Paracoccidioides brasiliensis: Attenuation of yeast cells by gamma irradiation

Abstract

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The aim of this study was to attenuate the yeast form of P. brasiliensis by gamma irradiation for further studies on vaccine research. Paracoccidioides brasiliensis (strain Pb 18) cultures were irradiated at doses between 0.5 and 8.0 kGy. After each dose the viability, reproductive ability and protein metabolism were evaluated. The comparison between the antigenic profile of irradiated and control yeast was made by Western blot and the virulence evaluated by the inoculation in C(57)Bl/J6 mice. At 6.5 kGy the yeast lost its reproductive capacity. The viability and the incorporation of [L-(35)S]-methionine were the same in control and up to 6.5 kGy irradiated cells, but 6.5 kGy-irradiated yeast secreted 40% less proteins. The Western blot profile was clearly similar in control and 6.5 kGy-irradiated yeast. No colony-forming unit (CFU) could be recovered from the tissues of the mice infected with the radioattenuated yeast. We concluded that for P. brasiliensis yeast it is possible to find a dose in which the pathogen loses its reproductive ability and virulence, while retaining its viability, metabolic activity and the antigenic profile.

Full text

Paracoccidioides brasiliensis: attenuation of yeast cells by
gamma irradiation
Marina Cortez Demicheli,
1
Bernardo Sgarbi Reis,
2
Alfredo Miranda Goes
2
and Antero Silva Ribeiro de
Andrade
1
1
Laborato
´rio de Radiobiologia, Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG and
2
Departamento de Bioquı´mica e
Imunologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil
Summary Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent
mycosis in Latin America, and currently there is no effective vaccine. The aim of this
study was to attenuate the yeast form of P. brasiliensis by gamma irradiation for further
studies on vaccine research. Paracoccidioides brasiliensis (strain Pb 18) cultures were
irradiated at doses between 0.5 and 8.0 kGy. After each dose the viability, reproductive
ability and protein metabolism were evaluated. The comparison between the antigenic
profile of irradiated and control yeast was made by Western blot and the virulence
evaluated by the inoculation in C
57
Bl/J6 mice. At 6.5 kGy the yeast lost its
reproductive capacity. The viability and the incorporation of [L-
35
S]-methionine
were the same in control and up to 6.5 kGy irradiated cells, but 6.5 kGy-irradiated
yeast secreted 40% less proteins. The Western blot profile was clearly similar in control
and 6.5 kGy-irradiated yeast. No colony-forming unit (CFU) could be recovered from
the tissues of the mice infected with the radioattenuated yeast. We concluded that for
P. brasiliensis yeast it is possible to find a dose in which the pathogen loses its
reproductive ability and virulence, while retaining its viability, metabolic activity and
the antigenic profile.
Key words: Paracoccidioides brasiliensis, gamma irradiation, attenuation, vaccine.
Introduction
Paracoccidioides brasiliensis is a thermally dimorphic
fungal agent of paracoccidioidomycosis, a deep-seated
systemic infection of humans. The disease has a high
incidence in Latin American countries, especially Brazil,
Venezuela, Colombia and Argentina. It is estimated that
approximately 10 million people are infected, although
the most of them do not show clinical symptoms.
1
Airborne fungal propagules are thought to initiate the
infection in the lungs after conversion to the yeast
phase. The infection can proceed either as a mild, self-
limited process, or be severe, eventually fatal, spreading
to other organs and tissues.
2
As other fungal pathogens, protective immunity
against P. brasiliensis has been correlated with an
exuberant cell-mediated immune response. Neither
vaccine nor protective immunogen has still been repor-
ted, although the major diagnostic antigen gp43 has
shown to be a promise as potential protective antigen.
3
Paracoccidioides brasiliensis antigens fractionated by
anion-exchange chromatography, termed F0 and FII,
have also been demonstrated to confer protection in
mice.
4
Ionising radiation has been successfully used to
attenuate parasites and micro-organisms for vaccine
development and research.
5–11
Irradiated pathogens
frequently lost their reproductive ability or virulence,
but retain the metabolic activities and morphology, and
are able to stimulate a specific immune response. In
Correspondence: Dr. Antero Silva Ribeiro de Andrade, Laborato
´rio de
Radiobiologia, Centro de Desenvolvimento da Tecnologia Nuclear (CDTN),
Rua Prof. Mario Werneck, s/n, Pampulha, Campus de UFMG, Caixa postal
941, CEP 30123-970, Belo Horizonte, MG, Brazil.
Tel: +55-31-3499 3182. Fax: +55-31-3499 3380.
E-mail: antero@cdtn.br
Accepted for publication 12 January 2006
Original article
184 2006 Blackwell Publishing Ltd •Mycoses,49, 184–189

some cases, the radioattenuated pathogens are more
immunogenic than the normal counterparts.
12
An
instructive example is the Schistosoma mansoni radi-
ation-attenuated cercariae vaccine, which stimulates a
level of immunity and protection that surpasses any
generated by purified or recombinant molecules.
5
Although this schistosome vaccine has not been tested
in humans, primarily for safety reasons, it has allowed
that the mechanisms of induction of the protective
immunity and the protective antigens were known.
13
The analogous sporozoite radiation-attenuated malaria
vaccine was tested in human volunteers and elicited
protective immunity.
14
The paradigm for a successful
radiation-attenuated vaccine is that employed for Dicty-
ocaulus viviparous, which causes parasitic bronchitis in
cattle. This vaccine has been used with widespread
success since its introduction in 1958.
15
Efforts were
also accomplished to develop irradiation-attenuated
vaccines against Toxoplasma gondii,
6
Leishmania major,
8
Eimeria maxima,
10
Fasciola hepatica,
11
among others.
In the present study, the effects of gamma irradiation
on the reproductive ability, metabolic activity, antigenic
profile and virulence of the yeast form of P. brasiliensis
were analysed. The purpose was to attenuate the
pathogen for further studies on immunity in the
paracoccidioidomycosis and vaccine research.
Material and methods
Culture conditions
Paracoccidioides brasiliensis, strain Pb 18, was main-
tained in the yeast form, at 35 C, in brain infusion agar
medium (BHIA) supplemented with 1% glucose. The
yeast cells were subcultured every 10 days.
Yeast cell counting
The cells were collected and diluted in a counting
solution (0.9% NaCl, 4% formaldehyde and 4% Tween-
20). The mixture was vigorously mixed up in order to
disperse the aggregated cells. The counting was per-
formed in a Neubauer chamber (BOECO, Hamburg,
Germany).
Gamma irradiation
Cultures of P. brasiliensis, in solid medium, were
irradiated at doses ranging from 0.5 to 8.0 kGy, in the
presence of oxygen and at room temperature. The
irradiation was performed in a uniform source of
60
Co
gamma rays, at dose rate of 950 Gy h
)1
. Adequate
controls were maintained outside the source. After each
dose the growth, viability and metabolic activity were
estimated.
Yeast growth analysis
Control and irradiated cells were transferred to phos-
phate-buffered saline (PBS) solution, vortexed and
counted. The suspensions, containing 10
2
to 10
7
colony-forming units (CFU), were spread on Petri dishes
with a high plating efficiency medium. The plates were
incubated at 35 C for 7 days and colonies counted
under a magnifying glass. The mean colony counting
was obtained from triplicate determinations.
Agar plates with high plating efficiency were prepared
adding 20 g of agar to 910 ml of distilled water. The
solution was autoclaved for 15 min at 121 C, and
cooled to 56 C. Then, 40 ml of fetal bovine serum and
50 ml of water extract of P. brasiliensis were added. The
water extract was obtained by culturing the strain Pb
18, at 35 C for 5 days, on BHIA supplemented with 1%
glucose. About 5 ml of the pelleted yeast cells were
suspended in 45 ml of distilled water and autoclaved.
The suspension was left to rest for 24 h at room
temperature and centrifuged at 1000 gfor 15 min. The
supernatant was used as the extract.
16
Yeast viability analysis
The viability was determined by using the modified vital
dye Janus green method
16
and the methylene blue
method.
17
In brief, for Janus green, 20 ll of 0.05%
solution of the dye was added to an equal volume of the
fungal cell suspension. The methylene blue test was
performed by adding 20 ll of methylene blue solution
(0.05 mmol l
)1
in PBS, pH 7.2) to the same volume of
fungal cells suspension. The suspensions were allowed to
interact with the dye for 10 min. The counting was
performed in a Neubauer chamber. Viable cells remained
unstained and dead cells stained blue for both methods.
Protein synthesis assay
Controls and irradiated yeast cells were transferred to
10 ml of liquid medium (brain heart infusion, BHI), at
an initial inoculum density of 1 ·10
6
cells ml
)1
.To
each sample were added 10 lCi of [L-
35
S]-methionine
and cells were incubated for 12 h at 35 C. The cells
were harvested, washed in PBS and disrupted using
glass beads. After the centrifugation at 10 000 g,
the protein concentration in the supernatant was
determined by the Lowry method. Then, a volume
Gamma irradiation of Paracoccidioides brasiliensis
2006 Blackwell Publishing Ltd •Mycoses,49, 184–189 185

containing 0.3 mg of protein was placed in scintillation
vials and 4.5 ml of scintillation fluid was added. Counts
were determined in a liquid scintillation analyzer (Insta-
Gel Plus, Perkin Elmer, Wellesley, MA, USA) and
expressed in counts per minute (cpm).
Protein secretion
The control and the irradiated yeast cells were transferred
to 10 ml of 0.65% yeast nitrogen-based medium (Sigma,
St Louis, MO, USA), supplemented with 2% glucose. The
initial inoculum density was 1 ·10
6
cells ml
)1
. After
4 days the cell densities were determined, the cultures
pelleted and the supernatants harvested. The superna-
tants were lyophilised and the resulting powders dis-
solved in 0.5 ml of 0.01 mol l
)1
borate buffer (pH 8.0).
These solutions were dialysed against the same buffer
using a 1000 Da cut-off membrane. After that, protein
concentrations were estimated by the Lowry method. The
total secreted protein was normalised by the growth
factor of each individual culture.
Western blotting
The controls and the 6.5 kGy-irradiated yeast (16 h
after irradiation) were disrupted using glass beads. After
the centrifugation at 10 000 g, the supernatants were
collected and 20 lg of each sample submitted to sodium
dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) in a 12.5% running gel, with subsequent
transfer to nitrocellulose membrane. The membrane
was incubated in 0.3% Tween-20 in PBS, for 1 h, and
then twice in 0.05% Tween-20 in PBS, for 30 min, and
stored (dried) until being used. Appropriately dilute sera,
from P. brasiliensis-infected patients, were incubated for
90 min at room temperature with the membrane. The
antibodies were detected by sequential incubation with
peroxidase-conjugated antibodies, for 1 h at room
temperature, and incubated with the staining solution
(0.6 mg ml
)1
4-cloro-1-naphtol and 0.03% hydrogen
peroxide in PBS). After development the membrane was
washed in distilled water and dried.
Virulence assay
C
57
Bl/J6 mice were inoculated by ocular artery with
50 ll of PBS containing 10
5
radioattenuated yeast cells.
Controls were inoculated with the same number of
viable virulent cells. Organ CFUs were determined after
1 month of infection in the lung, spleen and liver. The
organs were removed, weighed, homogenised and
washed three times in PBS by centrifugation. The final
suspensions in PBS were plated in the medium described
in Yeast growth analysis section. The plates were
incubated at 35 C and examined after 20 days. The
results were expressed as the number of P. brasiliensis
CFUs per gram of tissue per mouse in each experimental
group (n¼3).
Results
The reproductive ability of irradiated cells was monit-
ored by their capacity to form colonies in a high plating
efficiency solid medium. The survival curve was pre-
sented in Fig. 1. The number of surviving cells falls off
with increasing doses of radiation. A typical tailoring
effect, or a tail, was observed in the curve after 2.0 kGy.
This behaviour has been well documented with radi-
ation and other environmental stresses
18
and it repre-
sents a yeast population that exhibits higher radiation
resistance. A reduction of 5 log
10
cycles in the number
of colonies was achieved at 6.0 kGy and at 6.5 kGy, no
colonies could be recovered, even if large inoculum and
incubation time (40 days) were used. The 6.5 kGy-
irradiated yeast cells were also unable to grow in liquid
BHI and PYG (Bactopeptone, yeast extract, glucose)
media (data not shown).
The viability of irradiated cells was first evaluated
using the Janus green and methylene blue dyes (Fig. 1).
The vital dye exclusion test with Janus green is a very
popular method for the determination of cell viability of
fungi and useful for estimating cell viability of yeast
form of P. brasiliensis.
16
The analysis by this method
showed that up to 6.5 kGy the cells were viable,
retaining the membrane permeability control. A similar
result was obtained with methylene blue that evaluates
the status of the oxidative metabolism. Although
Figure 1 Effect of gamma irradiation on survival and viability of
yeast cells of Paraccocicioides brasileinsis. Yeast cells were irradiated
with increasing doses of external gamma radiation and the fraction
of surviving cells determined ( ). The viability was measured by
Janus green ( ) and methylene blue (d). The bars represent the
standard deviations of triplicate determinations.
M. C. Demicheli et al.
186 2006 Blackwell Publishing Ltd •Mycoses,49, 184–189

unreliable when used with P. brasiliensis, the methylene
blue test can be used for crude viability estimation. A
good agreement was obtained by the two methods. The
irradiated yeast cells remain viable for 3 weeks (data not
shown).
The protein synthesis was analysed by the incorpor-
ation of [L-
35
S]-methionine (Fig. 2). The results showed
that the yeast cells, irradiated up to 6.5 kGy, main-
tained a similar synthetic protein metabolism level than
controls. The protein secretion was another metabolic
parameter evaluated (Fig. 3), as proteins secreted by
P. brasiliensis have important roles in the establishment
of the infection, in the stimulation of the immune
system and include some virulent factors.
19, 20
A
decrease of 40% in the protein secretion occurred at
6.5 kGy.
The comparison between the antigens recognised by
the sera from patients with paracoccidioidomycosis, in
6.5 kGy-irradiated yeast and control yeast, was realised
by Western blot (Fig. 4). The profile was clearly similar
in both cases, indicating that the irradiated yeast
retained the expression of the same antigens that are
present in the wild yeast.
The 6.5 kGy gamma-irradiated yeast of P. brasiliensis
fails in producing infection in C
57
Bl/J6 mice. Table 1
shows the number of CFUs recovered by each organ
from mice infected with the irradiated yeast in relation
to controls. No CFU could be recovered from mice
Figure 2 Evaluation of protein synthesis after gamma irradiation.
Yeast cells were irradiated with increasing doses of external
gamma radiation and the metabolism of protein synthesis monit-
ored by the incorporation of [L-
35
S]-methionine. The negative
control (C)) was cells killed by heating. The positive control (C+)
was no irradiated cells. The values represent the median and
standard deviations of three independent experiments.
Figure 3 Gamma irradiation effect on protein secretion. The neg-
ative control (C)) was cells killed by heating. The positive control
(C+) was no irradiated cells. The values represent the median and
standard deviations of three independent experiments.
Figure 4 Comparison between antigenic profiles of irradiated and
control yeast by Western blot. Sera from Paracoccidioides brasilien-
sis-infected patients were used. P, molecular weight standards;
1, control yeast; 2, 6.5 kGy-irradiated yeast. The same protein
bands were recognised in control and 6.5 kGy-irradiated yeast.
A 12.5% acrylamide gel was used for electrophoresis protein
separation.
Table 1 Recovery of CFUs from mice infected with the
radioattenuated yeast of Paracoccidioides brasiliensis.
Organ Control yeast (CFU
1
) Radioattenuated yeast (CFU
1
)
Lung 4750 (±1041) 0
Spleen 417 (±158) 0
Liver 123 (±44) 0
1
Colony-forming units (CFUs) per gram of tissue per mouse
(n¼3).
Gamma irradiation of Paracoccidioides brasiliensis
2006 Blackwell Publishing Ltd •Mycoses,49, 184–189 187

infected with the radioattenuated yeast, indicating that
the pathogen was attenuated.
Discussion
Paracoccidioides brasiliensis yeast cells, like many fungi,
exhibited high radioresistance relative to other micro-
organisms and mammalian cells.
21
A dose of several
thousand grays was required to kill, in the reproductive
sense, the yeast cells whereas the lethal dose for
mammalian cells is only a few grays. The mechanisms
responsible for these differences are not well understood,
but could involve more efficient mechanisms of DNA
repair of the damage caused by gamma rays. The
inability of 6.5 kGy-irradiated cells to divide results from
unrepaired double DNA breaks, leading to a cell cycle
arrest or unbalanced chromatin exchange in daughter
cells and consequent mitotic death.
22, 23
However, these
lesions do not impair the protein synthesis, likely
because most of the breaks occur outside operons,
usually a minor part of the genome, allowing adequate
functions of the genes until cell division. The lost of the
reproductive ability seems irreversible. No colonies were
observed in the plating experiment, despite of long
incubation times, and no CFU could be recovered from
infected mice. However, P. brasiliensis has the ability to
initiate the infection after prolonged period of dor-
mancy. For example, it is not uncommon for patients to
fall ill more than a decade after leaving an endemic
region.
1
As a consequence, the hypothesis that the
radioattenuated yeast may, after long times, recover the
cell division capacity cannot be excluded. This possibil-
ity could limit the utilisation of a radioattenuated yeast
vaccine in humans and confine its utilisation for
experimental paracoccidioidomycosis on animal models.
Among the metabolic parameters evaluated, only
protein secretion was changed with the gamma irradi-
ation dose that eliminated cell division. Now, we are
evaluating if the secretion of antigens and virulent
factors was negatively affected.
The 6.5 kGy dose do not disrupt the expression of the
known antigens of P. brasiliensis. This is a very
favourable result that indicates that the radioattenuated
yeast is able to stimulate the host immune system at
least as the native yeast. So, the irradiated yeast could
be a valuable tool for the evaluation if the interaction of
multiple antigens is necessary for an effective immune
response.
We concluded that for the yeast cells of P. brasiliensis,
it is possible to find an absorbed dose (6.5 kGy) in which
the pathogen loses its reproductive ability and virulence,
while retaining its viability, metabolic activity and
antigenic profile. This behaviour allows the immune
system to recognise the irradiated yeast as a viable
agent without risk of progressive infection. The radio-
attenuated yeast provides a novel tool for immunologi-
cal studies in experimental paracoccidioidomycosis and
vaccine research, because ÔliveÕvaccines can expose
their antigens sequentially to the host, as in a natural
infection, avoiding problems associated with single
immunisation schedules. Currently, there are no
approved vaccines for the prevention or treatment of
medically important fungi infection in humans. This
failure determines the need to explore new alternatives
and there are no studies using radioattenuated fungi for
this purpose. Now, we are evaluating the capacity of the
radioattenuated yeast to stimulate a protective immu-
nity against a P. brasiliensis infection.
Acknowledgments
This research was supported by Centro de Desenvolvi-
mento da Tecnologia Nuclear/Comissa˜o Nacional de
Energia Nuclear (CDTN/CNEN). The authors gratefully
thank the support of Mr Ricardo Ferracini Corre
ˆa from
the Gamma Irradiation Laboratory of CDTN/CNEN.
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