protease liberated by these organisms in the growth medium or prepared the enzyme extract from the crushed mycelium of the fungus. The proteolytic enzyme activity of different species of dermatophytes during different growth phases (vegetative and sporulation) has also been studied earlier. [4] However, a comparative study of the enzyme profile of different ecological groups of dermatophytes viz. geophilic, zoophilic and anthropophilic during the vegetative growth phase and sporulation phase and their enzyme activity at different time intervals of growth (days) have not been studied in detail so far. Hence, it was planned to study the proteolytic enzyme profile of different species of dermatophytes during vegetative (mycelial) and artificially induced sporulation phase, and correlate the role of proteases in different ecological groups to the severity and chronicity of human infections caused by them.


   Materials and Methods Top


The following dermatophytes belonging to different ecological categories were used in the present study:

a. Anthropophilic

Trichophyton rubrum (6), T. mentagrophytes (6), T. tonsurans (6), T. violaceum (4), Epidermatophyton flococcum (4), (All of clinical origin)

b. Geophilic

Microsporum gypseum (2 clinical, 2 soil origin)

c. Zoophilic

Microsporum canis (2 clinical (human), 2 animal (pet dogs) origin)

The nature (chronic/non chronic), clinical type and severity (mild, moderate, severe, very severe) of infection of these isolates were documented. The persistence of lesion for more than one year with or without remission and recurrence, with or without treatment failure to any anti fungal agent was designated as chronic infection. The cases that did not fit in to this category of definition were defined as nonchronic.

Protease assay

Proteolytic enzyme assay on different ecological groups of dermatophytes (six isolates each of Trichophyton rubrum, T. mentagrophytes, T. tonsurans, four isolates each of T. violaceum and Epidermophyton floccosum, Microsporum gypseum and M. canis) was carried out with slight modification of the method described by Joo et al. [5] In brief, 0.5 ml of culture filtrate was incubated with one ml of one per cent vitamin free casein in Tris buffer (pH 7.5) at 45 °C for 20 minutes. After incubation, enzyme activity was arrested and the protein was precipitated by 5 ml of 20% trichloroacetic acid (TCA) and filtered through Whatman No.1 filter paper. The tyrosine in the filtrate was read spectrophotometrically at 280 nm. Similarly, a control was run in an identical manner with the culture filtrate being added after precipitation with TCA. The optical density value for tyrosine in the test was compared with the standard tyrosine graph and the enzyme activity was calculated using the standard procedure.

One unit of proteolytic enzyme activity was the amount of the enzyme which liberated one m mol of tyrosine/ml/minute under assay condition. The enzyme activity for each of the test organisms at different time intervals (days) of growth was represented in a graph by plotting the protease activity in the 'Y' axis and the time interval in 'X' axis. The enzyme activity of each of the dermatophytes in the two phases - artificially induced sporulation and vegetative, were studied for comparison using standard procedure. [4] The enzyme activity was also compared with severity of the lesion produced by the clinical isolates.

The enzyme activity for each of the test organisms at different time intervals (days) of growth was represented in a graph by plotting the protease activity in the 'Y' axis and the time interval in 'X' axis. The data obtained was analyzed statistically using Tukey-HSD test (SPSS version 11). Enzyme activity was also compared with the severity of the lesion produced by the clinical isolates.
   Results Top


Nature and Severity of Infection of Clinical Isolates

The details of nature and severity of infection caused by these clinical isolates have been tabulated in [Table 1] and [Table 2]a and b.

All six isolates of T. rubrum were isolated from patients who had chronic history of dermatophytoses (two cases each of tinea cruris and tinea corporis, one case each of tinea pedis and tinea unguium) while one/six isolate of T. mentagrophytes was isolated from patient with chronic history of tinea cruris. All the other isolates of dermatophytes were from non-chronic cases [Table 1].

Enzyme Activity of T. rubrum

The enzyme activity of the isolates of T. rubrum recorded at sporulation phase was much lower than the activity at the vegetative phase. During the sporulation phase four/six isolates recorded peak enzyme activity on day 18, while the remaining two isolates recorded peak activity on days 14 and 16 respectively.

The enzyme activity of T. rubrum isolates in vegetative phase showed variable patterns. Two/six isolates showed peak activity on day 22. The other four isolates exhibited peak values on dyas 14, 16, 18 and 20 respectively.

Enzyme Activity of T. mentagrophytes

The enzyme activity of the isolates of T. mentagrophytes recorded at sporulation phase was marginally lower than thr activity at the vegetative phase. Two/six isolates showed highest enzyme activity on day18, while the other three isolates showed enzyme peaks on days 16, 20 and 22. All the isolates of T. mentagrophytes recorded peak activities between 16 and 18 days during the vegetative phase.

Enzyme Activity of T. tonsurans

Five of the six isolates showed peak values between days 18 and 20, while a single isolate showed high enzyme activity on day 22 during the sporulation phase.

All the six isolates recorded peak values of enzyme activity between 18 and 20 days. The peaks recorded for each of the isolate in the vegetative phase is relatively higher than that recorded during their sporulation phase.

Enzyme Activity of T. violaceum

In the sporulation phase, two/four isolates recorded peak values on day 18, the remaining two/four isolates exhibited high enzyme activity on day 20; while all the four isolates exhibited highest enzyme activity on the day 20 during the vegetative phase. Further, the protease activity was higher in all the isolates during the vegetative phase

Enzyme Activity of E. floccosum

During the vegetative phase three/four isolates showed peak enzyme production while one isolate recorded highest peak on day 22, while the activities at the sporulation phase were varying for each isolate (14, 16, 18 and 20 days). However, all the isolates recorded low enzyme profile during the sporulation phase

Enzyme Activity of M. gypseum

All the four isolates and three out of four isolates showed peak values of enzyme activity between days 16 and 18 during sporulation and vegetative phases respectively [Table 9]. However, in all the four isolates the enzyme activity was relatively similar in the sporulation and vegetative phase

Enzyme Activity of M. canis

Two of the four isolates showed peak activity on day 18 and the remaining two/four isolates showed peak enzyme activity of day 20 at sporulation phase. During the vegetative phase, three isolates showed peak activity on day 20 while one isolate recorded peak activity on day 18 [Table 10]. However, in all the four isolates the enzyme activity was relatively similar in the sporulation and vegetative phase.

Statistical Analysis

Multiple Range Tests (Tukev- HSD test) were applied to compare protease activity of the different isolates of dermatophytes during sporulation and vegetative stages. The results indicate that the activity levels between the sporulation and the vegetative phase are comparable in M. gypseum and M. canis isolates where as the activity recorded during the vegetative phase was much higher than that recorded in the sporulation phases of T. rubrum, T. mentagrophytes, T. tonsurans, T. violaceum and E. floccosum. A similar co relation in enzyme activities recorded at different days at the vegetative and sporulation phases could also be recorded in the isolates of M. gypseum and M. canis [Table 3],[Table 4],[Table 5],[Table 6],[Table 7],[Table 8],[Table 9],[Table 10], [Figure 1].


   Discussion Top


The roles of protease in the pathogenesis of many microorganisms have been described. [6] For the hydrolysis of structural proteins of skin, hair and nails, dermatophytes require and therefore elaborate certain protein hydrolyzing enzymes. The roles of these enzymes in pathogenesis of the disease have been well established. [1],[2],[3],[7]

We studied the enzyme activity of all the dermatophytes between 10 and 22 days. The growth rate of fungi differs in different species. However, on an average, most of the isolates of the different species recorded peak enzyme activity between 16-20 days. Further, in the present study, high enzyme activity was seen during the vegetative growth phase of all the species of anthropophilic dermatophytes studied. The enzyme activity of Microsporum gypseum (geophile) and M. canis (zoophile) isolates were found to be high and comparable during both the vegetative and sporulation phase. Zoophilic and geophilic species usually evoke a severe inflammatory response in humans on infection and is almost and always severe. [8] Whether the ability to produce high levels of protease during sporulation phase by M. gypseum and M. canis as noted in our present study is the cause for the severe nature of infection when they clinically manifest in their unusual host (man) warrants a detailed study. However, a possible co relation between the ability to produce high levels of proteolytic enzymes during both sporulation and vegetative phases of growth to the severity of infection may not be ruled out. The lowest enzyme activity among the anthropophilic group was recorded in all the strains of Trichophyton rubrum during sporulation phase when compared to vegetative growth phase in all the isolates. Rippon [9] reported enhanced sporulation during parasitism. The low level of enzyme production during sporulation in Trichophyton rubrum might be the reason for the mild lesions produced in the host. Some researchers had already reported differences in proteolytic activity among strains isolated from different types of lesions. Strains from acute, inflamed and deep lesions were usually more proteolytic in vitro. [10],[11],[12],[13],[14] Of the four isolates of M. gypseum used, two were of clinical origin and two of soil origin. However, their potential to produce protease was similar under sporulation and vegetative conditions. Similarly, out of the four M. canis isolates two were isolated from dogs and two from human infection. The geophilic and zoophilic isolates [irrespective of their clinical (human/animal)/ soil origins] showed statistically comparable protease activity in sporulation and vegetative phases of growth. There seems to be no moderation of activity between these two growth phases in these organisms. Further, the clinical manifestation of the human isolates of M. gypseum and M. canis had been severe and extensive which is suggestive of a possible role for proteases. Severity of the lesions produced by Trichophyton rubrum is less and chronic when compared to other species of dermatophytes. It is strikingly evident in our present study as we find that all the clinical isolates of T. rubrum were from chronic cases and the case history of three isolates indicate the persistence of lesions for more than five years (data not included). Because of noninflammatory mild lesions, early lesions were untreated/neglected by the patients. The protease production is highly host specific showing reduced physiological activity when growing on their preferred host. [9] This would explain the well-established anthropization of these dermatophyte species.

The medium used to study the enzyme activity during sporulation was Takashio broth (1/10 diluted Sabouraud's dextrose broth with KH 2 PO 4 and MgSO 4 ). The spores obtained in Takashio broth were asexual conidia, but during parasitism, the organism produces more of arthroconidia. The study of the enzyme activity of Trichophyton rubrum during arthroconidia formation (produced during parasitism) was not possible because of the non availability of techniques to induce arthroconidial formation in vitro. Therefore, the low levels of protease activity of Trichophyton rubrum during sporulation phase cannot be directly correlated with pathogenesis.

Previous workers have reported correlation between fungal biomass and protease production in Trichophyton verrucosum. [15] However, in the present study, the total biomass of all the test fungi during their sporulation and vegetative growth phases was not estimated. Perhaps the recorded low levels of enzyme activity in Trichophyton rubrum during sporulation might also be due to differences in the biomass of the fungi grown in Takashio and Sabouraud's dextrose broth. The enzyme activity in Trichophyton rubrum and other dermatophytes in Takashio medium, which prevent pleomorphism of many dermatophytes, had been studied earlier. [16] Pleomorphic changes were observed during vegetative growth phase in Sabouraud's dextrose broth. The correlation between enzyme production and pleomorphic changes remains to be established. However, Lu [17] has reported that the pleomorphic strains of Trichophyton mentagrophytes perforated hair much faster than the non-pleomorphic strains.

Nevertheless it is really intriguing to know the reason for low protease production during sporulation in all the anthropophilic group of dermatophytes when the geophilic and zoophilic organisms showed almost statistically comparable levels of protease production during both phases of growth.

Trichophyton infection is now considered a major public health problem. [18],[19],[20],[21] Anthropophization in certain dermatophytes like T. rubrum is well-established with a total loss of certain geophilic characteristics such as conidial abundance and ornamentation, osmotolerence, hetrothallic mating, etc. [22] We presume that the moderation of protease activity in all the isolates of anthropophilic dermatophyte species during sporulation observed in the present study could also be an evolutionary adaptation of welldefined anthropization.


   Conclusion Top


This research study postulates that enzyme moderation could also be an evolutionary adaptation of the anthropization of certain dermatophytes. However, a detailed study on the moderation of different individual proteases viz. keratinase, elastase etc with relationship to the different ecological groups of dermatophytes would enable us in better understanding of the evolution of dermatophytes in general and emergence of obligate parasitism in anthropophilic dermatophytes in particular. It would also be intriguing to include isolates from HIV patients for future studies.

 
   References Top

1.Apodaca G, McKerrow JH. Regulation of Trichophyton rubrum proteolytic activity. Infect Immun 1989a;57:3081-90.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Apodaca G, McKerrow JH. Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum. Infect Immun 1989b;57:3072-80.   Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Abdel-Rahman SM. Trichophyton tonsurans exocellular protease expression: correlation with clinical presentation in tinea capitis. Clin Exp Dermatol 2002;27:268-71.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
4.Ranganathan S, Ranjith MS, Gokul Shankar S, Arun Mozhi Balajee S et al. Protease production in dermatophytes during sporulation and vegetative phase- its role in pathogenesis and mating type associated virulence. Indian J Dermatol 2000;45:174-81.  Back to cited text no. 4    Medknow Journal  
5.Joo HS, Kumar CG, Park GC, Kim KT, Paik SR, Chang CS. Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Process Biochem 2002;38:155-9.  Back to cited text no. 5      
6.Ogawa H, Nozawa Y, Rojanavanich V, Tsuboi R, Yoshiike T, Banno Y, et al. Fungal enzymes in the pathogenesis of fungal infections. J Med Vet Mycol 1992;30:189-96.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Tanaka S, Summerbell RC, Tsuboi R, Kaaman T, Sohnle PG, Matsumoto T, et al. Advances in dermatophytes and dermatophytosis. J Med Vet Mycol 1992;30:29-39.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Fisher F, Cook NB. Dermatophytoses: In Fisher F, Cook NB, editors Fundamentals of Diagnostic Mycology. Philadelphia: WB Saunders Company; 1998. p. 118-56.  Back to cited text no. 8      
9.Rippon JW. Dermatophytosis and dermatomycosis. In: Rippon JW, editor. Medical Mycology, the Pathogenic fungi and Pathogenic Actionmycetes. 3rd ed. Philadelphia: W.B. Saunders Company; 1988. p. 169-275.  Back to cited text no. 9      
10.Wawrzkiewicz K, Rubaj B, Ziolkowska G. Elastolytical activity in vitro and in vivo of the mycelial and spore forms of the Trichophyton Verrucosum strains. Mykosen 1978;21:236-44.  Back to cited text no. 10  [PUBMED]    
11.Minocha Y, Pasricha JS, Mohapatra LN, Kandhari KC. Proteolytic activity of dermatophytes and its role in the pathogenesis of skin lesions. Sabouraudia 1972;10:79-85.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Rippon JW, Garber ED. Dermatophyte pathogenicity as a function of mating type and associated enzymes. J Invest Dermatol 1969;53:445-8.   Back to cited text no. 12  [PUBMED]    
13.Skorepová M, Hauck H. Extracellular proteinases of Trichophyton rubrum and the clinical picture of tinea. Mykosen. 1987;30:25-7.  Back to cited text no. 13      
14.Samdani AJ, Dykes PJ, Marks R. The proteolytic activity of strains of Trichophyton mentagrophytes and T. rubrum isolated from tinea pedis and tinea unguium infections. J Med Vet Mycol 1995;33:167-70.  Back to cited text no. 14      
15.Grzywnowicz G, Lobarzewski J, Wawrzkiewicz K, Wolski T. Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trichophyton verrucosum. J Med Vet Mycol 1989;27:319-28.  Back to cited text no. 15  [PUBMED]    
16.Takashio M. Sexual reproduction of some Arthroderma and Nannizzia on diluted Sabouraud agar with or without salts. Mykosen 1972;15:11-7.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]  
17.Lu YC. A new method for the study of hair digestion by dermatophytes. Mycopathologia 1962;17:225-35.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]  
18.Fuller LC, Child FC, Midgley G, Higgins EM. Scalp ringworm in south-east London and an analysis of a cohort of patients from a paediatric dermatology department. Br J Dermatol 2003;148:985-8.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]  
19.Ghannoum M, Isham N, Hajjeh R, Cano M, Al-Hasawi F, Yearick D, et al. Tinea capitis in Cleveland: Survey of elementary school students. J Am Acad Dermatol 2003;48:18-93.  Back to cited text no. 19      
20.Gupta AK, Summerbell RC. Increased incidence of Trichophyton tonsurans tinea capitis in Ontario, Canada between 1985 and 1996. Med Mycol 1998;36:55-60.  Back to cited text no. 20  [PUBMED]  [FULLTEXT]  
21.Hay RJ, Robles W, Midgley G, Moore MK; European Confederation of Medical Mycology Working Party on Tinea Capitis. Tinea capitis in Europe: New perspective on an old problem. J Eur Acad Dermatol Venereol 2001;15:229-33.  Back to cited text no. 21  [PUBMED]  [FULLTEXT]  
22.Weitzman I, Summerbell RC. The dermatophytes. Clin Microbiol Rev 1995;8:240-59.  Back to cited text no. 22  [PUBMED]  [FULLTEXT]  

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Correspondence Address:
S Gokulshankar
Microbiology Unit, Faculty of Medicine, AIMST University, Jalan Bedong, Semeling 08100, Kedah
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.59191

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]

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