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Glycobiology vol. 13 no. 11 pp. 743±747, 2003
DOI: 10.1093/glycob/cwg069
Structural differences between the alkali-extracted water-soluble cell wall
polysaccharides from mycelial and yeast phases of the pathogenic dimorphic
fungus Paracoccidioides brasiliensis
Oussama Ahrazem2, Alicia Prieto2, Gioconda San-Blas3,
Juan Antonio Leal2, Jes
us Jimenez-Barbero2, and Manuel
1,4
Bernabe
2
Centro de Investigaciones Biol
ogicas, CSIC, Vel
azquez 144,
28006-Madrid, Spain; 3Centro de MicrobiologõÂa y BiologõÂa Celular,
IVIC, P.O. Box 21827, Caracas 1020A, Venezuela; and 4Instituto de
QuõÂmica Organica, CSIC, Juan de la Cierva 3, 28006-Madrid, Spain
Received on January 13, 2003; revised on March 13, 2003; accepted on
March 13, 2003
Paracoccidioides brasiliensis is a pathogenic dimorphic fungus
causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure
of the alkali-extracted water-soluble cell wall polysaccharides
(F1SS) from both mycelial and yeast phases of this fungus by
using chemical analysis and NMR spectroscopic techniques.
The F1SS polysaccharide from the mycelial phase consists
of a trisaccharidic repeating unit of ! 6)-[a-Galf -(1 ! 6)a-Manp-(1 ! 2)]-a-Manp-(1 ! . The F1SS polysaccharide
of the yeast phase maintains 10% of the structure of the
mycelium phase, but the main structure contain a disaccharide
repeating unit of ! 6)-[-a-Manp-(1 ! 2)]-a-Manp-(1 ! ,
alternating with a trisaccharide repeating block of ! 6)-[bGalf -(1 ! 6)-a-Manp-(1 ! 2)]-a-Manp-(1 ! .
Key words: cell wall polysaccharides/fungi/NMR
spectroscopy/Paracoccidioides brasiliensis
Introduction
Paracoccidioides brasiliensis is a pathogenic dimorphic fungus, which at environmental temperatures (20±26 C) grows
in a mycelial form while transforming to a yeast phase at the
temperature of the mammalian host (36±37 C). It is endemic
to the region between the south of Mexico and the north of
Argentina, causing paracoccidioidomycosis, the most
widespread systemic mycosis in this geographical region
(San-Blas et al., 2002), which invades lungs and afterward
disseminates to almost any organ. If not treated, it is
almost always fatal. Different aspects of the pathobiology
and dimorphism of P. brasiliensis have been reviewed (SanBlas et al., 2002; Borges-Walmsley et al., 2002; Franco,
1987; San-Blas, 1982; San-Blas and San-Blas, 1977).
P. brasiliensis has been subjected to a number of chemical
studies intended to analyze cell wall components (proteins,
glycoproteins, glycolipids, and oligo- or polysaccharides) or
1
To whom correspondence should be addressed; e-mail:
[email protected]
metabolites whose presence would modulate host±parasite
relationships through interactions with the host immune
system (Toledo et al., 1999; Levery et al., 1998, Almeida
et al., 1996; Azuma et al., 1974; Kanetsuna and Carbonell,
1970; Kanetsuna et al., 1969).
The alkali-extractable and water-soluble fungal polysaccharides (F1SS), which are minor components of the
cell wall (2±8%), are the glycosidic moieties of peptidopolysaccharides. They differ in composition and structure
among genera and, in certain cases, among species within a
genus (Leal et al., 2001). These polysaccharides are antigenically relevant (Ahrazem et al., 2000a; Domenech et al.,
1996, 1999; Mischnick and De Ruiter, 1994; Latge et al.,
1991; De Ruiter et al., 1991) and are probably involved in
cell±cell and/or cell±host recognition mechanisms.
The aim of the present study was to ascertain whether the
structures of P. brasiliensis polysaccharides F1SS differ in
the mycelial and yeast phases, a fact that might have pathobiological relevance.
Results
Polysaccharide F1SS from the mycelial phase
The analyses of the crude alkali-extractable water-soluble
cell wall polysaccharidic material from the mycelial phase of
P. brasiliensis gave glucose (10%), galactose (30%), and
mannose (60%). After purification through Sepharose CL6B, the main component (polysaccharide F1SS) consisted
of mannose (70%) and galactose (30%), as shown by gasliquid chromatography (GLC) of the alditol acetates. The
absolute configuration was shown to be D for both sugars
(Gerwig et al., 1979). Methylation analysis allowed to
deduce the linkage types summarized in Table I.
Both polysaccharides are polydisperse, with an average
molecular mass around 70 kDa as determined by gel permeation chromatography on Sepharose CL6B.
Table I. Linkage types (%) deduced from methylation analyses of the
polysaccharides F1SS of the mycelial and yeast phases of P. brasiliensis
Linkage type
Manp-(1 !
Mycelial
Yeast
2.7
21.3
27.3
12.6
! 2)-Manp-(1 !
9.3
10.1
! 6)-Manp-(1 !
28.7
20.7
! 2,6)-Manp-(1 !
31.7
35.0
Galf-(1 !
Glycobiology vol. 13 no. 11 # Oxford University Press 2003; all rights reserved.
743
O. Ahrazem et al.
The 1 H±nuclear magnetic resonance (NMR) spectrum
contained three anomeric signals at 5.08, 5.04, and
5.02 ppm (Figure 1a) in the proportion 1:1:1, as deduced
from integration. The corresponding residues were labeled
A±C, from low to high field. A and C appeared as slightly
broad singlets (J1,2 2 Hz), and B exhibited a clear
doublet, J1,2 ˆ 4.4 Hz, that could be indicative of a
galactofuranose with a-configuration (see also the 13 C d
information later and compare with b-configuration, J1,2 2 Hz) (Cyr and Perlin, 1979).
The 13 C-NMR spectrum (Figure 1b) showed 18 singlets,
3 of them anomeric, at 103.0, 101.2, and 99.0 ppm. A series
of 2D homo-NMR (double quantum filtered homonuclear
correlated spectroscopy [DQCOSY], total correlation spectroscopy [TOCSY]) and hetero- (heteronuclear multiplequantum coherence [HMQC], heteronuclear single quantum
coherence [HSQC]-TOCSY) NMR experiments allowed the
assignment of all the proton and carbon signals of the
three residues (see Table II). Comparison of the chemical
shifts values with those reported by Bock and Pedersen
(1983) permitted us to deduce the glycosylation positions.
Fig. 1. (a) 1 H-NMR (500 MHz) and (b) 13 C-NMR (125 MHz) spectra in
D2O at 40 C for the cell wall F1SS polysaccharide from the mycelial form
of P. brasiliensis. The anomeric protons have been labeled A±C.
Thus, A is 2,6-di-O-substituted mannopyranose; B,
terminal galactofuranose; and C, 6-O-substituted mannopyranose, which is in accordance with the methylation results.
Concerning the anomeric configuration of the mannose
residues, a carbon-coupled HMQC experiment allowed the
measurement of their 1 JC-1,H-1 values, giving 173 Hz for
units A and C, which are indicative of a-configurations
for the mannose residues (Bock and Pedersen, 1974).
To find the connections among residues, we performed a
heteronuclear multiple bond correlation (HMBC) experiment, which gives cross-peaks between a proton and the
carbons placed at two or three bonds from it. In addition to
expected intraring connections, peaks corresponding to H1A/C-6A0 , H-1B/C-6C, and H-1C/C-2A could be observed.
We denote a second unit of A as A0 .
The NMR spectral data, together with those of the
methylation analyses, suggest the following main structure
for the cell wall polysaccharide of the mycelial form of
P. brasiliensis.
The small quantities (510%) of terminal and 2-Osubstituted mannopyranoses observed in the methylation
analyses were not detected in the NMR spectra. Therefore,
to further investigate those minor components, we selectively hydrolyzed the galactofuranose residues, taking
advantage of the lability of the glycosylic linkages of the
furanoid rings, as compared with those of the pyranoid
rings. Treatment of the polysaccharide with 0.05 M sulfuric
acid gave a new polysaccharide composed exclusively of
mannose. Methylation analysis gave terminal mannopyranose (34%); 2-O-substituted, 3-O-substituted, and
6-O-substituted mannopyranoses (23.5%, 1.1%, and 9%,
respectively); and 2,6-di-O-substituted mannopyranose
Table II. 1 H- and 13 C-NMR chemical shifts (d) for the alkali-extractable
water-soluble cell wall polysaccharide F1SS isolated from the mycelial
form of P. brasiliensis
Proton or Carbon
Units
A
1
H
C
B
H
C
C
H
C
1
5.08
99.0
5.04
101.2
5.02
103.0
4.01
80.0
4.15
77.4
4.08
70.9
3
3.94
71.4
4.22
75.2
3.83
71.3
4
3.87
67.1
3.84
82.1
3.81
67.3
5
6a
3.80
72.2
3.79
72.8
3.91
72.6
13
Fig. 2. (a) H-NMR (300 MHz) and (b) C-NMR (75 MHz) spectra in
D2O at 40 C for the partially hydrolyzed mycelial form of P. brasiliensis.
744
2
Numbers in boldface type represent glycosylation sites.
4.04
6b
3.74
66.4
3.72
3.63
63.4
4.05
67.0
3.66
Cell wall polysaccharides in phases of P. brasiliensis
Table III. 1 H- and 13 C-NMR chemical shifts (d) for the
alkali-extractable water-soluble cell wall polysaccharide F1SS
isolated from the yeast form of P. brasiliensis
Proton or Carbon
Units
1
D
H
E
H
C
C
F
H
C
Fig. 3. (a) 1 H-NMR (500 MHz) and (b) 13 C-NMR (125 MHz) spectra in
D2O at 40 C for the cell wall F1SS polysaccharide from the yeast form of
P. brasiliensis.
G
H
H
H
C
C
(32.4%). The 1 H-NMR spectrum (Figure 2) suggests the
presence of a small proportion of short chains of (1 ! 2)
and (1 ! 3) linked mannopyranoses and also small amounts
of unbranched mannopyranoses in the main chain, as
observed in several yeasts (see, for instance, Vinogradov
et al., 1998).
Polysaccharide F1SS from the yeast phase
The analysis of the alkali-extractable water-soluble yeast
cell wall polysaccharidic material from the yeast form gave
mannose (67.9%), galactose (22.1%), and glucose (9.9%).
After purification through Sepharose, the main component
(polysaccharide F1SS) gave mannose (77%) and galactose
(23%). The absolute configuration was shown to be D for
both sugars. Methylation analysis led to the results gathered
in Table I.
The 1 H-NMR spectrum (Figure 3a) showed a severely
crowded anomeric region, from which very little information could be inferred. The 13 C-NMR spectrum (Figure 3b)
contained four main and one small anomeric singlets. The
signal at 108.7 ppm suggests a b-galactofuranose moiety,
and those around 103 ppm are characteristic of mannopyranose units. The small singlet appeared at identical position
to that of the a-galactofuranose unit (101.2 ppm) found in
the spectrum of the mycelial phase. The 2D 1 H-13 C correlation (HMQC) spectrum contained six anomeric crosspeaks, which were labelled D±I. By using 2D homo- and
hetero-NMR experiments, we were able to assign most
proton and carbon signals of the six main residues present
in the polysaccharide (Table III). Again, by comparison of
the chemical shifts values with those of model compounds
(Bock and Pedersen, 1983) and consideration of the methylation results, we could deduce the glycosylation sites as
being D and E 2,6-di-O-substituted mannopyranoses; F and
G, terminal galactofuranoses; H, terminal mannopyranose;
and I, 6-O-substituted mannopyranose.
The configuration of the anomeric centers was deduced
from a carbon-coupled HMQC experiment, which allowed
the measurement of the 1 JC-1,H-1 values, giving 174.4, 174.3,
174.4, 175.9, 172.9, and 173.0, for units D to I, respectively,
which, in accordance with the values of the chemical shifts,
I
H
C
2
5.11
99.2
5.08
99.2
5.06
108.7
5.04
101.2
5.04
103.1
5.02
103.0
3
4.03
79.5a
4.03
79.9
a
4.14
81.8
4.15
77.5
4.07
70.9
4.07
70.8
3.94b
71.5
3.93b
71.5
4.07
77.8
4.22
75.2
3.81
71.5
3.83
71.5
4
5
3.85
67.7c
6a
~.78
72.2
3.87
~.81
c
~3.78
67.4
4.01
84.0
3.83
82.2
3.68
67.7
3.81
67.4
3.84
71.8
3.78
72.7
3.76
74.1
6b
4.02d 3.69
66.6e
4.03d 3.72
66.4e
3.73
3.66
63.8
3.73
3.64
63.4
3.88
3.77
62.0
4.03
3.67
67.4
Numbers in boldface type represent glycosylation sites.
a,b,c,d,e
These values may have to be interchanged within each respective row.
are demonstrative of b-configuration for the furanose unit
G and a for the rest of the residues (Bock and Pedersen,
1974; Cyr and Perlin, 1979).
To discriminate among the various possibilities of
arrangement of the different fragments, we recorded a
long-range proton±carbon correlation HMBC experiment
that, in addition to trivial cross-peaks, showed signals for
H-1D/C-6E (D0 ), H-1E/C-6D (E0 ), H-1F/C-6I, H-1G/C-6I,
H-1H/C-2D (E), and H-1I/C-2E (D). Second units of D and
E are labeled D0 and E0 .
All the NMR spectral data, in agreement with the methylation analyses, allow us to propose the main structure of
the galactomannan from the yeast phase of P. brasiliensis as
being n, m, and p in a proportion around 1:1:0.2, as
deduced from integration of the anomeric singlets of the
carbon spectrum.
In addition, a small proportion of short chains of (1 ! 2)
linked mannopyranoses is deduced from the methylation
analysis, analogously to that observed in the mycelium phase.
Discussion
P. brasiliensis grows as filamentous fungus when cultivated
at 23 C and is easily converted to the yeast phase by
simply changing the temperature to 37 C. This process is
745
O. Ahrazem et al.
reversible, as demonstrated by Nickerson (1948), who also
showed that this reversibility was exclusively due to the
temperature factor and was independent from other
changes in the culture medium.
It has been shown (Kanetsuna and Carbonell, 1970;
Kanetsuna et al., 1969) that both yeast and mycelial forms
have chitin as a common structural polysaccharide, but an
a-(1 ! 3) glucan was found in the yeast phase, whereas a
b-(1 ! 3) glucan was encountered in the mycelial form. This
difference has been suggested as a possible contribution to
the distinct morphology of both forms (San Blas and San
Blas, 1994). However, a study on the structure of glucans
and F1SS polysaccharides in Eupenicillium, Penicillium, and
Talaromyces species grown at 25 C (Leal and BernabeÂ,
1998) revealed that the mycelium from these microorganisms contained either a-(1 ! 3) or b-(1 ! 3) glucans,
regardless of their common mycelial shape. On the other
hand, studies on mutants of P. brasiliensis have suggested a
direct relationship between virulence and the presence of
variable amounts of a-(1 ! 3) glucan in the cell walls of the
mutant strains (San-Blas, 1982 and references therein).
Major quantities of this glucan determine enhancement of
the virulence, whereas a lower amount of the glucan results
in a decreased virulence. This behavior has been explained
as the result of a-glucan working as a protection mechanism
of the fungus against host defences (San-Blas, 1982).
Structural variations have also been observed by
MendoncËa et al. (1976) in the alkali-extractable cell wall
polysaccharides from both morphological types of the
dimorphic fungus Sporotrix schenckii. Nevertheless, they
dissociated the effect of the temperature on morphological
phase transition because 100% yeast was obtained in a
synthetic medium either at 25 C or 37 C. Therefore they
concluded that the variations in the structure of the polysaccharides observed must be due to differences in the
morphology of both phases and not to modification on
growth temperatures.
Nickerson (1948 and references therein) attributed the
changes in morphology to reversible denaturation or activation of enzyme processes due to the changes in temperature.
In this context, it has been shown that exogenous cAMP
inhibits the yeast to mycelial transitions, thus favoring the
pathogenic yeast form (Borges-Walmsley et al., 2002).
It is not currently possible to interpret the meaning and
evaluate the importance of the structural modifications of
F1SS polysaccharides observed in the transition from the
mycelial to the yeast phase of P. brasiliensis. The antigenic
relevance of the b-galactofuranose domains of polysaccharides from several pathogenic fungi (Latge et al., 1991;
Notermans et al., 1988; Azuma et al., 1974) is known, yet
little or nothing is known of the role of a-galactofuranoses
because these residues have only been described in cell wall
polysaccharides from a few species belonging to Onygenales
(Bernabe et al., 2002).
Material and methods
Strains and growth conditions
P. brasiliensis strain IVIC Pb73 (ATCC 32071) was grown
in peptone yeast glucose medium and incubated at either
746
23 C (mycelium form) or 37 C (yeast form) with continuous shaking.
Wall material preparation and fractionation
Cell walls from P. brasiliensis were prepared according to
Kanetsuna et al. (1969). Fractions and polysaccharides
F1SS were obtained and purified following Ahrazem et al.
(2000b).
Chemical analysis
For analysis of neutral sugars the polysaccharides were
hydrolyzed with 3 M trifluoracetic acid (TFA) (1 h at
121 C), converted into their corresponding alditol acetates
(Laine et al., 1972), and identified and quantified by GLC
using a SP-2380 fused silica column (30 m 0.25 mm ID 0.2 mm film thickness) with a temperature program (210 C
to 240 C, initial time 3 min, ramp rate 15 C min ÿ1 , final
time 7 min) and a flame ionization detector.
The monosaccharides released after hydrolysis were derivatized according to Gerwig et al. (1979) and their absolute
configuration was determined by gas chromatography mass
spectrometry of the tetra-O-TMSi-(‡)-2-butylglycosides
obtained.
Methylation analyses
The polysaccharides (1±5 mg) were methylated according to
the method of Ciucanu and Kerek (1984). The methylated
material was treated and processed according to Ahrazem
et al. (2000b), with the exception that the partially methylated polysaccharide was hydrolysed with TFA 1.5 M
(100 C, 30 min).
Partial hydrolysis of the polysaccharide F1SS from the
mycelial phase
Eighty milligrams of the polysaccharide were hydrolyzed as
described by Prieto et al. (2001).
NMR analysis
1D and 2D 1 H- and 13 C-NMR experiments were carried out
at 40 C on a Varian Unity 500 spectrometer with a reverse
probe and a gradient unit or a Varian INOVA-300 spectrometer (1 H, 300 MHz). Proton chemical shifts refer to residual HDO at d 4.61 ppm. Carbon chemical shifts refer to
internal acetone at d 31.07 ppm. The polysaccharides F1SS
(~20 mg) were dissolved in D2O (1 ml) followed by centrifugation (10,000 g, 20 min) and lyophilization. The
process was repeated twice, and the final samples were
dissolved in D2O (0.6 ml, 99.98% D).
The 2D NMR experiments (DQCOSY, TOCSY, HMQC,
HSQC-TOCSY, and HMBC) were performed by using the
standard Varian software.
Acknowledgments
The authors thank J. L
opez and Lic. B. Moreno for technical assistance. This work was supported by Grant BQU2000-1501-C02-01 from Direcci
on General de Investigaci
on
CientõÂfica y Tecnica.
Cell wall polysaccharides in phases of P. brasiliensis
Abbreviations
DQCOSY, double quantum filtered homonuclear correlated spectroscopy; GLC, gas-liquid chromatography;
HMBC, heteronuclear multiple bond correlation; HMQC,
heteronuclear multiple-quantum coherence; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; TFA, trifluoroacetic acid; TOCSY, total
correlation spectroscopy
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