Medical Mycology August 2007, 45, 419 427
Original Article
Putative structure and characteristics of a
red water-soluble pigment secreted by
Penicillium marneffei
SONIA BHARDWAJ*, ANSHUMAN SHUKLA$, SOURAV MUKHERJEE$, SWATI SHARMA$,
PURNANANDA GUPTASARMA$, ASIT K. CHAKRABORTI§ & ARUNALOKE CHAKRABARTI*
*Postgraduate Institute of Medical Education and Research, Chandigarh, India, $Institute of Microbial Technology, Chandigarh,
India, and §Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, S. A. S. Nagar,
Punjab, India
The dimorphic fungus, Penicillium marneffei, produces and secretes a brick red
pigment, during growth at temperatures below 308C. It generally diffuses into
commonly used media like Sabouraud dextrose agar and malt extract agar. The
pigment was purified by reverse-phase liquid chromatography and subjected to
structural determination by elemental and spectral analysis using atomic absorption
(AAS), ultra violet and visible (UV-VIS), fluorescence, infra red (IR), nuclear
magnetic resonance (NMR) and tandem mass spectrometry (MS-MS). The pigment
showed a buffering ability in aqueous solutions, maintaining an alkaline pH of 8.0.
It behaved as a colorimetric pH indicator over a wide acidic and alkaline pH range,
with discoloration occurring ostensibly through hydrolysis of key chemical groups
at extremely acidic pH ( 2.0). The pigment was found to have some structural
resemblance with the copper-colored pigment (herquinone) produced by Penicillium
herquei as both pigments contain the phenalene carbon framework. The notable
differences between herquinone and the pigment produced by P. marneffei are
(i) the latter’s apparent dimerization through a sulphur-sulphur (disulfide) bond
and (ii) the presence of 1,1,3,3-tetramethyl-2,3-dihydropyrrole moiety in the latter
instead of 2,3,3-trimethyl-2,3-dihydrofuran moiety found in the former. The
delineation of the structure of the pigment produced by Penicillium marneffei
may help in understanding certain aspects of the biology of this pathogenic fungus.
Keywords
Penicillium marneffei, pigment, chemistry, molecular structure
Introduction
Penicillium marneffei is the only known dimorphic
species among 200 described members of the genus
and is pathogenic to human and certain species of
Received 16 June 2006; Accepted 5 February 2007
Correspondence: Arunaloke Chakrabarti, Professor, Department of
Medical Microbiology, Postgraduate Institute of Medical Education
& Research, Chandigarh 160012, India. Tel: 91 172 2755156/
2755172; Fax: 91 172 2744401/2745078. E-mail: arunaloke@
hotmail.com
– 2007 ISHAM
bamboo rats [1,2]. This fungus is endemic in south East
Asia, and the disease that it produces has received
much attention as an AIDS-defining illness in the same
area [3]. P. marneffei produces a brick-red diffusible
water-soluble pigment when the fungus is allowed to
grow in media at 25 308C [4]. Interestingly, another
species, Penicillium herquei, also produces a diffusible
copper-colored pigment, indicating a possible commonality of pigment-producing metabolism between
the two species [5]. P. marneffei does not produce
pigment in observable amounts when the fungus is
DOI: 10.1080/13693780701261614
420
Bhardwaj et al.
cultured at temperatures higher than 308C [6] and
consequently, the possibility of pigment production
during tissue invasion has not been studied.
In recent years pigment production by fungi has
drawn attention of scientists especially after demonstration of the role of melanin in many pathogenic
fungi with respect to virulence, protection against
antifungal drugs, and promoting survival in the environment [7]. Though a role of the diffusible pigment of
P. marneffei has not yet been demonstrated in tissue
invasion, it assists in the identification of this species.
The unique pigment production by this fungus
prompted us to characterize the pigment as it may
help to better understand certain biological aspects of
this pathogenic fungus.
Materials and Methods
(Millipore). The filtered solution was loaded on to the
HPLC column and eluted with acetonitrile water
gradient. In the second method, the unresolved pigment mixture was subjected to RP-HPLC on a C18
column in conjunction with tandem mass spectrometry
(MS-MS) under similar conditions. The eluent was
taken and subjected to MS-MS analysis. As some
minor contaminants were still present at this stage,
only the dominant mass peak of first stage of MS was
further fragmented during the MS-MS experiment.
Solubility
The lyophilized red pigment powder was tested for its
solubility in water, methanol, ethanol, propanol and
non-polar solvents such as carbon tetrachloride. The
powder (10 mg) was added to the solvent (10 ml)
separately and observed for solubility.
Pigment extraction
P. marneffei ATCC (American Type Culture Collection, Rockville, Maryland), 201700 was maintained in
our laboratory at 708C, until subcultured for these
studies on malt extract agar (MEA, Hi-media, Mumbai, India), and incubated at 268C for five days. The
media containing diffusible red pigment was sliced out
approximately 1.5 cm away from the growing colony
and minced into very small pieces (approximately 1
2 mm diameter) with sterile precautions. These pieces
were then placed in about 50 ml of warm sterile
distilled water at 508C until the pigment diffused into
the water and the minced pieces became almost colorless. Water containing pigment was separated from the
solid pieces of agar by centrifugation at 40,000 rpm for
40 min. Finally, the water containing pigment was
filtered through 0.22 mm filters and lyophilized for
further experiments.
Pigment purification
The HPLC grade acetonitrile (E. Merck, India) and
double distilled water were filtered through 0.45 m
membrane filter (Millipore, India) before each use.
The pigment was purified, using reverse-phase high
performance liquid chromatography (RP-HPLC) following two methods. The first involved separation
employing a C18 column (2.1 100 mm, 5 m particle
size, using ultraviolet and visible UV-VIS detector at
500 nm) on a Pharmacia SMART system (Pharmacia),
using a gradient of acetonitrile-water that involved
100% water to 100% acetonitrile in 30 min with a flow
rate of 1 ml/min. The crude sample of the pigment was
dissolved in minimum volume of acetonitrile and the
solution was passed through 0.45 m membrane filter
Changes of color with pH and buffering action
To study the pH of the pigment solution and its
buffering capacity, 10 ml of the pigment solution
(1 mg/ml) was taken in a sterile test tube and 2 ml of
NaOH (1 M) or HCl (1 M) was added slowly to it. Any
effective change in color was monitored. After each
addition of the basic and alkaline solution, the pH of
the solution was measured.
Siderophore activity
To examine whether the pigment was a siderophore, the
fungus was grown with iron supplementation in growth
media. Ferric ammonium chloride was added as an
iron source to the MEA (Hi-Media, India) at concentrations of 56, 93, and 150 mM with the expectation
that pigment synthesis would be noticeably suppressed.
In addition, the same medium without iron supplement
served as control. The pigment production in all media
was observed for a period of three to five days.
Spectroscopic characterization
To determine the structure, the pigment was subjected
to various analyses such as UV-VIS absorption [Shimadzu UV160A], UV-VIS fluorescence [Perkin Elmer
LS-50B], atomic absorption (AA) [Analytic Jena
AS5EA], Fourier transform infrared (FT-IR) spectra
[Nicolet IMPACT 410], nuclear magnetic resonance
(1H NMR) [Bruker 300 MHz DPX], CHNS elemental
analysis [Elementar], liquid chromatography-coupled
electrospray ionisation mass (LC-MS) [Finnigan Matt
LCQ]. All studies used water or water-methanol or
water-acetonitrile as media.
– 2007 ISHAM, Medical Mycology, 45, 419 427
Characteristics of a red pigment secreted by P. marneffei
The UV absorption, fluorescence, FT-IR and elemental analyses were carried out using the sample
obtained from the RP-SMART dried elution and
the mass fragmentation analysis employed RP-HPLC
purified material, which was further purified for mass
fragmentation analysis through separation of mass
peaks in the first stage of MS-MS. Mass spectral
analyses of the pigment was carried out by electrospray
ionization-based liquid chromatography-coupled tandem mass spectrometry (ESI-LC-MS-MS) in which the
dominant species obtained in a mass spectrum could be
selectively fragmented and analyzed through a series of
such fragmentations. The Finnigan Matt LCQ mass
spectrometer was used for this study. The various
instrumental parameters were set at 4.28 V spray
voltage, 5.19 V capillary voltage, 1.66 Amp spray
current, 2808C capillary temperature, 60 ml/min sheath
gas flow, 20 ml/min auxiliary gas flow and 40% relative
collusion energy (in case of MS-MS). An initial full
mass spectrum was recorded using the ESI-LC-MS to
collect information about the major ion peaks corresponding to each component. The most abundant ion
in the ESI-LC-MS chromatogram was subjected to
further fragmentation using the MS-MS operation.
421
buffering action of some kind, possibly mediated
through ionization of some chemical groups.
Pigment purification
Fig. 1A represents the HPLC profile of the pigment on
reverse phase C18 column, using a water acetonitrile
gradient. The eluents bearing the color of the pigments
were collected and the HPLC profile of the purified
fraction is represented in Fig. 1B.
UV-VIS spectroscopic characterization
In the UV-VIS region, the pigment displayed the
following three distinct absorption maxima; 273, 418
and 493 nm (Fig. 2A). Each absorption maximum also
corresponded to excitation-deexcitation electronic transitions associated with fluorescence (Fig. 2B). The
excitation at a wavelength close to the 273 nm showed
a low-intensity fluorescence at three different emission
maxima; 425, 495 and 515 nm. As the excitation
wavelength was increased to the absorption maximum
Results and Discussion
Solubility and changes of color with pH
The pigment was readily soluble in water though
saturation level was not determined due to insufficient
quantity of pigment powder. In addition, it was soluble
in methanol and partly soluble in ethanol but not in
any other organic solvents such as in carbon tetrachloride. The solubility of the pigment in water
indicated the presence of charged groups which indicated a polar nature to the molecule. Thus, we
examined the pH and buffering action of the red
pigment in solution. We noted that the color changed
from red to orange to yellow as the pH was increased
through the addition of NaOH (1 M) from 8.0 through
10.0 to 13.0. On the other hand, when the pH was made
more acidic through the use of hydrochloric acid, there
was initially no change in the pigment’s color, but, at
pH B2.0, the color abruptly disappeared and solution
became almost colorless.
Buffering of solutions
We found that regardless of the pigment’s concentration in water, the pH of aqueous solutions was always
maintained at 8.0, even after addition of solutions of
different pH. It appeared that the pigment could have a
– 2007 ISHAM, Medical Mycology, 45, 419 427
Fig. 1 (A) HPLC chromatogram of the isolated pigment without
further purification. (B) HPLC chromatogram of the major constituent obtained after HPLC purification of the crude isolate of the
pigment.
422
Bhardwaj et al.
This is due to the fact that most other colors present in
white light, barring the red color (wavelengths longer
than 625650 nm), tend to be absorbed to a greater
degree than red. This contention was supported by the
absorption spectrum of the pigment, which showed a
broad absorption envelope at short wavelengths in the
visible region. This absorption profile gave rise to a
greater reflectance of the red component of white light
from the pigment’s surface. Notably, high absorptivity
was seen at the relatively long wavelength of 493 nm,
together with high quantum yield of fluorescence seen
at 515 nm, as a result of absorption of 493 nm light.
Together, this suggested that the pigment probably
consisted of some sort of a conjugated ring system with
substantially delocalized electrons amenable to excitation with long-wavelength, low energy photons.
FTIR spectroscopic analysis
In the FTIR (Fig. 3A), the absorptions at 3429, 2952,
and 2841 cm 1 indicated the presence of NH/OH,
aromatic CH, and aliphatic CH groups, respectively.
The peak at1638 cm 1 suggested the pigment to be an
a, b unsaturated carbonyl compound (e.g., a quinonederivative). The absorptions 1450 and 1017 cm 1
indicated the existence of an ether group.
1
Fig. 2 (A) The UV-Visible absorption spectrum of the major
constituent obtained after HPLC purification of the crude isolate of
the pigment. (B) The fluorescence spectra of the major constituent
obtained after HPLC purification of the crude isolate of the pigment.
at 418 nm, the fluorescence was observed maximally
at 495 nm. With further increase of the excitation
wavelength (data shown up to 440 nm), the emission
at 515 nm could be observed. Peak emission at 515 nm
was seen when excitation was done at 493 nm (data not
shown in Fig. 2B). Thus it appeared that three different
‘excitation: emission’ fluorescence ‘wavelength maxima’
pairs exists in the pigment: 273:425 nm, 418:495 nm,
and 493:515 nm. Of the three, the one that showed the
highest quantum yield of fluorescence was the visible
emission at 515 nm, which resulted from the longest
excitation wavelength of 493 nm (also in the visible
range of the spectrum).
The pigment was naturally red and fluorescent under
visible light with a yellow-orange hue. However, this
fluorescence did not dominate the pigment’s perceived
color; rather, the pigment appeared to be brick red.
H NMR analysis
The signals at d 0.75, 1.12, 1.89 and 3.50 ppm in the 1H
NMR spectrum (Fig. 3B), are indicative of the presence
of C -CH3, C -CH3, N -CH3, and O -CH3 protons,
respectively. The signals that appeared in the range of
d 6.8 7.8 suggested the presence of aromatic protons.
However, the low integral value indicated that the
aromatic ring(s) were substituted.
Elemental analysis
The elemental analysis provided the value of C: H: N: S
of 29.5: 5.3: 4.0: 2.6. Calculations suggested that the
pigment had one nitrogen atom, and 2 3 sulphur
atoms. However, it appeared that the oxygen had
been overestimated, and the carbon had been underestimated, probably due to the high moisture content of
the sample. The calculated oxygen and carbon atom
contents are not presented here.
Atomic absorption spectral analysis
Atomic absorption spectra indicated the presence of
iron in 460 ppm by weight. This content was much
smaller than that required to establish the presence
of a covalently bonded iron atom in each pigment
molecule. Therefore, it appeared that iron existed in this
– 2007 ISHAM, Medical Mycology, 45, 419 427
Characteristics of a red pigment secreted by P. marneffei
423
Fig. 3 (A) The FTIR spectrum of the major constituent obtained after HPLC purification of the crude isolate of the pigment. (B) The 1H NMR
spectrum of the major constituent obtained after HPLC purification of the crude isolate of the pigment.
– 2007 ISHAM, Medical Mycology, 45, 419 427
424
Bhardwaj et al.
pigment only as a chelated metal atom in some form of
non-covalent association, with samples containing far
fewer bound iron atoms than molecules of the pigment.
The detected presence of chelated iron raised the
interesting possibility of the pigment being a siderophore of some kind. However, when we examined the
incorporation of iron into growth media we noted that,
even with 150mM concentration of iron, the pigment
production was not visibly reduced (no quantitative
estimation was made). Therefore, it may be concluded
that the presence of iron is merely incidental to the
pigment, and the pigment does not appear to be used
by the fungus as an ‘iron sink’.
Mass spectrometric analysis
In the MS (Fig. 4A), the pigment exhibited ion peaks at
m /z 679.3 (base peak), 680.3 and 681.3 which could be
assigned to the M-3, M-2, M -1, respectively,
giving rise to the molecular mass of 682Da. The MSMS study on the base peak (Fig. 4B) resulted in an ion
peak at m /z 343 which corresponds to the M-2 of the
daughter ion of size 341 Da. This suggested that the
parent molecule is a dimer of a molecule of size 341 Da.
The presence of an ion peak at m /z 452 in addition to
the daughter ion of 343 Da suggested that the parent
molecular ion undergoes fragmentation in two different
ways. The MS spectrum of the compound contained
other daughter ions of low abundance and the same
sets of mass fragmentation product ions were obtained
in higher abundance upon selective fragmentation of
the dominant mass species. Thus, these sets of daughter
ions constituted the mass fingerprint of the parent
molecule. The significant product ion peaks in the MS
and MS-MS spectra of the parent compound were at
m /z 681, 680, 679, 661, 452, 343, 326, 226, 209 and 175.
Structural analysis
The structure of the red pigment produced by
P. marneffei was deduced by analyzing the FTIR,
NMR and MS-MS data and by comparing it with
Fig. 4 (A) The ESI-MS spectrum of the major constituent obtained after HPLC purification of the crude isolate of the pigment. (B) The
MS-MS spectrum of the base peak of the ESI-MS of the major constituent obtained after HPLC purification of the crude isolate of the pigment.
– 2007 ISHAM, Medical Mycology, 45, 419 427
Characteristics of a red pigment secreted by P. marneffei
already known structure of herqueinone [8], a copper
coloured pigment produced by Penicillium harquei .
Considering the conservation of synthetic pathways
for metabolites in related species within the same
genera, we considered it likely that the red pigment
could have some similarities to herqueinone. The size of
one of the fragmentation products (the presumed
monomeric unit of the pigment) turned out to be close
to the size of herqueinone. In addition, the FT-IR and
NMR analyses suggested that the pigment has a
quinone-based structure, and that there are several
chemical groups within the pigment that are also
present in herqueinone. However, some clear differences were also observed such as the presence of suphur
and nitrogen. Structural comparison of the present
pigment with herqueinone is presented in Fig. 5. A schematic drawing of the pigment of P. marneffei deduced
from mass fragmentation pattern is presented in Fig. 6.
The pigment has a highly conjugated structure,
which explains the long wavelength absorptions. These
transitions result from the excitation transitions of
significantly delocalized electrons. Some of the masses
presented in Fig. 3B were not taken into account in
the structural analysis. It would appear to suggest that
the molecule of mass 682 also exists in alternate forms
in which the N -CH3 and O -CH3 groups underwent loss
of the CH3 moiety. Certainly, the -NH groups could
explain the fact that the pH of a pigment solution is
close to 8.0. Also the FTIR band at 3429 cm 1
indicates that a part of the population has either NH
or OH protons.
Several other aspects of the structure also explain
observations concerning its color and change in color
upon pH alteration. It is likely that the change in
color is due to a change from a hydroquinone structure
to a quinone structure with protonation giving rise to a
hydroquinone structure. Under protonation, there
would be delocalization of electrons over the three
six-membered rings of carbon atoms. The red color
would increase with acidification upon lowering of pH
and reduce with change to higher pH values, as has
been observed.
Herqueinone
CH3
Present Pigment
CH3
H
H3C
CH3
H
CH3
H3C
O
O
N
O
+
CH3
H3C
H
OH
OH
H
OCH 3
H
OCH 3
H
S
|
S
OH
H
H
H
H
H
OCH 3
CH3
O
N
+
H3C
CH3
Fig. 5 Structural resemblance of herquinone and the present pigment.
– 2007 ISHAM, Medical Mycology, 45, 419 427
425
CH3
H
426
Bhardwaj et al.
CH3
H
CH3
H3C
N
O
+
341 - 15 ≈ 326
CH3
341 - 99 ≈ 242
O CH 3
242 – 15 ≈ 226
242 - 67 ≈ 175
226 - 17 ≈ 209
S
|
S
226 + 226 ≈ 452
682 – 341 ≈ 341
226 - 17 ≈ 209
242 – 15 ≈ 226
242 - 67 ≈ 175
O CH 3
341 - 99 ≈ 242
O
CH3
N+
341 - 15 ≈ 326
H3C
CH3
CH3
H
Characteristic pigments are produced by wide variety of fungi and exhibit several biological activities.
However, the exact nature and function of the pigments
are not known in majority of fungi that form them.
Fungal pigments have drawn the attention of scientists
as many are used in the food and dye industry [9]. In
medically important fungi, the pigments are expected to
play an important role in host and pathogen interaction.
The physiochemical role of melanin in pathogenesis of
fungal infection has been extensively studied [7,10 12]
as its role as a virulence factor has been reported in an
increasing number of fungi. Melanin protects fungi
against a broad range of toxic insults and contributes to
fungal survival in the environment and human host
[7,1012]. The function of the pigment produced by P.
marneffei is still not known and the presence of this
pigment during infection has not been studied. However, the present investigation of the structure of the
pigment may be of assistance in helping us understand
biological aspects of P. marneffei . In future, it will be
important to study the presence of the pigment in the
Fig. 6 The deduced mass fragmentation pattern of the pigment.
yeast phase of this dimorphic fungus and its potential
role as a virulence factor during infection.
Acknowledgements
We thank the Indian Council of Medical Research for
sponsoring the project and Mr Vikas Grover, technician
at NIPER, for help with NMR, FTIR and MS spectra.
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Characteristics of a red pigment secreted by P. marneffei
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