FEMS Immunology and Medical Microbiology 43 (2005) 241–247
www.fems-microbiology.org
Humoral immune response against soluble and fractionate
antigens in experimental sporotrichosis
Rosana C. Nascimento, Sandro Rogério Almeida
*
Departamento de Análises Clı́nicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo,
Av. Prof. Lineu Prestes 580, Bloco 17, 05508-900, São Paulo, Brazil
Received 17 May 2004; received in revised form 26 July 2004; accepted 20 August 2004
First published online 11 September 2004
Abstract
Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the
prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix
schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis
by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of
IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected
mice.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Sporotrichosis; Humoral immune response; Antibody
1. Introduction
Sporotrichosis a chronic granulomatous mycosis
caused by the dimorphic fungus Sporothrix schenckii
which is widely distributed in nature and presents a
saprophytic mycelial form on plant debris and soil.
The traumatic inoculation of conidia and hyphae of S.
schenckii results in the development of this subcutaneous mycosis and within the infected tissue the fungus
differentiates into its yeast form and may spread to other
tissues. The systemic form of sporotrichosis may evolve
from an initial cutaneous lesion or be associated with
the inhalation of conidia [1–3]. Recently, more severe
clinical forms of this disease have been associated with
immunocompromised patients, such as human immuno*
Corresponding author. Tel.: +55 11 3091 3633; fax: +55 11 3813
2197.
E-mail address: [email protected] (S.R. Almeida).
deficiency virus (HIV)-infected patients, suggesting that
S. schenckii is an emerging opportunistic pathogen [4].
Cultivation and immunological techniques are of
great importance for the diagnosis of S. schenckii
infection. The latter techniques require an understanding of the antigenic structure of the etiological agent
[5]. Apparently, mono-rhamnosyl rhamnomannan is
the principal component causing cross reactivity. Predominant in the yeast phase of the fungus [6], this
component has not had, as of yet, its molecular
weight determined [7]. It has, however, been precipitated and purified from the yeast wall of S. schenckii
using Concanavalin A. Three principal subunits with
masses 84, 70 and 58 kDa have been identified [8].
Other investigations suggest that the surface polysaccharides vary during the morphological differentiation
of the fungus [6]. Additional studies of the yeast antigens describe a range of proteins with masses between
22 and 70 kDa [9].
0928-8244/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsim.2004.08.004
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The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully
understood. Previous studies have suggested that cellmediated immunity plays an important role in the protection of the host against this fungus. Tachibana
et al. [10] have recently demonstrated that acquired
immunity against S. schenckii is expressed by T-cellactive macrophages.
Even though many studies have investigated antibody (Ab) responses in fungal infections, critical features such as Ab fine specificity and/or Ab isotype
were seldom observed. Moreover, because many different clinical and experimental settings have been addressed, no general conclusion has yet been reached
[11]. Some Abs (for instance, the anticryptococcal ones)
may be disease enhancing. Nonetheless, recent data
strongly support the existence of protective Abs against
two of the major opportunistic agents of fungal disease.
In addition to strengthening the few significant findings
published previously, this new evidence is important because it highlights new protective antigens as well as
possible novel sources and mechanisms of Ab-mediated
protection [12–14].
The role that the humoral immune response plays in
sporotrichosis is until now uncertain. Characterisation
of Sporotrix antigens which are active in disease would
permit the understanding of the importance of antibody
response, which might be of value in forecasting the
prognosis and for the selection of an optimal therapeutic
regimen. In this study, we evaluated antibody production and determined the antigenic components involved
in the humoral immune response in experimental
infection.
2. Materials and methods
2.1. Organism
Sporothrix schenckii strain M-64, previously isolated
from a human case of sporotrichosis, was originally obtained from the Department of Dermatology of the Faculty of Medicine at the University of São Paulo. Yeast
forms of S. schenckii were grown for 3 days in brainheart infusion agar (BHI) at 37 C.
2.2. Animals
Twelve BALB/c female mice with 8–12 weeks of age
were used. Mice were obtained from the University of
São Paulo animal facilities.
2.3. Soluble antigen preparation
For the preparation of yeast-phase soluble antigen,
yeast cells were grown in yeast nitrogen-Casamino
Acids-glucose (YCG) medium consisting of (grams per
litre of distilled water); yeast nitrogen base, 6.7; Casamino acids, 2.5; glucose, 50; and 1 mL of a vitamin
mixture (containing [per 100 mL] thiamine hydrochloride, 50 mg; riboflavin, 50 mg; calcium pantothenate,
50 mg; nicotinic acid, 50 mg; pyridoxine hydrochloride,
10 mg; p-aminobenzoix acid, 10 mg; inositol, 10 mg;
folic acid, 1 mg; biotin, 0.4 mg) [9]. Cells from 48-hold cultures on BHI agar were suspended in BHI broth
and inoculated into tubes containing 20 mL of BHI
broth followed by incubation with agitation at 35 C
for 48 h. Yeast grown in BHI broth was inoculated into
200 mL of YCG in 1-L Erlenmeyer flasks. These starter
cultures were incubated for 3 days with shaking (100
rpm, orbital shaker, New Brunswick Scientific, Edison,
NJ, USA). Yeast cells were centrifuged and transferred
to 3 L of YCG medium (in 4-L Erlenmeyer flasks) and
incubated at 35 C with shaking (as before) for 7 days.
The yeast cells were removed by filtration and antigen
preparation was stocked at 20 C until use.
2.4. Animal infection
Groups of six BALB/c mice were infected intraperitoneally with 5 · 106 yeast forms of S. schenckii suspended in
PBS (2.65 g Na2HPO4, 0.35 g NaH2PO4.H2O and 8.18 g
NaCl in 1 L of distilled water). The control group received
PBS only. Blood samples were collected individually at 7,
14, 21 and 28 days after infection and their serum was
stocked at 20 C until use.
2.5. Fungal loads in the organs of infected animals
On the 7th, 14th, 21st and 28th day after infection,
animals were sacrificed and the fungal load in organs (liver and spleen) was determined by measuring colony
forming units (CFU). Briefly, the organs were collected,
weighed and disrupted using a tissue grinder. The cell
suspensions were plated on BHI agar. Colonies were
counted from the 4th day until no fungal growth was detected. The results were expressed in CFU/g of tissue.
3. Elisa
An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) was performed on serum samples
by standard methods as previously described [15]. An
optimal concentration of the soluble Sporothrix antigen
(0.5 lg/mL) (prepared as above) was added to a microplate well (in 0.2 mL) (Polystyrene microtiter plates,
Costar). This concentration of Sporothrix protein was
determined by checkerboard titration of twofold dilutions of antigen and high-titered infected mouse serum.
The plates were incubated overnight at 4 C and washed
in phosphate-buffered saline (PBS) containing 0.05%
R.C. Nascimento, S.R. Almeida / FEMS Immunology and Medical Microbiology 43 (2005) 241–247
Tween 20 (Sigma, St. Louis, MO) (PBS-T). Nonspecific
binding sites were blocked with PBS containing 10%
FCS (fetal calf serum). The sera from infected mice were
diluted to 1:100 in PBS and incubated for 2 h at 25 C.
Control wells were coated with non-infected mouse serum. The plates were washed in PBS-T and a peroxidaseconjugated anti-mouse (Bio-Rad) Immunoglobulin G
was added to the wells, and the plates were incubated
for 2 h at 25 C followed by washing in PBS-T. The enzyme substrate (o-phenylenediamine-OPD) was added
to all wells, followed by a further incubation for 30
min. The reaction was stopped with H2SO4, and absorbance at 492 nm was determined using a Multiskan
MCC/340 II EIA reader.
4. SDS–PAGE
Antigen samples (containing 2 lg of protein) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) were dissolved under reducing conditions
in electrophoresis sample buffer (62.5 mM Tris, 2% SDS,
5% 2-mercaptoethanol, 10% glycerol, and 0.01% phenol
blue, pH 6.8) and boiled for 2 min. Following solubilisation, the antigen samples were electrophoresed on 10%
polyacrylamide gels in the presence of SDS as described
by Laemmli [16]. The soluble antigen fractions were
applied to a vertical slab gel apparatus (Bio-Rad) and
electrophoresed at 30 mA for 1 h. Gels were stained or
processed for immunoblotting.
243
glycine in 20% methanol) at 200 mA for 4 h. After elution, the protein was dialyzed, lyophilised and resuspended in PBS. Protein content was determined by the
Bradford method [17] and purification was monitored
by SDS–PAGE [16].
4.3. ELISA assays for isotyping antigen-specific antibody
production
The Mouse Typer – Isotyping panel was purchased
from Bio-Rad Laboratories. Serum was collected as described above, diluted 1:100 in PBS and stored for this
assay. Polystyrene microtiter plates (Costar) were coated
with purified protein (0.5 lg/mL) or soluble antigen (0.5
lg/mL) for 1 h at room temperature (RT) and were then
washed in PBS three times. The remaining binding sites
were blocked with PBS containing 1% of bovine serum
albumin (BSA) (Sigma) for 1 h at RT. Diluted sera
(1:100) were added and incubated for 1 h at RT. Horseradish peroxidase-conjugated rabbit anti-mouse Ig,
IgG1, IgG2a, IgG2b and IgG3 diluted 1:2000 were
added to wells and the plates were incubated for 1 h at
37 C. The reaction was developed with a substrate solution containing 3.7 mM o-phenylenediamine and 3.52
mM hydrogen peroxide (H2O2) in 0.05 M citrate buffer
(pH 5.0) and plates were kept in the dark at RT for 15
min. The reaction was stopped by adding 4 N sulfuric
acid to each well. Absorbance at 492 nm was determined
using a Multiskan MCC/340 II EIA reader.
4.4. Statistics
4.1. Immunoblotting
Proteins from SDS–PAGE were electrotransferred
onto nitrocellulose membranes using a transblotting
chamber (Bio-Rad) with 25 mM Tris, 192 mM glycine,
pH 8.3, and 20% methanol (v/v). For immunoblot
assays, the nitrocellulose membranes were saturated
with 5% skimmed milk, 0.3% Tween 20 in 10 mM phosphate buffer, pH 7.2, and 0.15 M NaCl (PBS-M-T
0.3%). The membranes were incubated for 2 h at 21
C with sera diluted 1/100 in PBS-M-T 0.1%, washed
four times for 10 min each in PBS-T 0.1%, and finally
incubated in peroxidase conjugated anti-mouse immunoglobulin (H + L) (Bio-Rad). After washing four times
for 10 min each with PBS-T-0.1%, the peroxidase activity was developed with 5 mg 3,3 0 -diaminobenzidine in 10
mL PBS and 40 mL H2O2 (30%). After the development
of colour, the blots were rinsed in distilled water.
Statistical comparisons were made by analysis of variance (ANOVA) and by Tukey–Kramer test. All values
were reported as means ± standard error of the mean
(SEM).
5. Results
5.1. Analysis of soluble antigen by SDS–PAGE
Components of the soluble antigen were analyzed on
a 10% gel. Fifteen protein-containing bands ranging
from 20 to 96 kDa (20, 30, 35, 38, 43, 45, 50, 53, 56,
58, 60, 67, 70, 85 and 96 kDa) were identified in the antigen preparation by protein staining of the SDS–PAGE
strip of this isolate (Fig. 1).
5.2. Fungal loads in organs of the infected animals
4.2. Electroelution of proteins from SDS–PAGE gels
The antigen was subjected to SDS–PAGE in 10% linear gels, and a protein of Mr 70 kDa was located in
gels after Coomassie blue staining and eluted by electrophoresis in transfer buffer (0.025 M Tris, 0.192 M
The evolution of the disease in BALB/c mice was
monitored by CFU counts in the liver and spleen at different periods following infection. As shown in Fig. 2,
the number of fungus cells in the spleen and liver was
high in the early part of the infection (7 days). However,
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Fig. 1. Profiles of S. schenckii antigen preparation separated by SDS–
PAGE on 10% of acrylamide, followed by silver staining. Numbers at
left indicate molecular-mass markers in kilodaltons.
Fig. 2. Time course of infection of mice inoculated intraperitoneally
with 5 · 106 yeast forms of S. schenckii. Each bar represents the mean
of six animals. Range bars represent standard errors of the means.
*p < 0.05 when compared with 7 days of infection.
on weeks 14–28, the number of CFUs in the spleen and
liver were found to be significantly reduced.
5.3. Immunoblot of sera from mice infected with
S. schenckii
Sera from infected mice obtained 7, 14, 21 and 28
days after infection were used to identify the antigenic
component of the soluble antigenic preparation. The
results show that sera from immunized mice had antibodies against the 70 kDa protein. This reaction became evident 14 days after infection. No reaction
with other bands was detected. As a negative control,
serum from non-immunized mice was used and no
reaction was observed (Fig. 3). After identification
by immunoblotting, the antigenic component was
purified using the electroelution technique. Fig. 4
shows the SDS–PAGE of the purified 70 kDa molecule after electroelution.
Fig. 3. Immunoblot analysis of IgG response in infected mice at 7
(lane 1), 14 (lane 2), 21 (lane 3) and 28 days (lane 4) with 5 · 106 yeast
forms of S. schenckii. Numbers at left indicate molecular-mass marker
in kilodaltons.
Fig. 4. SDS–PAGE of soluble antigenic components on 10% acrylamide (A) and purified 70 kDa protein after electroelution (B). The gels
were stained by silver staining. Numbers at left indicate molecularmass marker in kilodaltons.
5.4. Total Ig detection by ELISA in the sera of infected
mice
Antibodies specific to S. schenckii were determined as
total Ig in the sera of infected mice. An ELISA-plate was
sensitized with soluble antigen (Fig. 5(a)) or purified
70 kDa protein (Fig. 5(b)) and total Ig was determined.
The level of Ig was low 7 days after infection. However,
the level of Ig against soluble antigen or purified protein
increased significantly 14 days after infection and was
maintained high for at least 28 days following infection.
5.5. Isotyping of IgG
The IgG1 and IgG3 isotypes were produced at earlier
stages of infection (from day 14 onwards) against the
soluble antigen (Fig. 6) or purified protein (Fig. 7).
The IgG1 isotype was significantly higher than IgGa,
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IgGb and IgG3 when soluble antigen was used (Fig. 6).
On the other hand, both IgG1 and IgG3 were significantly higher that IgG2a and IgG2b when purified
70 kDa protein was tested (Fig. 7).
6. Discussion
Fig. 5. Total Ig levels, in the course of infection, against antigen
preparation (a) or purified 70 kDa protein (b) in mice infected
intraperitoneally with 5 · 106 yeast forms of S. schenckii. The
horizontal dotted lane denotes mean of serum of non-infected mice.
*p < 0.05 when compared with 7 days of infection.
The importance of humoral immunity has been documented in the host defence against infection by various
fungi including Cryptococcus neoformans, Candida albicans and Paracoccidioides brasiliensis. Few reports have
described the importance of humoral immunity in protecting against infection with S. schenckii. Some authors
have shown that Sporotrix-specific antibodies have little
effect on host resistance to the disease [10]. However, the
data are few and the precise mechanisms of protection
have yet to be elucidated. This study has clearly shown
the importance of the humoral immune response and
has determined an important antigenic component in
experimental sporotrichosis.
The progress of infection of BALB/c mice monitored
by organ culturing revealed that fungal loading increased in the first week following infection and then decreased drastically 14 days after infection. The relatively
higher fungal load observed in the organs at the initial
Fig. 6. Specific antibody levels (IgG1, IgG2a, IgG2b and IgG3) against soluble antigen preparation in mice infected intraperitoneally with 5 · 106
yeast forms of S. schenckii. Each bar represents the mean of serum of six animals. Range bars represent standard errors of the means. The horizontal
dotted lane denotes mean of serum of non-infected mice. *p < 0.05 when compared with IgG2a, IgG2b and IgG3 isotypes.
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Fig. 7. Specific antibody levels (IgG1, IgG2a, IgG2b and IgG3) against purified 70 kDa protein in mice infected intraperitoneally with 5 · 106 yeast
forms of S. schenckii. Each bar represents the mean of serum of six animals. Range bars represent standard errors of the means. The horizontal
dotted lane denotes mean of serum of non-infected mice. *p < 0.05 when compared with IgG2a and IgG2b isotypes.
phases of the disease demonstrated that resistance to
S. schenckii is not linked to a natural capacity of the
host to clear the pathogen load from the organs, but
the acquired immunity later developed by the host could
be the main cause for this event.
A specific protein produced by S. schenckii that contributes to the pathogenic potential of this fungus has
not yet been described. Then, we analysed by immunoblotting the antibodies produced by infected mice to soluble antigenic preparation during infection. Our results
show that the sera of all mice tested had antibodies
reacting only with the 70 kDa antigen. This reaction
starts 2 weeks after infection and persists until at least
28 days after infection. Sera from mice infected subcutaneously or intravenously were also used and the same results were observed. Scott and Muchmore [9] using
immunoblotting techniques showed that sera from patients with sporotrichosis reacted with antigens of 40
and 70 kDa. These authors showed that the reaction
was specific, since no reaction with healthy individuals
or patients with other fungal diseases was observed.
The ELISA technique was used to quantify total Ig.
Plates were sensitized with soluble antigen or the
70 kDa molecule purified by electroelution. The results
showed that the total Ig concentration was high 14 days
after infection for both antigens tested and that this titer
was maintained throughout the entire period of infection. It is important to highlight that the increase of Ig
against soluble antigen or purified protein could be related to the decrease in CFU count. These results
showed that the high level of antibody found could be
involved with the specific immune response protection
in mice, especially against the 70 kDa molecule. Isotyping showed the predominance of IgG1 and IgG3 in both
antigens tested. The IgG2a level increased in the course
of infection especially when soluble antigen was used to
sensitize the plate.
The role that the antibody plays in sporitrichosis is
unknown. Our results show the possible participation
of specific antibodies in controlling the infection. The
high level of IgG1 observed could be an important
mechanism for antigen neutralisation, since this isotype
is involved in opsonisation, enhancing the efficiency of
phagocytosis. We can thus speculate that the high level
of IgG1 and IgG3 against soluble antigens or the
70 kDa molecule could facilitate the neutralisation of
these components, increasing macrophage efficiency
and consequently decreasing CFUs in the organs.
It is not known whether specific proteins or enzymes
produced by S. schenckii contribute to the pathogenic
potential of this fungus. However, in studies involving
Candida infections in humans, Candida albicans poly-
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peptide profiles by immunoblot analysis have shown
that antibodies to certain polypeptides predominate in
patients with systemic candidiasis, while antibodies to
mannan appear to predominate in the sera of patients
with superficial infections [18]. Moreover, other investigators have postulated that extracellular proteinases
produced by various fungal organisms (Microsporum
spp., Trichophyton spp., Candida spp.) may be linked
to their virulence and pathogenesis [19–22]. Tsuboi
and co-workers [23,24] have reported the production
of extracellular proteinases by S. schenckii, but these enzymes have not yet been linked to the pathogenesis of
this disease. Recently, Carlos and co-workers [25] have
shown that a lipid compound of the cell wall plays an
important role in the pathogenesis of Sporotrichosis.
These authors showed that lipids from S. schenckii inhibit the phagocytic process and induce the abundant liberation of NO and TNF-a in macrophage cultures.
Taken together, our results show that antigens from
the S. schenckii induced and specific humoral responses
in infected mice. Further studies for the characterisation
of the 70 kDa molecule are currently being carried out in
our laboratory, the results of which will soon become
available.
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