Microbiology Section DOI : 10.7860/JCDR/2018/31303.11535
Year : 2018 | Month : May | Volume : 12 | Issue : 05 Page : DC17 - DC19

The Lipase Activities of Malassezia Species Isolated from Seborrhoeic Dermatitis/Dandruff Patients

Prasanna Honnavar1, Arunaloke Chakrabarti2, Ghandam S Prasad3, Jillwin Joseph4, Sunil Dogra5, Sanjeev Handa6, Shivaprakash M Rudramurthy7

1 Lecturer, Department of Microbiology and Immunology, Xavier University School of Medicine, Aruba, Dutch Caribbean.
2 Professor and Head, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
3 Senior Scientist, Microbial Type Culture Collection and Genebank (MTCC), Institute of Microbial Technology, Council of Scientific and Industrial Research, Chandigarh, India.
4 PhD scholar, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
5 Additional Professor, Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
6 Professor and Head, Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
7 Professor, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.


NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Shivaprakash M Rudramurthy, Professor, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India.
E-mail: mrshivprakash@yahoo.com
Abstract

Introduction

Malassezia a commensal yeast, can become pathogenic leading to different clinical forms of dermatosis. Being an obligatory lipophilic, the lipolytic enzymes produced by them help in their growth by obtaining useful lipids from the environment further contributing to the pathogenesis of dermatosis. There are no comparative reports concerning divreted lipase activities of Malassezia species associated with Seborrhoeic Dermatitis/Dandruff (SD/D).

Aim

To analyse and compare lipase activities of Malassezia spp. isolated from SD/D patient’s lesional area and Healthy Controls (HC) and correlates this feature in pathogenesis of SD/D.

Materials and Methods

Lipase activities of Malassezia species isolated from lesional area of SD/D patients and HC were analysed and compared. Extracellular proteins of Malassezia spp. were extracted from the Leeming and Notman agar after removing and weighing the yeast cells from the agar surface. The lipase activity was measured using an assay based on hydrolysis of p-Nitrophenylpalmitate (pNPP). ANOVA test and GraphPad were used for statistical analysis.

Results

Malassezia furfur wet fungal weight was higher (1.1±0.13 g) compared to M. globosa (0.36±0.4 g) and M. restricta (0.26±0.4 g). The mean extracellular lipase activity of M. globosa (0.15 U) and M. restricta (0.18 U) was significantly higher than M. furfur (0.08 U) isolates (p<0.001). No significant difference was observed among M. globosa (SD/D-0.15 U vs. HC-0.14 U), M. restricta (SD/D-0.2 U vs. HC-0.16 U) and M. furfur (SD/D-0.08 U vs. HC-0.1 U) isolated from SD/D patient’s lesional area and HC.

Conclusion

The lipase activity of M. globosa and M. restricta was higher despite slow growth rate. Lipase activity alone may not play a role in the onset of SD/D. As the pathogenic mechanism of fungi is complex, it is difficult to explain the pathogenicity of Malassezia based only on lipase activities.

Introduction

Members of the genus Malassezia are commensal yeasts that reside on the skin of human and many warm-blooded animals. Under the influence of diverse environmental factors such as temperature and humidity or immunodeficiency it can become pathogenic leading to different clinical forms of dermatosis like pityriasis versicolor, seborrhoeic dermatitis/dandruff (SD/D), Malassezia folliculitis and some subsets of psoriasis and atopic dermatitis [1]. Malassezia can also cause serious infections such as catheter associated sepsis [2, 3]. There are totally 15 Malassezia spp. described till date based on molecular characterisation [4]. Lipolytic enzymes secreted by them could help them in utilising the lipids from the immediate environment further contributing to the pathogenesis of dermatoses. Lipolytic enzymes such as lipase, esterase, phospholipase, and lysophospholipase are considered to be closely associated with the virulence of Malassezia [5]. However, due to fastidious nature of Malassezia (especially M. globosa and M. restricta), research on analysis of these virulence factors are restrained.

The SD/D is a common skin disorders associated with Malassezia, occurring in at least 40-50% of the general population of immunocompetent adults [6,7]. Analysis of the complete genome of M. globosa and the partial genome of M. restricta have presented gene-encoding enzymes of the lipase and phospholipase families that could explain the lipid dependency of the genus [8]. It seems likely that these fungi would also require lipid for growth on human skin and that lipase-mediated digestion of sebum glycerides would be the first step in lipid consumption. There are no comparative reports concerning the secreted lipase activities of Malassezia species associated with SD/D. Hence, the aim of the present study was to analyse and compare lipase activities of Malassezia species isolated from lesional area of SD/D patients and HC and correlate this feature in pathogenesis of SD/D.

Materials and Methods

The prospective case-control study was conducted in villages of northwestern India (Punjab) and northern India (Haryana) during December 2011 to February 2013. The present study was conducted at the departments of Medical Microbiology and Dermatology, Venereology and Leprosy, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. Study population was categorised as SD/D patients or healthy individuals as per the previous conducted study [4]. Inclusion criteria were based on the presence of inflammation of the scalp in the form of red, scaly and flaking rash. Cases applying topical antifungals and/or steroid to the scalp preceding a month of sampling were excluded from the study. Specimen was collected using a blunt scalpel from relatively severely affected areas and vertex region of SD/D patients and healthy individuals scalp respectively. The specimen collection was carried out at convenience by random screening. The degree of SD/D severity was graded as mild, moderate and severe based on size, colour and surface of the flakes/scales [9]. All isolates were identified based on phenotypic and molecular technique as described earlier [10]. Total 30 clinical isolates of Malassezia isolated from SD/D patient’s lesional area and from HC were randomly selected and included in the study. Isolates include M. globosa, n=10 (5 SD/D, 5 HC); M. restricta, n=10 (5 SD/D, 5 HC) and M. furfur, n=10 (5 SD/D, 5 HC). The study protocol was approved by the Institute Ethics Committee of Postgraduate Institute of Medical Education and Research, Chandigarh, India. The samples were collected after obtaining informed consent of the subjects. All strains were grown on Leeming and Notman Agar (LNA) media and incubated at 34°C for seven days.

Each Malassezia isolate (20 CFU/mL) was cultured on five sets of LNA plates under the condition described above. Malassezia cells were scrapped from the surface of LNA plate and transferred to prewieghed eppendorf tube. The wet weight of Malassezia cells was noted. After scraping the Malassezia cells from the surface of agar medium, the surface was rinsed twice with distilled water. LNA medium was crushed in pestle-mortar. The resultant slurry was incubated in 100 mL protein extraction buffer at 4°C for 24 hour. The mixture was filtrated through Whatman filter paper followed by 0.2 mm membrane filter (Millipore Corporation, MA, USA). The resultant solution was concentrated using Amicon Ultra centrifugal filter, ultracel- 30K (Millipore corporation, MA, USA) [5].

Lipase activity was measured using an assay based on hydrolysis of p-Nitrophenylpalmitate (pNPP). Due to low solubility in water, pNPP (Sigma, MO, USA) was first dissolved in 2-propanol at 10 mM concentration then 1 mM pNPP was dissolved in 10 mM phosphate buffer (pH=6) and 1% TritonX-100. To this substrate, 30 μL of concentrated extracted extracellular protein was added and incubated at 37°C for one hour. Before measuring the absorbance at 405 nm, 200 mL of 1 M Tris buffer (pH=8.0) was added to stabilise the pH-dependent dye pNP (p-Nitrophenol). Release of pNP from pNPP was measured in an ELISA reader as the increase in absorption at 405 nm [11].

Statistical Analysis

Comparison of lipase activity among M. globosa, M. restricta and M. furfur was analysed by ANOVA (Tukey’s multiple comparisons) test and calculated using the GraphPad Prism version 6.01 (www.graphpad.com). p<0.05 was considered as statistically significant.

Results

After seven days of incubation on LNA media, M. furfur average wet fungal weight was higher (1.1±0.13 g), compared to M. globosa (0.36±0.4 g) and M. restricta (0.26±0.4 g) [Table/Fig-1]. The protein concentration was normalised, as the wet weight of M. furfur was ~3 and ~2 times higher than M. globosa and M. restricta. One unit of lipase activity is defined as the amount of enzyme that released 1 μM of p-nitrophenol per minute [11]. The mean extracellular lipase activity of M. globosa (0.15 U) and M. restricta (0.18 U) was significantly higher than M. furfur (0.08 U) isolates (p<0.0001) [Table/Fig-2,3]. No significant difference in the extracellular lipase activity was observed among M. globosa (SD/D-0.15 U vs. HC-0.14 U; p=0.5818), M. restricta (SD/D-0.2 U vs. HC-0.16 U; p=0.0671) and M. furfur (SD/D-0.08 U vs. HC-0.1 U; p=0.4067) isolated from SD/D patient’s lesional area and HC [Table/Fig-4]. As there was no statistical difference between isolates from SD/D patients and HC, degree of SD/D and lipase activity was not calculated.

Isolation source and wet weight (in grams) of different Malassezia isolates.

M. globosaM. restrictaM. furfur
SD/D patientsWeight in gmHealthy controlsWeight in gmSD/D patientsWeight in gmHealthy controlsWeight in gmSD/D patientsWeight in gmHealthy controlsWeight in gm
U100.44NU110.38U540.23NU550.32U51.12NU51.25
U660.36NU680.38U640.28NU630.29U601.14NU91.35
U710.38NU700.43U650.28NU650.32U621.32NU331.2
U770.37NU750.45U730.25NU780.3U821.34NU351.28
U600.39NU600.47U740.30NU800.27U891.3NU621.29

Abbreviation: SD/D, Seborrhoeic dermatitis/dandruff

U and NU areis just the lab number in which U indicates SD/D patient isolate and NU indicates HC isolates


Statistical analysis of the mean extracellular lipase activity of M. globosa, M. restricta and M. furfur isolates.

Tukey’s multiple comparisons testMean Diff.95% CI of diff.p-value
M. globosa vs. M. restricta-0.031870.06466 to 0.0009227ns
M. globosa vs. M. furfur0.061810.03025 to 0.09337<0.001
M. restricta vs. M. furfur0.093680.05924 to 0.1281<0.0001

Test employed: ANOVA (Tukey’s multiple comparisons) test. p<0.05 was considered as statistically significant


Lipase activity in extracted extracellular proteins of different Malassezia species. Error bars indicate the standard deviations of the background-subtracted values for three independent replicate experiments (* p<0.05- significant).

Lipase activity in extracted extracellular proteins of Malassezia species isolated from seborrhoeic dermatitis/dandruff patient’s lesional area and healthy controls.

Error bars indicate the standard deviations of the background-subtracted values for three independent replicate experiments.

Discussion

The pathobiology of Malassezia in SD/D is not clear, due to the complex interaction of the commensal yeast with the skin. Malassezia shows lipid dependency due to the absence of fatty acid synthase gene. Malassezia yeast secretes the extracellular lipolytic enzymes to obtain saturated fatty acids from the environment [8]. The lipolytic enzymes (lipase, phospholipase, esterase and lysophospholipase) may be associated with the virulence of Malassezia in SD/D.

Juntachai W et al., compared the extracellular lipase activity of standard strains of seven Malassezia spp. M. globosa (CBS 7986) had the highest lipase activity compared to the other six Malassezia spp. (M. pachydermatis, M. restricta, M. furfur, M. slooffiae, M. sympodialis and M. obtusa) [5]. However, there are no relative reports regarding secreted lipase activities of Malassezia species associated with SD/D. In the present study the lipase activity of M. globosa and M. restricta was higher despite slow growth rate (the wet weight of species indicate the growth rate; higher the wet weight higher the growth rate) contrasting the results of previous study [5]. This difference may be due to source of isolation. In the previous study the standard strains were used to compare the lipase activity, whereas in the present study the isolates were from SD/D patients and HC [5]. This is the first in-vitro study to investigate the lipase activity between the Malassezia strains isolated from SD/D patient’s lesional area and healthy controls. Recently Park M et al., characterised MrLip1, lipase gene of M. restricta, isolated from a SD patient. However, it would have been interesting to characterise expression of this gene in M. restricta isolated from healthy individual [12].

Ran Y et al., for the first time studied the role of lipase and its significance in M. furfur. The lipase was detected in the insoluble fraction of the cells and was acidic in nature. Association between cell growth and lipase activity was observed [13]. Brunke S et al., in 2006 cloned and characterised MfLIP1 (gene encoding extracellular lipase of M. furfur) [11]. Conserved lipase motif of C. albicans has shown to be similar to MfLIP1. In the same year, Shibata N et al., cloned and characterised M. pachydermatis extracellular lipase gene [14]. But both M. furfur and M. pachydermatis have least association in the causation of SD/D. Many reports including the previous study have shown that M. globosa and M. restricta are the species most commonly associated with SD/D [9]. Even the whole genome sequence of M. globosa has revealed the presence of 14 lipase-encoding genes, of which 13 were predicted secreted proteins [8]. However, only two genes (MgLIP1 and MgLIP2) have been isolated and characterised [15,16]. It also contained conserved lipase motif. Late log phase of M. globosa expressed higher MgLIP1. Due to fastidious nature of M. restricta, its lipase activity was not detected. In addition, recently MgLIP1 expression was detected on the human scalp [17].

In the present study, in a given species no difference has been observed in the lipase activity among the strains isolated from lesional area of SD/D patients and HC. These results suggest that lipase activity alone may not play a role in the onset of SD/D. However, lipase may help in metabolising lipids and integrating the products on cell wall leading to growth and survival in the environment. As the pathogenic mechanism of fungi is complex, it is difficult to explain the pathogenicity of Malassezia based on lipase activity alone. Other virulence factors of Malassezia species may play a role in pathogenesis either alone or in combinations. The activity of phospholipase is an important virulence that has been detected in M. pachydermatis and implicated in the occurrence of skin lesions [18]. In addition, secreted phospholipase activities of M. furfur were detected in vitro by the egg-yolk method [19]. Quantitative analysis and comparison of phospholipase activities of M. globosa and M. restricta isolated from SD/D patient’s lesional area and HC may provide more insight on the pathogenesis of SD/D.

Limitation

The limitation of present study was the low number of Malassezia strains studied, due to difficulty in cultivating these fastidious species. Due to small number of strains we couldn’t comment on the degree/severity of SD/D and lipase activity. Storage of the Malassezia strains for longer period may have reduced the enzyme activity.

Conclusion

Despite slow growth rate of M. globosa and M. restricta, the lipase activity was higher than M. furfur. No difference was observed in the lipase activity among the strains isolated from lesional area of SD/D patients and HC. Malassezia pathogenesity is a complex mechanism. Hence, lipase activity alone may not play a role in the causation of SD/D.

Abbreviation: SD/D, Seborrhoeic dermatitis/dandruffU and NU areis just the lab number in which U indicates SD/D patient isolate and NU indicates HC isolatesTest employed: ANOVA (Tukey’s multiple comparisons) test. p<0.05 was considered as statistically significant

References

[1]Velegraki A, Cafarchia C, Gaitanis G, Iatta R, Boekhout T, Malassezia infections in humans and animals: pathophysiology, detection, and treatment PLoS Pathog 2015 11(1):e100452310.1371/journal.ppat.100452325569140  [Google Scholar]  [CrossRef]  [PubMed]

[2]Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL Jr, Skin diseases associated with Malassezia species J Am Acad Dermatol 2004 51(5):785-98.10.1016/j.jaad.2003.12.03415523360  [Google Scholar]  [CrossRef]  [PubMed]

[3]Boekhout T, Kamp M, Gueho E, Molecular typing of Malassezia species with PFGE and RAPD Med Mycol 1998 36(6):365-72.10.1080/0268121988000058110206745  [Google Scholar]  [CrossRef]  [PubMed]

[4]Honnavar P, Prasad GS, Ghosh A, Dogra S, Handa S, Rudramurthy SM, Malassezia arunalokei sp. nov., a novel yeast species isolated from seborrheic dermatitis patients and healthy individuals from India J Clin Microbiol 2016 54(7):1826-34.10.1128/JCM.00683-1627147721  [Google Scholar]  [CrossRef]  [PubMed]

[5]Juntachai W, Oura T, Murayama SY, Kajiwara S, The lipolytic enzymes activities of Malassezia species Med Mycol 2009 47(5):477-84.10.1080/1369378080231482518798119  [Google Scholar]  [CrossRef]  [PubMed]

[6]Ashbee HR, Evans EG, Immunology of diseases associated with Malassezia species Clin Microbiol Rev 2002 15(1):21-57.10.1128/CMR.15.1.21-57.200211781265  [Google Scholar]  [CrossRef]  [PubMed]

[7]Warner RR, Schwartz JR, Boissy Y, Dawson TL Jr, Dandruff has an altered stratum corneum ultrastructure that is improved with zinc pyrithione shampoo J Am Acad Dermatol 2001 45:897-903.10.1067/mjd.2001.11784911712036  [Google Scholar]  [CrossRef]  [PubMed]

[8]Xu J, Saunders CW, Hu P, Grant RA, Boekhout T, Kuramae EE, Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens Proc Natl Acad Sci USA 2007 104(47):18730-35.10.1073/pnas.070675610418000048  [Google Scholar]  [CrossRef]  [PubMed]

[9]Rudramurthy SM, Honnavar P, Dogra S, Yegneswaran PP, Handa S, Chakrabarti A, Association of Malassezia species with dandruff Indian J Med Res 2014 139(3):431-37.  [Google Scholar]

[10]Rudramurthy SM, Honnavar P, Chakrabarti A, Dogra S, Singh P, Handa S, Association of Malassezia species with psoriatic lesions Mycoses 2014 57(8):483-88.10.1111/myc.1218624655111  [Google Scholar]  [CrossRef]  [PubMed]

[11]Brunke S, Hube B, MfLIP1, a gene encoding an extracellular lipase of the lipid-dependent fungus Malassezia furfur Microbiology 2006 152(pt 2):547-54.10.1099/mic.0.28501-016436442  [Google Scholar]  [CrossRef]  [PubMed]

[12]Park M, Jung WH, Han SH, Lee YH, Lee YW, Characterisation and expression analysis of MrLip1, a class 3 family lipase of malassezia restricta Mycoses 2015 58(11):671-78.10.1111/myc.1241226404462  [Google Scholar]  [CrossRef]  [PubMed]

[13]Ran Y, Yoshiike T, Ogawa H, Lipase of Malassezia furfur: some properties and their relationship to cell growth J Med Vet Mycol 1993 31(1):77-85.10.1080/026812193800000818483059  [Google Scholar]  [CrossRef]  [PubMed]

[14]Shibata N, Okanuma N, Hirai K, Arikawa K, Kimura M, Okawa Y, Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis FEMS Microbiol Lett 2006 256(1):137-44.10.1111/j.1574-6968.2006.00106.x16487331  [Google Scholar]  [CrossRef]  [PubMed]

[15]DeAngelis YM, Saunders CW, Johnstone KR, Reeder NL, Coleman CG, Kaczvinsky JR Jr, Isolation and expression of a Malassezia globosa lipase gene, LIP1 J Invest Dermatol 2007 127(9):2138-46.10.1038/sj.jid.570084417460728  [Google Scholar]  [CrossRef]  [PubMed]

[16]Juntachai W, Oura T, Kajiwara S, Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa Microbiology 2011 157(pt 12):3492-99.10.1099/mic.0.054528-022016565  [Google Scholar]  [CrossRef]  [PubMed]

[17]Patino-Uzcategui A, Amado Y, Cepero de Garcia M, Chaves D, Tabima J, Motta A, Virulence gene expression in Malassezia spp from individuals with seborrheic dermatitis J Invest Dermatol 2011 131(10):2134-36.10.1038/jid.2011.17821697882  [Google Scholar]  [CrossRef]  [PubMed]

[18]Cafarchia C, Otranto D, Association between phospholipase production by Malassezia pachydermatis and skin lesions J Clin Microbiol 2004 42(10):4868-69.10.1128/JCM.42.10.4868-4869.200415472366  [Google Scholar]  [CrossRef]  [PubMed]

[19]Riciputo RM, Oliveri S, Micali G, Sapuppo A, Phospholipase activity in Malassezia furfur pathogenic strains Mycoses 1996 39(5-6):233-35.10.1111/j.1439-0507.1996.tb00131.x8909036  [Google Scholar]  [CrossRef]  [PubMed]