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Medical Mycology, 2016, 00, 1–5
doi: 10.1093/mmy/myw058
Original Article
Original Article
β-Endorphin enhances the phospholipase
activity of the dandruff causing fungi Malassezia
globosa and Malassezia restricta
Prasanna Honnavar1, Arunaloke Chakrabarti1, Ghandam S Prasad2,
Pankaj Singh1, Sunil Dogra3and Shivaprakash M Rudramurthy1,∗
1Mycology Division, Department of Medical Microbiology, Postgraduate Institute of Medical Education
and Research, Chandigarh, India, 2Microbial Type Culture Collection and Gene Bank, Institute of Mi-
crobial Technology, Council of Scientific and Industrial Research, Chandigarh, India and 3Department
of Dermatology, Venerology and Leprosy, Postgraduate Institute of Medical Education and Research,
Chandigarh, India
∗To whom correspondence should be addressed. Shivaprakash M Rudramurthy, Professor, Mycology Division,
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh
-160012, India. Tel: +91 172 2755162; Fax: +91 172 2744401; E-mail: mrshivprakash@yahoo.com
Received 5 March 2016; Revised 12 May 2016; Accepted 26 May 2016
Abstract
β-Endorphin is known to stimulate phospholipase production by Malassezia pachyder-
matis during canine dermatoses. The role of β-endorphin in Malassezia infection in hu-
mans is not well studied. The present study compares the influence of β-endorphin on
Malassezia globosa and Malassezia restricta isolated from patients with seborrhoeic der-
matitis/dandruff (SD/D) and healthy controls. Malassezia isolates (five each of the two
species from patients and healthy controls) were grown on modified Dixon’s agar with
or without 100 nmol/L β-endorphin. Phospholipase activity was quantified based on its
ability to hydrolyze L-α-phosphatidylcholine dimyristoyl (phospholipid substrate). Free
fatty acid was measured by a colorimetry method. In isolates from patients, the phos-
pholipase activity significantly increased after exposure to β-endorphin (M. globosa, P =
.04; M. restricta, P =.001), which did not occur in isolates from healthy controls. More-
over, after β-endorphin exposure the patient isolates had significantly higher (P=.0004)
phospholipase activity compared to the healthy control isolates. The results suggest that
isolates of M. globosa and M. restricta from patients may differ from those of healthy
humans.
Key words: Malassezia, dandruff, phospholipase activity, β-endorphin.
Introduction
Malassezia species are known to cause seborrhoeic der-
matitis/dandruff (SD/D) in humans. The fungi are unable
to synthesize myristic acid, an essential fatty acid required
for its growth.1Various lipolytic enzymes, including lipase,
esterase, phospholipase, and lysophospholipase, produced
C
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2Medical Mycology, 2016, Vol. 00, No. 00
by Malassezia species help in the utilization of essential
fatty acids from exogenous lipid sources.2Among these en-
zymes, phospholipases are considered as a virulent factor.
Malassezia pachydermatis isolated from lesional areas of
dogs produces higher amount of phospholipase compared
to those isolated from non-lesional area.3The β-endorphin
present in skin of dog with dermatoses stimulates phos-
pholipase production from M. pachydermatis.4The nerve
endings, keratinocytes, sebocytes, and melanocytes contain
β-endorphin μ-opioid receptors. The β-endorphin activa-
tion modulates immunological and chemical activity of the
skin.5β-Endorphin also affects lipogenesis of long-chain
fatty acids and pigmentation.6
Malassezia globosa and Malassezia restricta are com-
monly isolated from patients with SD/D.7The role of
β-endorphin in modulating phospholipase production by
these species during human infection is not well understood
or explored. Hence, the present study was conducted to ana-
lyze and compare phospholipase activity of M. globosa and
M. restricta isolated from patients with SD/D and healthy
controls before and after stimulation with β-endorphin.
Materials and methods
Specimen collection and strains cultivation
Specimens were collected using a blunt scalpel from severely
affected scalp lesions of SD/D patients and from vertex of
healthy control scalp. The sampling was done at conve-
nience of patients and controls by random screening. All
isolates were cultured on Leeming and Notman agar (LNA)
plates and incubated at 34◦C for 2–4 weeks. The isolates
were identified based on phenotypic and molecular tech-
nique as described earlier.8Ten isolates of Malassezia (five-
M. globosa and five- M. restricta) isolated from the lesional
area of SD/D patients and the same number from healthy
controls were included in the study. The study protocol was
approved by the Institute Ethics Committee of Postgraduate
Institute of Medical Education and Research, Chandigarh.
The samples were collected after obtaining the informed
consent of each subject.
Phospholipase activity of Malassezia before
and after β-endorphin exposure
Malassezia isolates were inoculated on modified Dixon’s
agar plate with or without 100 nmol/L β-endorphin (Sigma
Aldrich, St. Louis, Missouri, USA) and incubated for seven
days.9Extraction of extracellular protein was performed
by the method described earlier with few modifications.10
Each isolate was cultured on five sets of LNA plates at
34◦C for seven days. The cells were collected by scraping
the agar surface using a sterile scalpel. After scraping the
cells from the surface of the agar medium, the surface was
rinsed twice with distilled water. LNA medium was crushed
in a pestle-mortar and the resultant slurry was collected in
100 ml protein extraction buffer kept at 4◦C for 24 h.10 The
mixture was filtrated through Whatmann filter paper fol-
lowed by 0.2 mm membrane filter (Millipore corporation,
MA, USA). The resultant solution was concentrated with
Amicon ultra centrifugal filter, ultracel-30 K (Millipore cor-
poration, MA, USA). Phospholipase activity was quantified
by the assay based on the hydrolysis of phospholipid sub-
strate, L-α-phosphatidylcholine dimyristoyl. The assay was
performed at 30◦Cfor1handterminatedbyadding1.25ml
methanol.11 The released fatty acids were extracted by the
rapid single step method.12 The concentration of free fatty
acid was measured by a colorimetry method using the half
micro test kit (Roche, Basel, Switzerland).11 The phospholi-
pase activity on the basal media (LNA) was deducted from
the value of the tests and controls to remove the background
activity of the basal media. Each strain was tested in tripli-
cate. One unit of phospholipase activity was defined as the
amount of enzyme that released 1 μM of free fatty acid per
minute.
Statistical analysis
The impact of β-endorphin exposure on phospholipase ac-
tivity of M. globosa and M. restricta isolated from SD/D
patients and healthy controls were compared by employing
the unpaired t-test using the software GraphPad Prism, ver-
sion 6.01. P<.05 was considered statistically significant.
Results
The source of the isolates and its phospholipase activity
before and after stimulation with β-endorphin is provided
in Table 1and in Supplementary Table S1. For both M.
globosa and M. restricta isolates from SD/D patients, phos-
pholipase activity significantly increased after stimulation
with β-endorphin (M. globosa, P =.04; M. restricta, P =
.001). In addition, after β-endorphin stimulation the over-
all phospholipase activity of the isolates from SD/D patients
was significantly higher compared to isolates from healthy
controls (Fig. 1b, P=.0004), indicating the presence of pos-
sible pathogenic strains within these two Malassezia species.
The phospholipase activity after β-endorphin stimulation
of M. globosa isolated from SD/D patients was significantly
higher when compared to the isolates from healthy controls
(Fig. 1d, P=.022). Similarly, phospholipase activity after
β-endorphin stimulation of M. restricta isolated from SD/D
patients was significantly higher compared to the isolates
from healthy controls (Fig. 1f, P=.017). No significant
difference of phospholipase activity between isolates from
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Honnavar et al.3
Ta b l e 1 . Phospholipase activity of M. globosa and M. restricta isolated from SD/D patients and healthy controls, before and
after stimulation of β-endorphin.
Seborrhoeic dermatitis/dandruff
patients Healthy controls Pvalue
After After
Before β-endorphin Before β-endorphin
β-endorphin (100 nmol/L) β-endorphin (100 nmol/L)
stimulation (A) stimulation (B) stimulation (C) stimulation (D) A v/s C B v/s D A v/s B C v/s D
M. globosa 1.232 ±0.072 1.535 ±0.1 1.277 ±0.031 1.230 ±0.038 .586 .022∗.041∗.367
M. restricta 1.277 ±0.031 1.561 ±0.049 1.233 ±0.072 1.171 ±0.119 .586 .017∗.001∗.673
All Malassezia 1.255 ±0.038 1.548 ±0.053 1.255 ±0.038 1.201 ±0.059 .999 .0004∗.0003∗.454
One unit of phospholipase activity is defined as the amount of enzyme that released 1 μM of free fatty acid per minute. Phospholipase activity expressed in mean
±SEM, ∗P<0.05 was considered statistically significant
patients and control was noted without β-endorphin stim-
ulation
Discussion
In the present study, a significant increase in phospholi-
pase activity after β-endorphin exposure was noted in pa-
tient isolates compared to those from healthy controls,
whereas similar difference was not observed without β-
endorphin stimulation.The finding was similar for both
M. globosa and M. restricta isolates from SD/D patients.
The whole genome sequence of M. globosa has revealed
the presence of six phospholipase-C encoding genes, which
are predicted to be transcribed and secreted enzymes,1
and two phospholipase-D genes and one phospholipase-B
gene, which are not known to be secreted.1Besides, many
genes encoding lipases have been identified and charac-
terized namely, MfLIP1 in M. furfur;13 phospholipase D
in M. pachydermatis;14 MgLIP1 and MgLIP2 in M. glo-
bosa;15,16 MrLIP1, MrLIP2,andMrLIP3 in M. restricta.17
The comparative genomics delineated the importance of
lipases, phospholipases, peptidases and aspartyl proteases
and their potential role in virulence and Malassezia niche-
specificity.18 Phospholipase of M. pachydermatis has been
reported as a potential virulence factor in canine der-
matoses.3M. sympodialis strains isolated from pityriasis
versicolor patients showed higher phospholipase activity
than strains derived from healthy controls.19 These data
suggest that phospholipase plays a major role in the patho-
genesis of Malassezia-associated diseases. The occurrence of
pathogenic subtypes has been described within M. furfur,
M. globosa and M. restricta on the basis of molecular typ-
ing.20,21 Depending on the β-endorphin action (switch-on
and switch-off phenomena) in a susceptible individual, the
potential pathogenic subtypes may get activated to release
higher amounts of phospholipase leading to dysfunction of
the stratum corneum in SD/D. In concordance with our
observation, Vlachos et al.showed recently that phospho-
lipase activity of Malassezia isolated from SD lesions was
stimulated by β-endorphin.9
However, in the majority of the earlier studies phospholi-
pase activity was measured by the semi-quantitative method
using egg yolk plate. This technique is not specific as the
presence of nonspecific substrates in the egg yolk along with
triglycerides and phospholipids interfere with the assay. In
addition, the formation of a zone of white precipitate ren-
ders its interpretation difficult. Hence in the present study,
the phospholipase activity was quantitatively measured us-
ing a specific substrate following the technique described
by Juntachai et al.with few modifications.10 In that study,
they compared the extracellular phospholipase activity of
standard reference strains of seven Malassezia species. M.
pachydermatis (CBS 1891) had the highest phospholipase
activity compared to six other Malassezia spp. (M. furfur,
M. globosa, M. slooffiae, M. sympodialis, M. obtusa, and
M. restricta). Both the standard strains of M. globosa (CBS
7966T, CBS 7986, CBS 8745) and M. restricta (IFM 15950,
IFM 15951) had very low phospholipase activity.10
Serum and skin concentrations of β-endorphin have been
shown to increase in psoriasis, atopic dermatitis, and other
inflammatory dermatoses.5A pathogenic link has also been
established between the increasing level of β-endorphin me-
diated stress and acne.6β-endorphin activates the μ-opiate
receptor present on the cell wall of M. pachydermatis to
overproduce the phospholipase enzyme.22 Similar mecha-
nisms may exist in pathogenic strains of M. restricta and
M. globosa causing SD/D. We propose the following hy-
potheses; in a susceptible individual, alteration in the mi-
croenvironment (physical/chemical/immunological factors)
of the skin, such as stress, pH, salts, bacterial microbiota,
keratinocytes, sebocytes, and melanocytes, may induce β-
endorphin resulting in the stimulation of receptors present
on the cell wall of pathogenic strains of M. restricta and
M. globosa. This stimulus may cause the abundant release
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4Medical Mycology, 2016, Vol. 00, No. 00
Figure 1. Comparison of phospholipase activity of strains isolated from seborrhoeic dermatitis/dandruff patients and healthy controls. (a) Background
phospholipase activity; (b) Phospholipase activity after β-endorphin stimulation; (c) Background phospholipase activity of M. globosa; (d) Phospho-
lipase activity of M. globosa after β-endorphin stimulation;(e) Background phospholipase activity of M. restricta; (f) Phospholipase activity of M.
restricta after β-endorphin stimulation.
of phospholipase. The phospholipase may degrade the in-
tercellular lipid of the stratum corneum resulting in dys-
function of the stratum corneum and exfoliations leading
to SD/D. Future in vivo studies are warranted to prove this
hypothesis.
Acknowledgments
We duly acknowledge the Indian Council of Medical Research
(ICMR), New Delhi, India for the financial support (Grant number
5/3/3/26/2012-ECD-I). The study was presented at the 11th national
conference of Society for Indian Human and Animal Mycologists,
Shimla. India.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
Supplementary material
Supplementary material is available at Medical Mycology online
(http://www.mmy.oxfordjournals.org/).
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