Mycopathologia (2015) 180:143–151 DOI 10.1007/s11046-015-9900-7
Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur Weerapong Juntachai . Susumu Kajiwara
Received: 31 December 2014 / Accepted: 11 May 2015 / Published online: 15 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity.
W. Juntachai (&) Department of Biology, Faculty of Science and Technology, Chiang Mai Rajabhat University, 202 Chang Phuak Road, Muang, Chiang Mai 50300, Thailand e-mail: [email protected] S. Kajiwara Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Keywords Malassezia Hyphae Extracellular enzymes Lipase Protease
Introduction Lipophilic Malassezia species are part of the microflora which commonly found on human and mammal skin and associated with several skin diseases, such as atopic dermatitis, dandruff, Malassezia folliculitis, pityriasis versicolor and seborrheic dermatitis [1–3]. Based on molecular studies, fourteen Malassezia species are now recognized (Malassezia furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. sympodialis, M. dermatis, M. nana, M. japonica, M. yamatoensis, M. equina, M. caprae and M. cuniculi) [4]. The dimorphism of Malassezia species has been known by the observation of both yeast and mycelial forms in the affected sites of pityriasis versicolor. Since the hyphal growth of Malassezia species is connected to the skin lesions, the hyphal formation is thought to be associated with their pathogenicity [5, 6]. However, the mechanism to produce the mycelial phase of Malassezia species is not well understood, and it has long been thought that morphological change of the yeast occurs only in vivo [7]. Despite the fastidious growth of Malassezia species, many groups have challenged to induce morphological change of the fungi in vitro by varying culture conditions and media. Nazzaro-Porro et al. [8] used agar containing cholesterol and cholesterol esters
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and were able to induce mycelia in four strains of Malassezia, up to 20 % of total cells. Other approaches were focused on various conditions for mycelial induction, such as salts, pH, CO2 concentration, nitrogen source and lipid sources [9–11]. Saadatzadeh et al. [12] modified the mycelial culture medium that was able to generate mycelial form in approximately 40 % of the total cells of M. furfur. The induction of pure hyphal form has been unsuccessful until Youngchim et al. [13] demonstrated that M. furfur yeast was completely transformed to hyphae when cultured on the minimal medium (MM) agar supplemented with L-3,4-dihydroxyphenylalanine (L-DOPA) and kojic acid. However, the information about an effective protocol to grow the hyphae of Malassezia under liquid culture condition and characteristic investigation of the two phases has not been published thus far. The purpose of this study was to demonstrate the induction of hyphal growth in vitro of M. furfur under both liquid and solid conditions and to compare the extracellular enzymatic activities between the two forms of the fungus. In addition, invasive ability of the hyphal form was also characterized.
To culture under liquid condition, the cells were incubated in a shaking incubator at 120 rpm. All the cultures from solid and liquid media were observed for mycelial production under light microscopy at 4009 magnification.
Materials and Methods
Preparation of Extracellular Proteins
Strains and Media The yeast strains used in this study were M. furfur CBS 1878T (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) and Saccharomyces cerevisiae S288c (ATCC 204508). The strains were routinely maintained by subculture in the following manner: S. cerevisiae strain and M. furfur strain on yeast peptone dextrose (YPD) agar (1 % yeast extract, 2 % bacto peptone, 2 % glucose and 2 % agar). The M. furfur strain was supplemented with 0.1 % Tween 40, 0.1 % Tween 80 and 1 % olive oil at 30 °C.
The extracellular proteins of M. furfur were prepared from the supernatant of the culture. The yeast cells were removed from YPD broth and IM broth after cultivation at 30 °C for 4 days by centrifugation at 2000g, 4 °C for 5 min. Then, the supernatant of the culture was filtered using a 0.2 lm membrane filter (Advantec, Japan) to remove the remaining cells, and the secretory proteins were concentrated by ultrafiltration with a molecular mass cutoff of 30 kDa (Millipore). The amount of proteins in the samples was determined by Bradford protein assay, using bovine serum albumin (BSA) as the standard.
Induction of Mycelial Growth in M. furfur
Lipase Activity Assay
To induce mycelial growth, the strain was cultured in induction medium (IM) (0.27 % glucose, 0.25 % MgSO47H2O, 0.4 % KH2PO4, 1 % glycine, 0.1 % Tween 40 and 0.1 % Tween 80, pH 5.5) modified from the liquid nutrient medium described previously [13, 14], or inoculated on IM agar (IM with 2 % agar) at 30 °C for an appropriate period.
The lipase activity was determined as described previously [16]. Briefly, the assay mixture contained 0.5 % Triton X-100, 100 mM citrate buffer or 100 mM phosphate buffer with a pH range of 3.0–7.0, 0.5 mM 4-nitrophenyl palmitate (4-NPP) (Sigma) as lipase substrate and 5 lg of the extracellular proteins in a total volume of 0.3 ml. The assay
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Plate-Washing Assay The plate-washing assay was performed as described by [15]. The yeasts were pre-cultured in YPD broth supplemented with 0.1 % of each Tween 40 and Tween 80 at 30 °C for 2 days. The cells were washed twice with distilled water and collected by centrifugation at 2000g, 4 °C for 5 min. Concentration of the cell suspensions was measured by spectrophotometer at 600 nm and adjusted for an appropriate concentration. The cell suspension was spotted on YPD agar and IM agar, respectively. After incubation at 30 °C for 4 days, the plates were washed with distilled water and rubbed gently with a spreader to remove cells that did not invade the agar. The invasive growth was observed as a cell mark on the plate. Invasive growth of S. cerevisiae was observed on YP (glucose-free YPD) and YPD agar, which were the positive and negative controls, respectively.
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mixture was incubated at 30 °C for 1 h. Then, two volumes of 1 M Tris–HCl (pH 8.0) were added to the assay mixture to terminate the reaction. The lipase activity was measured spectrophotometrically based on releasing of 4-nitrophenol (4-NP) from 4-NPP, which resulted in the increase in absorption at 405 nm. Finally, the concentration of released 4-NP was determined using a standard solution of 4-NP.
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Protease Activity Assay The proteolytic enzymes in the aspartic protease family are generally active at acidic pH [17]. Therefore, in this study, the assay of extracellular protease activity in yeast and hyphal form was performed under acidic condition. The protease activity of the fungal extracellular proteins was assayed by the method modified from Uehara et al. [18]. A 1 ml volume of the assay mixture contained 50 mM citrate buffer with a pH range of 3.0–5.0, 0.2 % BSA as substrate and 15 lg of the protein sample. The assay mixture was incubated at 30 °C for 4 h; then, the reaction was terminated by adding 500 ll of 15 % trichloroacetic acid (TCA). The solution was kept on ice for 30 min and then centrifuged to remove the precipitates of undegraded proteins. The absorbance of the supernatant was measured with a spectrophotometer at 280 nm. In the blank, TCA was added before adding the protein sample. Statistical Analysis The statistical analysis of quantitative results was performed using SPSS statistics version 17.0 for Windows (IBM, New York, USA). Differences were considered as statistical significance at p \ 0.05.
YPD
Fig. 1 Cell morphology of M. furfur cultured in YPD and IM media, respectively, at 30 °C for 3 days (magnification 9400). Bars represent 5 lm
IM, compared to the solid one. However, neither IM agar nor IM broth showed a change in color during incubation. Under light microscopy, the cells cultured in the IM broth tended to form cell clumps, and concurrently, the cell morphology switched to the hyphal form, where the branched, septate hyphae were seen around the clumps (Fig. 1). The cells in the yeast form were scarcely observed in the IM culture. Although almost all of the cells transformed to hyphal phase, the rate of hyphal formation could not be calculated, because the exact number of cells in the cell clumps was indeterminate. Intriguingly, morphological change of the cells was reversible; all hyphae turned back into normal yeast form after moved into the YPD, and the hyphal form was no longer observed (data not shown). Effect of Serum on Mycelial Growth of M. furfur
Results Induction of the Mycelial Growth of M. furfur To induce the mycelial phase of M. furfur, the cells were cultured in the IM broth and agar, respectively. The fungal hyphae were observed under both culture conditions. Compared to the growth in the YPD medium, the growth of the fungal cells in the IM was slow, and the density of cell culture was not visually as high as that in the YPD, since the IM was not an enriched medium. M. furfur grew quickly in the liquid
In the pathogenic dimorphic fungus Candida albicans, it has been known that serum is effectively able to induce hyphal formation [19]. To investigate the effect of serum on the hyphal formation of M. furfur, fetus bovine serum (FBS) was added into the IM broth at a final concentration of 10 % (v/v). Under the presence of FBS, filamentous elements consisting of elongated yeast cells and short hyphae were observed. During the course of cultivation, the cells gradually reversed to normal yeast form. None of hyphae were observed on the day 7 (Fig. 2).
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2d
5d
7d
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Fig. 2 Effect of serum on cell morphology of M. furfur. The cells were cultured in IM broth at 30 °C. The cell morphology was observed by microscopy (magnification 9400). Bars represent 5 lm
Invasive Growth of M. furfur The ability of invasion into the agar was detected by observing remaining cells on the agar after cell removal by washing the plate surface. After induction of hyphae on IM agar, the appearance of the colony was smooth and relatively white in color compared to that on YPD agar. However, the elevation of the colonies grown on IM and YPD agar was similar. The microscopic examination of M. furfur cultured on IM agar revealed a mixture of short hyphae and elongated cells (Fig. 3a). However, the constitutive invasion into the agar of the filamentous form of M. furfur was not observed (Fig. 3b). Moreover, an increase in the concentration of the inocula did not influence a change in invasive growth of the fungus (Fig. 3c).
optimum pH range, and the values of the activity were approximately 1.5–3 times higher than those of YPD, but about the same at the peak with no statistically significant difference (p \ 0.05; Fig. 4). Extracellular Protease Activity Protease activities of the samples from both YPD and IM were observed in the range of pH from 3–4, with the optimum pH at around 3.5. The activities in both samples decreased rapidly when the pH was above 4.5. Extracellular protease activity of the IM protein sample was about two times greater than that of YPD in the active pH range (Fig. 5).
Discussion Extracellular Lipase Activity To investigate the extracellular lipase activity of the fungus, secretory proteins from culture supernatants were isolated and concentrated, and the lipase activity was examined by using colorimetric method. Extracellular proteins both from IM and YPD similarly showed lipase activity at a pH range of 3.5–6.0, with a peak of activity at around pH 4.5, while inactivity was observed at pH above 6.5. The lipase activity of the extracellular proteins from IM indicated a wider
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To explain the role of the hyphal form of Malassezia in the pathogenicity, it is required to induce the hyphae and investigate them in vitro. However, the fungus reproduces only in the vegetative form by budding without formation of hyphae when cultured on standard laboratory media. Though the molecular mechanism of morphological change of the yeast is not well understood at present, successful prior attempts offer useful information to induce filamentous elements of the fungus in vitro. Mayser et al. [11] noted that
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(B)
(C) YPD
IM
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(D)
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Fig. 3 Hyphal growth of M. furfur did not cause invasive growth. a Cell morphology of M. furfur grown on YPD and IM agar at 30 °C for 4 days (magnification 9400). Bars represent 5 lm. Equal concentration of cells was spotted onto IM agar and grown at 30 °C for 4 days. The plates were photographed,
washed with water and photographed again; b approximately 2.5 9 104 cells, and c approximately 5.0 9 105 cells. d Invasive growth of S. cerevisiae (approximately 2.5 9 104 cells per spot) grown on YP and YPD agar at 30 °C for 3 days
morphological alteration in M. furfur is not due to overgrowth of the cells, but rather depends on changing amino acids in the nutritional environment. Moreover, among the amino acids, glycine was preferentially metabolised and seemed to be effective in the induction of hyphae in some clinical strains of
Malassezia, which are now classified as M. furfur and M. obtusa [8, 9, 11, 12]. Similarly, in Candida albicans, a mixture of amino acids in the synthetic medium was able to induce germ tube and hyphae [20]. According to these reports, therefore, the induction medium (IM) used in this study was modified to
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*
Lipase activity (U)
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*
YPD-Citrate IM-Phosphate
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40
YPD-Phosphate
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0 2
3
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pH Fig. 4 Lipase activity of extracellular proteins of M. furfur cultured in YPD and IM broth, respectively. The results represent the mean ± SD of the activity. One unit of lipase activity is defined as the amount of 4-nitrophenol nmol h-1 released from 4-nitrophenyl palmitate at 30 °C. The experiments were performed in triplicate, and the data were analyzed by paired-samples t test and are shown as the mean ± SD (*p \ 0.05)
Proteolytic activity (U)
120 100
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80 60 40
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20 0 3
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pH Fig. 5 Protease activity of extracellular proteins of M. furfur cultured in YPD and IM broth, respectively. The results represent the mean ± SD of the activity. One unit of protease activity was defined as the amount of enzyme which increased 0.001 of the absorbance at 280 nm per hour, at 30 °C. The experiments were performed in triplicate, and the data were analyzed by paired-samples t test and are shown as the mean ± SD (*p \ 0.05)
be a chemically defined medium, whose chemical composition is simpler than those used in the previous studies. The medium contains glycine and Tween as the only nitrogen and lipid sources, respectively, which are minimally sufficient for the growth of the
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fungus. The IM used in this study had a composition similar to that of the MM used by Youngchim et al. [13], but without NH4Cl, thiamine, L-DOPA and kojic acid. However, the medium was also successfully able to induce hyphal growth under both solid and liquid culture conditions without the addition of any special reagents. Furthermore, the yeast–hyphal phase of M. furfur was reversibly converted in vitro by changing only the culture media, without the necessity of controlling other culture conditions such as CO2 or temperature, or the addition of any special reagents. In S. cerevisiae, depletion of glucose or nitrogen can cause a morphological transition from round and budding yeast cells to elongated form and invasive pseudohyphal growth [21, 22]. Hypothetically, the morphological change in M. furfur may occur under a similar condition and may be related to adaption to the host environment for survival. Among Malassezia species, M. furfur and M. pachydermatis have been reported as pathogens causing systemic fungal infection, particularly in pediatric patients [23]. Parenteral nutrition through vascular catheter is thought to stimulate fungal growth resulting in Malassezia-related fungemia [3]. Previously, the hemolytic activity of six Malassezia species against sheep erythrocytes in vitro has been identified [24]. However, the role of blood components in the fungal growth and morphological change of these species has not yet been investigated. In the dimorphic fungus C. albicans, serum regularly causes morphological transition from yeast to various filamentous forms such as germ tube, pseudohyphae and hyphae forms [25, 26]. Contrary to C. albicans, it was found that serum did not stimulate the hyphal formation in M. furfur, but instead it stimulated the reversion from hyphae to yeast form, or the formation of unusual cell clumps. Therefore, the data suggest that M. furfur has a response to the blood serum distinctive from that of C. albicans, and the blood components did not the hyphal formation in the fungus. The observation of clinical specimens showed various patterns of yeast–hyphal communities of Malassezia, where the hyphae were found more significant in the deeper part of stratum corneum than those in the superficial layer [27]. Accordingly, the presence of hyphae in the deeper layers of the skin could be due to invasive hyphal growth, or perhaps the environment of this part of the skin could be suitable to form hyphae without correlation with the invasive
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ability. However, in vitro investigation of Malassezia hyphae may help to elucidate this matter and provide understanding about its nature. The present data suggest that the hyphae induced in vitro do not possess the invasive ability into the agar medium. Secretory hydrolytic enzymes such as phospholipases, lipases and proteases have been assumed to be one of the virulent factors in various pathogenic fungi. Most of these enzymes relate to invasive fungal infections or development of inflammation [28–30]. In C. albicans, it has been reported that the extracellular phospholipase activity produced by the invasive blood isolates was significantly higher than that of the noninvasive strains isolated from healthy volunteers [31]. Although the result of invasive growth of Malassezia in the present study was negative, genome analysis of the organisms in the genus Malassezia revealed an abundance of lipases and aspartic protease genes encoded in their genomes [32, 33]. This work may be the first study to identify the secretory lipase and protease in both yeast and hyphal forms of Malassezia. Consequently, information about these secretory enzymes of Malassezia species such as optimum pH, substrates and the analytic method to detect the activity has been very limited. Therefore, the secretory lipase and protease activities were measured in a broad pH range to prevent the false-negative result of enzymatic activity assay, which may be observed at an inactive pH. The extracellular lipase activity of M. furfur seems to have an active pH range in acidic conditions (approximately 3.5–6.0) similar to that of M. globosa secretory lipase, LIP2 [16]. This range is consistent with the human skin surface pH, which has ranged from pH 4.0 to 7.0 (average pH 4.7) [34]. In regard to the lipase activity, the correlation between extracellular lipase activity and cell morphology has been proposed in some dimorphic yeasts. In dimorphic strains of C. paralipolytica (syn. Yarrowia lipolytica) apparently producing high lipase activity, the correlation between mycelial form and lipase production has been expected [35]. However, a latter study revealed that there was no evidence supporting the correlation between lipase production and cell morphology in the dimorphic lipolytic yeast Y. lipolytica [36]. In the current study, despite the culture in YPD and IM having the same lipid source condition, the results revealed a broader active pH range of the hyphal phase of M. furfur, suggesting that other lipases may be secreted in this form.
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In terms of extracellular protease activity, it was difficult to control the nitrogen source of the two media, because the composition of nitrogen sources used in these media was different. Nevertheless, the data showed that the extracellular protease activity of M. furfur in the hyphal stage is more prominent, compared to that of the yeast form. The significant extracellular protease activity of M. furfur may be related to the morphological form, subsequently being involved in the development of pathogenicity. The hypothesis is in agreement with the finding that some isolates of the dimorphic fungus Paracoccidioides brasiliensis similarly showed a different specificity of proteolytic enzyme secretion between mycelial phase and yeast phase and that elastinolytic activity was apparently produced only in the mycelial phase [37]. In C. albicans, the regulation pathway of morphogenesis and secretion of aspartic protease gene under nitrogen-deficient environment has been described [38]. However, the correlation between these factors in Malassezia has not yet been investigated. In conclusion, this study demonstrated that M. furfur can produce hyphae under both solid and liquid condition with the IM medium. The comparison analysis of some extracellular hydrolase activities between the yeast and mycelial phases of M. furfur highlighted a significant difference between the two forms. Additionally, the invasive ability of the hyphal form was also described. This evidence may contribute to a better understanding of the characteristics of the filamentous form of the fungus. To clarify the molecular mechanism of dimorphism and its roles in the pathogenesis of Malassezia, further investigation should be carried out in the induction of mycelial growth in other Malassezia species, while other potentially differing characteristics, such as gene expression between the two phases, should also be examined. Acknowledgments This work was supported in part by a research Grant (2012) from Chiang Mai Rajabhat University, Chiang Mai, Thailand.
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Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur Weerapong Juntachai . Susumu Kajiwara
Received: 31 December 2014 / Accepted: 11 May 2015 / Published online: 15 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity.
W. Juntachai (&) Department of Biology, Faculty of Science and Technology, Chiang Mai Rajabhat University, 202 Chang Phuak Road, Muang, Chiang Mai 50300, Thailand e-mail: [email protected] S. Kajiwara Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Keywords Malassezia Hyphae Extracellular enzymes Lipase Protease
Introduction Lipophilic Malassezia species are part of the microflora which commonly found on human and mammal skin and associated with several skin diseases, such as atopic dermatitis, dandruff, Malassezia folliculitis, pityriasis versicolor and seborrheic dermatitis [1–3]. Based on molecular studies, fourteen Malassezia species are now recognized (Malassezia furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. sympodialis, M. dermatis, M. nana, M. japonica, M. yamatoensis, M. equina, M. caprae and M. cuniculi) [4]. The dimorphism of Malassezia species has been known by the observation of both yeast and mycelial forms in the affected sites of pityriasis versicolor. Since the hyphal growth of Malassezia species is connected to the skin lesions, the hyphal formation is thought to be associated with their pathogenicity [5, 6]. However, the mechanism to produce the mycelial phase of Malassezia species is not well understood, and it has long been thought that morphological change of the yeast occurs only in vivo [7]. Despite the fastidious growth of Malassezia species, many groups have challenged to induce morphological change of the fungi in vitro by varying culture conditions and media. Nazzaro-Porro et al. [8] used agar containing cholesterol and cholesterol esters
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and were able to induce mycelia in four strains of Malassezia, up to 20 % of total cells. Other approaches were focused on various conditions for mycelial induction, such as salts, pH, CO2 concentration, nitrogen source and lipid sources [9–11]. Saadatzadeh et al. [12] modified the mycelial culture medium that was able to generate mycelial form in approximately 40 % of the total cells of M. furfur. The induction of pure hyphal form has been unsuccessful until Youngchim et al. [13] demonstrated that M. furfur yeast was completely transformed to hyphae when cultured on the minimal medium (MM) agar supplemented with L-3,4-dihydroxyphenylalanine (L-DOPA) and kojic acid. However, the information about an effective protocol to grow the hyphae of Malassezia under liquid culture condition and characteristic investigation of the two phases has not been published thus far. The purpose of this study was to demonstrate the induction of hyphal growth in vitro of M. furfur under both liquid and solid conditions and to compare the extracellular enzymatic activities between the two forms of the fungus. In addition, invasive ability of the hyphal form was also characterized.
To culture under liquid condition, the cells were incubated in a shaking incubator at 120 rpm. All the cultures from solid and liquid media were observed for mycelial production under light microscopy at 4009 magnification.
Materials and Methods
Preparation of Extracellular Proteins
Strains and Media The yeast strains used in this study were M. furfur CBS 1878T (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) and Saccharomyces cerevisiae S288c (ATCC 204508). The strains were routinely maintained by subculture in the following manner: S. cerevisiae strain and M. furfur strain on yeast peptone dextrose (YPD) agar (1 % yeast extract, 2 % bacto peptone, 2 % glucose and 2 % agar). The M. furfur strain was supplemented with 0.1 % Tween 40, 0.1 % Tween 80 and 1 % olive oil at 30 °C.
The extracellular proteins of M. furfur were prepared from the supernatant of the culture. The yeast cells were removed from YPD broth and IM broth after cultivation at 30 °C for 4 days by centrifugation at 2000g, 4 °C for 5 min. Then, the supernatant of the culture was filtered using a 0.2 lm membrane filter (Advantec, Japan) to remove the remaining cells, and the secretory proteins were concentrated by ultrafiltration with a molecular mass cutoff of 30 kDa (Millipore). The amount of proteins in the samples was determined by Bradford protein assay, using bovine serum albumin (BSA) as the standard.
Induction of Mycelial Growth in M. furfur
Lipase Activity Assay
To induce mycelial growth, the strain was cultured in induction medium (IM) (0.27 % glucose, 0.25 % MgSO47H2O, 0.4 % KH2PO4, 1 % glycine, 0.1 % Tween 40 and 0.1 % Tween 80, pH 5.5) modified from the liquid nutrient medium described previously [13, 14], or inoculated on IM agar (IM with 2 % agar) at 30 °C for an appropriate period.
The lipase activity was determined as described previously [16]. Briefly, the assay mixture contained 0.5 % Triton X-100, 100 mM citrate buffer or 100 mM phosphate buffer with a pH range of 3.0–7.0, 0.5 mM 4-nitrophenyl palmitate (4-NPP) (Sigma) as lipase substrate and 5 lg of the extracellular proteins in a total volume of 0.3 ml. The assay
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Plate-Washing Assay The plate-washing assay was performed as described by [15]. The yeasts were pre-cultured in YPD broth supplemented with 0.1 % of each Tween 40 and Tween 80 at 30 °C for 2 days. The cells were washed twice with distilled water and collected by centrifugation at 2000g, 4 °C for 5 min. Concentration of the cell suspensions was measured by spectrophotometer at 600 nm and adjusted for an appropriate concentration. The cell suspension was spotted on YPD agar and IM agar, respectively. After incubation at 30 °C for 4 days, the plates were washed with distilled water and rubbed gently with a spreader to remove cells that did not invade the agar. The invasive growth was observed as a cell mark on the plate. Invasive growth of S. cerevisiae was observed on YP (glucose-free YPD) and YPD agar, which were the positive and negative controls, respectively.
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mixture was incubated at 30 °C for 1 h. Then, two volumes of 1 M Tris–HCl (pH 8.0) were added to the assay mixture to terminate the reaction. The lipase activity was measured spectrophotometrically based on releasing of 4-nitrophenol (4-NP) from 4-NPP, which resulted in the increase in absorption at 405 nm. Finally, the concentration of released 4-NP was determined using a standard solution of 4-NP.
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Protease Activity Assay The proteolytic enzymes in the aspartic protease family are generally active at acidic pH [17]. Therefore, in this study, the assay of extracellular protease activity in yeast and hyphal form was performed under acidic condition. The protease activity of the fungal extracellular proteins was assayed by the method modified from Uehara et al. [18]. A 1 ml volume of the assay mixture contained 50 mM citrate buffer with a pH range of 3.0–5.0, 0.2 % BSA as substrate and 15 lg of the protein sample. The assay mixture was incubated at 30 °C for 4 h; then, the reaction was terminated by adding 500 ll of 15 % trichloroacetic acid (TCA). The solution was kept on ice for 30 min and then centrifuged to remove the precipitates of undegraded proteins. The absorbance of the supernatant was measured with a spectrophotometer at 280 nm. In the blank, TCA was added before adding the protein sample. Statistical Analysis The statistical analysis of quantitative results was performed using SPSS statistics version 17.0 for Windows (IBM, New York, USA). Differences were considered as statistical significance at p \ 0.05.
YPD
Fig. 1 Cell morphology of M. furfur cultured in YPD and IM media, respectively, at 30 °C for 3 days (magnification 9400). Bars represent 5 lm
IM, compared to the solid one. However, neither IM agar nor IM broth showed a change in color during incubation. Under light microscopy, the cells cultured in the IM broth tended to form cell clumps, and concurrently, the cell morphology switched to the hyphal form, where the branched, septate hyphae were seen around the clumps (Fig. 1). The cells in the yeast form were scarcely observed in the IM culture. Although almost all of the cells transformed to hyphal phase, the rate of hyphal formation could not be calculated, because the exact number of cells in the cell clumps was indeterminate. Intriguingly, morphological change of the cells was reversible; all hyphae turned back into normal yeast form after moved into the YPD, and the hyphal form was no longer observed (data not shown). Effect of Serum on Mycelial Growth of M. furfur
Results Induction of the Mycelial Growth of M. furfur To induce the mycelial phase of M. furfur, the cells were cultured in the IM broth and agar, respectively. The fungal hyphae were observed under both culture conditions. Compared to the growth in the YPD medium, the growth of the fungal cells in the IM was slow, and the density of cell culture was not visually as high as that in the YPD, since the IM was not an enriched medium. M. furfur grew quickly in the liquid
In the pathogenic dimorphic fungus Candida albicans, it has been known that serum is effectively able to induce hyphal formation [19]. To investigate the effect of serum on the hyphal formation of M. furfur, fetus bovine serum (FBS) was added into the IM broth at a final concentration of 10 % (v/v). Under the presence of FBS, filamentous elements consisting of elongated yeast cells and short hyphae were observed. During the course of cultivation, the cells gradually reversed to normal yeast form. None of hyphae were observed on the day 7 (Fig. 2).
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2d
5d
7d
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Fig. 2 Effect of serum on cell morphology of M. furfur. The cells were cultured in IM broth at 30 °C. The cell morphology was observed by microscopy (magnification 9400). Bars represent 5 lm
Invasive Growth of M. furfur The ability of invasion into the agar was detected by observing remaining cells on the agar after cell removal by washing the plate surface. After induction of hyphae on IM agar, the appearance of the colony was smooth and relatively white in color compared to that on YPD agar. However, the elevation of the colonies grown on IM and YPD agar was similar. The microscopic examination of M. furfur cultured on IM agar revealed a mixture of short hyphae and elongated cells (Fig. 3a). However, the constitutive invasion into the agar of the filamentous form of M. furfur was not observed (Fig. 3b). Moreover, an increase in the concentration of the inocula did not influence a change in invasive growth of the fungus (Fig. 3c).
optimum pH range, and the values of the activity were approximately 1.5–3 times higher than those of YPD, but about the same at the peak with no statistically significant difference (p \ 0.05; Fig. 4). Extracellular Protease Activity Protease activities of the samples from both YPD and IM were observed in the range of pH from 3–4, with the optimum pH at around 3.5. The activities in both samples decreased rapidly when the pH was above 4.5. Extracellular protease activity of the IM protein sample was about two times greater than that of YPD in the active pH range (Fig. 5).
Discussion Extracellular Lipase Activity To investigate the extracellular lipase activity of the fungus, secretory proteins from culture supernatants were isolated and concentrated, and the lipase activity was examined by using colorimetric method. Extracellular proteins both from IM and YPD similarly showed lipase activity at a pH range of 3.5–6.0, with a peak of activity at around pH 4.5, while inactivity was observed at pH above 6.5. The lipase activity of the extracellular proteins from IM indicated a wider
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To explain the role of the hyphal form of Malassezia in the pathogenicity, it is required to induce the hyphae and investigate them in vitro. However, the fungus reproduces only in the vegetative form by budding without formation of hyphae when cultured on standard laboratory media. Though the molecular mechanism of morphological change of the yeast is not well understood at present, successful prior attempts offer useful information to induce filamentous elements of the fungus in vitro. Mayser et al. [11] noted that
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(B)
(C) YPD
IM
YPD
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(D)
YPD
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Fig. 3 Hyphal growth of M. furfur did not cause invasive growth. a Cell morphology of M. furfur grown on YPD and IM agar at 30 °C for 4 days (magnification 9400). Bars represent 5 lm. Equal concentration of cells was spotted onto IM agar and grown at 30 °C for 4 days. The plates were photographed,
washed with water and photographed again; b approximately 2.5 9 104 cells, and c approximately 5.0 9 105 cells. d Invasive growth of S. cerevisiae (approximately 2.5 9 104 cells per spot) grown on YP and YPD agar at 30 °C for 3 days
morphological alteration in M. furfur is not due to overgrowth of the cells, but rather depends on changing amino acids in the nutritional environment. Moreover, among the amino acids, glycine was preferentially metabolised and seemed to be effective in the induction of hyphae in some clinical strains of
Malassezia, which are now classified as M. furfur and M. obtusa [8, 9, 11, 12]. Similarly, in Candida albicans, a mixture of amino acids in the synthetic medium was able to induce germ tube and hyphae [20]. According to these reports, therefore, the induction medium (IM) used in this study was modified to
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*
Lipase activity (U)
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IM-Citrate
*
YPD-Citrate IM-Phosphate
*
40
YPD-Phosphate
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0 2
3
4
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pH Fig. 4 Lipase activity of extracellular proteins of M. furfur cultured in YPD and IM broth, respectively. The results represent the mean ± SD of the activity. One unit of lipase activity is defined as the amount of 4-nitrophenol nmol h-1 released from 4-nitrophenyl palmitate at 30 °C. The experiments were performed in triplicate, and the data were analyzed by paired-samples t test and are shown as the mean ± SD (*p \ 0.05)
Proteolytic activity (U)
120 100
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* IM
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YPD
80 60 40
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20 0 3
3.5
4
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5
pH Fig. 5 Protease activity of extracellular proteins of M. furfur cultured in YPD and IM broth, respectively. The results represent the mean ± SD of the activity. One unit of protease activity was defined as the amount of enzyme which increased 0.001 of the absorbance at 280 nm per hour, at 30 °C. The experiments were performed in triplicate, and the data were analyzed by paired-samples t test and are shown as the mean ± SD (*p \ 0.05)
be a chemically defined medium, whose chemical composition is simpler than those used in the previous studies. The medium contains glycine and Tween as the only nitrogen and lipid sources, respectively, which are minimally sufficient for the growth of the
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fungus. The IM used in this study had a composition similar to that of the MM used by Youngchim et al. [13], but without NH4Cl, thiamine, L-DOPA and kojic acid. However, the medium was also successfully able to induce hyphal growth under both solid and liquid culture conditions without the addition of any special reagents. Furthermore, the yeast–hyphal phase of M. furfur was reversibly converted in vitro by changing only the culture media, without the necessity of controlling other culture conditions such as CO2 or temperature, or the addition of any special reagents. In S. cerevisiae, depletion of glucose or nitrogen can cause a morphological transition from round and budding yeast cells to elongated form and invasive pseudohyphal growth [21, 22]. Hypothetically, the morphological change in M. furfur may occur under a similar condition and may be related to adaption to the host environment for survival. Among Malassezia species, M. furfur and M. pachydermatis have been reported as pathogens causing systemic fungal infection, particularly in pediatric patients [23]. Parenteral nutrition through vascular catheter is thought to stimulate fungal growth resulting in Malassezia-related fungemia [3]. Previously, the hemolytic activity of six Malassezia species against sheep erythrocytes in vitro has been identified [24]. However, the role of blood components in the fungal growth and morphological change of these species has not yet been investigated. In the dimorphic fungus C. albicans, serum regularly causes morphological transition from yeast to various filamentous forms such as germ tube, pseudohyphae and hyphae forms [25, 26]. Contrary to C. albicans, it was found that serum did not stimulate the hyphal formation in M. furfur, but instead it stimulated the reversion from hyphae to yeast form, or the formation of unusual cell clumps. Therefore, the data suggest that M. furfur has a response to the blood serum distinctive from that of C. albicans, and the blood components did not the hyphal formation in the fungus. The observation of clinical specimens showed various patterns of yeast–hyphal communities of Malassezia, where the hyphae were found more significant in the deeper part of stratum corneum than those in the superficial layer [27]. Accordingly, the presence of hyphae in the deeper layers of the skin could be due to invasive hyphal growth, or perhaps the environment of this part of the skin could be suitable to form hyphae without correlation with the invasive
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ability. However, in vitro investigation of Malassezia hyphae may help to elucidate this matter and provide understanding about its nature. The present data suggest that the hyphae induced in vitro do not possess the invasive ability into the agar medium. Secretory hydrolytic enzymes such as phospholipases, lipases and proteases have been assumed to be one of the virulent factors in various pathogenic fungi. Most of these enzymes relate to invasive fungal infections or development of inflammation [28–30]. In C. albicans, it has been reported that the extracellular phospholipase activity produced by the invasive blood isolates was significantly higher than that of the noninvasive strains isolated from healthy volunteers [31]. Although the result of invasive growth of Malassezia in the present study was negative, genome analysis of the organisms in the genus Malassezia revealed an abundance of lipases and aspartic protease genes encoded in their genomes [32, 33]. This work may be the first study to identify the secretory lipase and protease in both yeast and hyphal forms of Malassezia. Consequently, information about these secretory enzymes of Malassezia species such as optimum pH, substrates and the analytic method to detect the activity has been very limited. Therefore, the secretory lipase and protease activities were measured in a broad pH range to prevent the false-negative result of enzymatic activity assay, which may be observed at an inactive pH. The extracellular lipase activity of M. furfur seems to have an active pH range in acidic conditions (approximately 3.5–6.0) similar to that of M. globosa secretory lipase, LIP2 [16]. This range is consistent with the human skin surface pH, which has ranged from pH 4.0 to 7.0 (average pH 4.7) [34]. In regard to the lipase activity, the correlation between extracellular lipase activity and cell morphology has been proposed in some dimorphic yeasts. In dimorphic strains of C. paralipolytica (syn. Yarrowia lipolytica) apparently producing high lipase activity, the correlation between mycelial form and lipase production has been expected [35]. However, a latter study revealed that there was no evidence supporting the correlation between lipase production and cell morphology in the dimorphic lipolytic yeast Y. lipolytica [36]. In the current study, despite the culture in YPD and IM having the same lipid source condition, the results revealed a broader active pH range of the hyphal phase of M. furfur, suggesting that other lipases may be secreted in this form.
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In terms of extracellular protease activity, it was difficult to control the nitrogen source of the two media, because the composition of nitrogen sources used in these media was different. Nevertheless, the data showed that the extracellular protease activity of M. furfur in the hyphal stage is more prominent, compared to that of the yeast form. The significant extracellular protease activity of M. furfur may be related to the morphological form, subsequently being involved in the development of pathogenicity. The hypothesis is in agreement with the finding that some isolates of the dimorphic fungus Paracoccidioides brasiliensis similarly showed a different specificity of proteolytic enzyme secretion between mycelial phase and yeast phase and that elastinolytic activity was apparently produced only in the mycelial phase [37]. In C. albicans, the regulation pathway of morphogenesis and secretion of aspartic protease gene under nitrogen-deficient environment has been described [38]. However, the correlation between these factors in Malassezia has not yet been investigated. In conclusion, this study demonstrated that M. furfur can produce hyphae under both solid and liquid condition with the IM medium. The comparison analysis of some extracellular hydrolase activities between the yeast and mycelial phases of M. furfur highlighted a significant difference between the two forms. Additionally, the invasive ability of the hyphal form was also described. This evidence may contribute to a better understanding of the characteristics of the filamentous form of the fungus. To clarify the molecular mechanism of dimorphism and its roles in the pathogenesis of Malassezia, further investigation should be carried out in the induction of mycelial growth in other Malassezia species, while other potentially differing characteristics, such as gene expression between the two phases, should also be examined. Acknowledgments This work was supported in part by a research Grant (2012) from Chiang Mai Rajabhat University, Chiang Mai, Thailand.
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