Photon Journal of Microbiology. Photon 109 (2016) 262-275 https://sites.google.com/site/photonfoundationorganization/home/photon-journal-of-microbiology Original Research Article. ISJN: 5349-7294: Impact Index: 4.13

Photon Journal of Microbiology

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Effects of in vitro variation in nutrients on growth and extracellular protease production in Trichophyton rubrum, a human dermatophyte Madhulika Srivastavaa*, Shakti K. Prabhujib a b

Department of Botany, M.G. Post Graduate College, Gorakhpur-273001, India Biotechnology and Molecular Biology Centre, M.G. Post Graduate College, Gorakhpur-273001, India

Article history: Received: 27 October, 2015 Accepted: 30 October, 2015 Available online: 04 April, 2016 Keywords: BSM, Protease, Trichophyton rubrum Abbreviations: BSM: Basal Synthetic Medium Corresponding Author: Srivastava M.* Assistant Professor Email: madhulika.4299 (at) gmail (dot) com Prabhuji S.K. Associate Professor

Abstract Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in stratum corneum, nails and hairs. Work done on different strains of Trichophyton rubrum was studied for their proteolytic enzyme activity which accounts to be one of the main reasons for virulence. Effects of variation in nutrients of Basal Synthetic Medium (BSM) on growth and extracellular protease production in Trichophyton rubrum have been studied. Glucose supported the best growth of the variants of the organism but it showed a very significant decline in the protease activity even at low concentration. Disaccharides like sucrose and lactose were least favorable carbon sources for the growth as well as for

the protease activity. Mannitol and glycerol were utilized well as compared to disaccharides and promoted growth of the variants of T. rubrum but could not equally support for a better protease activity. However, starch promoted moderate growth in all the three variants and induced a better protease activity as compared to other carbohydrates. In inorganic nitrogen compounds best growth was seen when basal media was supplemented with NaNO3. KNO3 too promoted good growth and high protease activity in all the three variants. Moderate growth was seen in NH4NO3, NH4Cl and (NH4)2SO4 but did not have any inducing effect on enzyme activity. Among organic nitrogen sources Asparagine favoured the best growth for all the three variants. Lysine, Valine, Aspartic acid, Glutamic acid and Glutamine supported good growth and Glycine, Alanine, Methionine, Histidine and Tyrosine favoured moderate growth. Decline in protease activity was seen in all the amino acids tested. Four vitamins pyridoxine, thiamine, inositol and ascorbic acid individually or in combination were found to have little or no inhibitory effect on the growth and protease production. Citation: Srivastava M.*, Prabhuji S.K., 2016. Effects of in vitro variation in nutrients on growth and extracellular protease production in Trichophyton rubrum, a human dermatophyte. Photon Journal of Microbiology. Photon 109, 262-275 All Rights Reserved with Photon. Photon Ignitor: ISJN53497294D822404042016

1. Introduction Trichophyton rubrum is the most common causative agent of dermatomycosis which occur worldwide mainly occupying human feet, skin and finger nails. It is a keratinophilic filamentous fungus adapted to the utilization of proteins as the sole source of nutrition. Though very little is known about the mechanism of invasion and pathogenicity, this fungal pathogen’s ability to produce and secrete proteolytic enzymes is a major virulence factor (Chen et al., 2010). This enzyme could play a prominent role not only in growth and multiplication but also in pathogenesis. Ph ton

The diversified line of research on T. rubrum and its related species primarily centered around the isolation, identification and pure cultures of the organism in vitro, however, isolated attempts (Meevootisom and Niederpruem, 1979; Brasch et al., 1991; Brasch and Zaldua, 1994; Apodaca and Mckerrow, 1990) have been made to correlate pattern of nutrition or supply of nutrients with exocellular proteolytic activity of T. rubrum. These fungi tend to utilize amino- acids, peptides and proteins as carbon sources even in presence of sugars. All the three variants of T. rubrum grew 262

well and showed maximum proteolytic activity in BSM with casein as a sole source of C, N and vitamins. During the present investigations effects of variation in nutrients of basal synthetic medium (BSM) on growth and extracellular protease production in Trichophyton rubrum have been studied.

for each variant was run with BSM containing only casein as a source of carbon and nitrogen. Flasks were inoculated and left undisturbed for incubation o at 30 C. Dry weight of the mycelia and protease were calculated according to the method of Anson th th (1938) on every alternate day from 6 to 14 day of the incubation.

Objective of Research Dermatophytes like T. rubrum exhibit large degree of adaptability to parasitic habit; they can easily be grown on wide variety of nutrients saprophytically under laboratory conditions (Howard, 1985). The study of nutritional pattern of pathogenic fungi like T. rubrum serves three purposes: 1. To design media that would be selectively useful in cultivation of pathogen from its host and from natural sources. 2. To know nutritional requirements for growth of fungi that can be used as a means for classification of morphologically similar and identical forms. 3. To find out explanations of particular traits such as dimorphism and tissue tropism. The knowledge of these points can help understand the aggressiveness of the pathogen which is manifested clinically in the form of tinea (Brasch et al., 1991) due to assimilation and digestion of host tissue by secreting enzymes. In the present work, a systematic approach has been made to link the variation of any single nutrient source with the production of protease enzyme by the variants of T. rubrum. The result can help to understand the reasons why intensity of pathogenic implications of T. rubrum appears sporadically during particular seasons of the year. Deletions/ Avoidance of such nutrients can easily be used to exclude dermatophytic diseases. 2. Materials and Methods 2.1 Carbon assimilation studies For carbon assimilation pattern the modified Basal Synthetic Media (BSM) was prepared (Table-1). These constituents were added in their fixed proportion in 500 ml of distilled water. The carbon sources to be tested were added to the BSM in 0.5% concentration to know their effect on growth and extracellular protease production of the variants of T. rubrum. The carbon sources were glucose, sucrose, lactose, starch, mannitol and glycerol. Finally the volume was made to 1000 ml and the pH of the medium were adjusted to 7.0. 25 ml of this medium was dispensed in 250 ml Erlenmeyer flask and sterilized. A control Ph ton

2.2 Nitrogen assimilation studies For nitrogen assimilation study, BSM was supplemented with different organic and inorganic sources of nitrogen at 0.2% concentration. The various N sources tested were: Inorganic sources: KNO3, NaNO3, NH4NO3, NH4Cl, (NH4)2 SO4 and Organic sources : Glycine, Alanine, Valine, Methionine, Cysteine, Aspartic acid, Glutamic acid, Lysine, Leucine, Histidine, Tyrosine, Asparagine, Arginine and Glutamine. 2 g of N sources was added to 500 ml of BSM, the pH of the medium was adjusted to 7.0 and volume made to 1l by distilled water. One test set for each variant was run with only casein as a source of C & N. In nitrogen assimilation studies the same general procedure was followed as mentioned in C assimilation studies for determination of growth and extracellular protease by the strains of the organism. 2.3 Vitamin requirement studies The same procedure (as described in above 2 sections) was followed in the vitamin requirement test with slight variation. Vitamin free casein hydrolysate was used as a nitrogen source and the basal medium was boiled with activated charcoal (5g/l) in order to render it vitamin free. The charcoal was removed by filtration. The volume of the medium was made 1l by distilled water. The medium was dispensed in 250 ml Erlenmeyer flasks in 30ml amounts and flasks were autoclaved at 12lb/in2 steam pressure. The vitamins tested were Pyridoxine H Cl: 100µ g/l, Inositol: 100µ g/l, Ascorbic acid: 100µ g/l, Thiamine: 100µ g/l and combination of above four vitamins. Thiamine was sterilized separately and was added to the flasks which contained 1ml less the required amount (30ml of the medium). One control set for each variant was taken without any vitamin source in the medium. Table 1: Composition of Basal Synthetic Medium (g/l). Casein : 10.0 KH2PO4 : 0.7 K2HPO4 : 0.3 MgSO4.7H2O : 0.5 Yeast extract : 1.0

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Figure 1: Effect of Carbon Source on the growth of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 2: Effect of Carbon source on the protease activity of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 3: Effect of Inorganic Nitrogen source on the growth of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 4: Effect of Inorganic Nitrogen source on the protease activity of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 5: Effect of Organic Nitrogen source on the growth of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 6: Effect of Organic Nitrogen source on the protease activity of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia

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Figure 7: Effect of Vitamin source on the growth of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia.

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Figure 8: Effect of Vitamin source on the protease activity of the variants of T. rubrum at different days of incubation in (units) per mg dry weight of the mycelia.

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3. Results and Discussion 3.1 Carbon assimilation Hexose sugar like glucose supported the best growth of the variants of the organism but on the contrary it showed a very significant decline in the protease activity even at such a low concentration of 0.5% (Fig 1, 2). Disaccharides like sucrose and lactose were least favourable carbon sources for the growth as well as for the protease activity. Maltose too showed very feeble growth and enzyme activity (Fig 1, 2 ). Mannitol and glycerol were utilized well as compared to disaccharides. They promoted growth of the variants of T. rubrum but could not equally support for a better protease activity. The activity was seen to decline significantly (Fig 1, 2 ). Starch promoted moderate growth in all the three variants of T. rubrum and induced a better protease activity as compared to other carbohydrates (Fig 1, 2). 3.2 Nitrogen assimilation Both organic and inorganic nitrogen sources did not help the organism in their growth and protease production excepting a few variants (Fig 3, 4; 5, 6). In inorganic nitrogen compounds best growth was seen when basal media was supplemented with NaNO3. KNO3 too promoted good growth and high protease activity in all the three variants. Moderate growth was seen in NH4 NO3, NH4Cl and (NH4)2 SO4 but did not have any inducing effect on enzyme activity (Fig 3, 4). The protease activity was best seen in KNO3 as compared to other inorganic forms in all the three variants in the 10th day of incubation. NH4Cl also supported a good protease activity. (NH4)2SO4, NH4NO3 and NaNO3 did not have any inducing effect on the activity (Fig 4). Among organic nitrogen sources asparagine favoured the best growth for all the three variants. Lysine, valine, aspartic acid, glutamic acid and glutamine supported good growth of the organism. Glycine, alanine, methionine, histidine and tyrosine favoured moderate growth (Fig 5). No growth was seen with cysteine. Decline in protease activity was seen in all the amino acids tested as against the control media which showed that these amino acids had no inducing effect on protease production (Fig 6 ). Remarkable decline in enzyme activity was seen in valine, glycine, aspartic acid and glutamic acid. 3.3 Vitamin requirement Individual addition of the vitamins tested had little effect on the growth and the protease production of the organism. All the variants could grow well on the control medium which lacked any vitamin source (Fig 7, 8). Pyridoxine Ph ton

and thiamine had little effect on the protease production too and showed slight increase in enzyme activity (Fig.8). The activity was maximum on 10th day of incubation in all the variants. Inositol had no effect on the metabolic process of the organism. Inhibitory effect in the protease activity and growth was seen in ascorbic acid and combination of all the vitamins tested. The influence of nutrients on the enzyme release of T. rubrum has been studied systematically before. The various C & N sources were used here as substrates because of easy biochemical assimilability and can be used by the fungus for its parasitic growth. The carbon sources used in the synthetic medium to evaluate the growth and exocellular protease of T. rubrum were in 0.5% concentration. The reason for not using carbon sources at normally used concentration of 1% is that it curtailed the enzyme extrusion to a great extent. It has already been observed that disaccharides like maltose, sucrose and lactose are not good promoters of growth and retarded the extracellular protease production. Glucose, the only monosaccharide used although induced good growth yet it also significantly retarded the enzyme production. (Fig 1,2). Maltose sugar on addition neither promoted good growth of the organism nor accounted for any increase in protease production. It was observed that all strain of T. rubrum utilized sugar alcohol, glycerol and mannitol very efficiently but could not equally support the better protease production. Polysaccharide starch was assimilated slowly promoting moderate growth of T. rubrum but the enzyme production was still better. The enzyme being inducible one, the occurrence of mono and disaccharide repressed its production but when starch was used in the medium the carbon sources might have been used in many alternative pathways. Hence repressive effect was not expressed to a greater extent. However, with starch as sole source of carbon under aeration the enzyme production could not be accelerated. The organism being aerobic, aeration might have helped in easy assimilation of starch in to mono and disaccharides which might have prevented protease enzyme extrusion. Meevootisom and Niederpruem, 1979 reported that glucose in the concentration above 0.5% (w/v) inhibited the production of extracellular protease in T. rubrum. Previous work has demonstrated that the expression of proteolytic enzymes is reduced when these organisms are grown in rich medium or one that contains easily metabolized substrates such as glucose, amino acids, nitrates or ammonium (Cohen, 1972; 1973; Drucker, 1972; Matile, 1965). In contrast 272

nutrient depletion or growth as stationary phase cultures increases production of proteinases Matile, 1965. Apodaca and McKerrow, 1990 cultured T. rubrum in Sabouraud’s broth reported partial inhibition of proteolytic activity by glucose and the effect depends on time and conditions of the culture. In the initial stages of infection T. rubrum grew logarithmically. In this state proteolytic activity is depressed whenever C, N or Sulphur is lacking in the fungal milieu as observed in present investigation. Stationary phase cultures of T. rubrum secreted all proteolytic activities constitutively. These activities fell in carbon, nitrogen and phosphorus depleted media but remained higher in sulphur depleted medium. Disease may occur when fungus reaches stationary phase when proteinases are secreted constitutively resulting in inflammatory manifestations. (Brasch et al., 1991) also suggested that glucose containing dermatophytic media is not suitable for the purpose of enzymatic studies with pathogenic relevance. A repression by suitable sources of C & N (glucose and amino acids) was also described by (Ziegler et al., 1964; Grzywnowicz and Lobarzewski., 1989; Singh., 1997). (Apodaca and McKerrow, 1989) observed that amino acids are inhibitory repressing proteinase expression except in the case of isoleucine, serine and complex amino acids. (Meevootisom and Niederpruem, 1979) have also observed that in T. rubrum and T. mentagrophytes various amino acids were easily assimillable but there was appreciable decline in extracellular protease activity. In the present investigation it was observed that all amino acids inhibited protease production to lesser or greater extent. None of the amino acids tested were found to be required for the better growth and enzyme production. Tyrosine and histidine, as well as heterocyclic amino acids had less retarding effect on growth and protease production whereas cysteine, a sulphur-containing amino acid was completely inhibitory for the organism (Cochrane., 1958; Zussman et al., 1961; Kunert., 1981; 1985a, b; Danew, 1981; Malviya et al., 1991). There is paucity of reports in linking nutritional aspect of the organism with enzyme production. Meevootisom and Niederpruem., 1979 have observed that ammonium ions did not cause any repression. A better protease activity was seen when the synthetic medium was supplemented with KNO3 and NH4Cl. In contrast to their observation (NH4)2SO4 was not found to promote protease production which may be due to wide variability of the nature of organism under the influence of environmental and nutritional parameters as proposed by Sanyal et al., 1985.

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This may be the reason for KNO3 becoming a good inducer of enzyme production here. Four vitamins pyridoxine, thiamine, inositol and ascorbic acid individually or in combination were found to have little or no inhibitory effect on the growth and protease production of all the three variants of T. rubrum. On the basis of nutritional requirements they were arranged in the order pyridoxine> thiamine> inositol>ascorbic acid. The results indicate that all the three strains are not only independent to vitamins tested but also to the extrusion of protease production. 44 morphologically typical strains were isolated and studied by ( Georg and Camp., 1956) and all the strains were able to grow well in vitamin free casein media. No stimulation in any of water soluble vitamins was observed. Conclusion Dermatophytes are adapted to use the proteins, peptides and amino acids as sources of carbon and nitrogen. These compounds are preferentially utilized from the medium even in presence of glucose. Proteins may serve as sole source of organic nutrition. Supplementation with glucose however accelerates the growth and increases maximum dry mass but does not account for better protease production. As no relevant amount of glucose is known to be found in the epidermis of the skin this further might help the invasion of inducible protease enzyme in the skin. The results of carbon and nitrogen assimilation studies showed that as easily metabolized substrates are made available to T. rubrum protease activity is not induced, in contrast nutrient depletion or growth as stationary phase cultures increases the production of protease. Moderate to good growth was seen in all the strains of T. rubrum in some of the inorganic and organic nitrogen sources tested but that too did not account to the increase in protease activity. Inhibitory effect on growth and protease production was seen using vitamins too in the growth medium. Acknowledgement We owe our sincere and deep sense of gratitude to all our seniors (MS) and colleagues of the Department of Botany for their kind co-operation in completion of this research work. Research Highlights The strains of T. rubrum grew well and showed high proteolytic activity in control medium with only casein as sole source of C and N as against hexose sugar, disaccharides or any polysaccharides 273

thereby suggesting that protein may serve as sole source of nutrition for this dermatophyte.

enzyme synthesis. The Journal of Bacteriology, 110, 1041-1049.

Both inorganic and organic nitrogen sources supplemented in BSM did not help the organism in growth and protease production.

Georg L.K., Camp L.B., 1956. Routine nutritional tests for the identification of dermatophytes. The Journal of Bacteriology, 74, 113.

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