Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction.

Abstract

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.

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RESE AR CH LET TER
BiochemicalpropertiesofCandidaparapsilosisecto-
50-nucleotidaseandthepossibleroleofadenosinein
macrophageinteraction
Thais Russo-Abraha ˜o1,2, Daniela Cosentino-Gomes1,2, Marta T. Gomes1,2,3, Daniela S. Alviano3,
Celuta S. Alviano3, Angela H. Lopes3& Jos´ e Roberto Meyer-Fernandes1,2
1Laborat´ orio de Bioqu´ ımica Celular, Instituto de Bioqu´ ımica M´ edica, Centro de Cie ˆncias da Sau ´de, Universidade Federal do Rio de Janeiro, UFRJ,
Cidade Universit´ aria, Rio de Janeiro, RJ, Brazil;2Instituto Nacional de Cie ˆncia e Tecnologia de Biologia Estrutural e Bioimagem (INCTBEB), CCS,
Cidade Universit´ aria, Rio de Janeiro, RJ, Brazil; and3Instituto de Microbiologia Professor Paulo de G´ oes, Universidade Federal do Rio de Janeiro,
CCS, Cidade Universit´ aria, Rio de Janeiro, RJ, Brazil
Correspondence: Jos´ e Roberto Meyer-
Fernandes, Laborat´ orio deBioqu´ ımica Celular,
Instituto de Bioqu´ ımica M´ edica, Centro de
Cie ˆncias da Sau ´de, Universidade Federal do
Rio de Janeiro, UFRJ, Cidade Universit´ aria, Ilha
do Funda ˜o, 21941-590, Rio de Janeiro, RJ,
Brazil. Tel.: 15521 25626781; fax: 15521
22708647; e-mail: meyer@bioqmed.ufrj.br
Received 29 November 2010; revised 21
December 2010; accepted 21 December 2010.
Final version published online 1 February 2011.
DOI:10.1111/j.1574-6968.2011.02216.x
Editor: Albert Descoteaux
Keywords
Candida parapsilosis; ecto-50-nucleotidase;
CD73; adenosine acquisition; host–yeast
interaction.
Abstract
Candida parapsilosis is considered to be an emerging fungal pathogen because it is
associated with an increasing range of infections. In this work, we biochemically
characterized ecto-50-nucleotidase activity on the surface of living, intact C.
parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 50-AMP at a
rate of 52.44?7.01nmolPih?110?7cells. 50-AMP, 50-IMP and 50-UMP were
hydrolyzed at similar rates, whereas 50-GMP and 50-CMP hydrolyzed at lower
rates. Enzyme activity was increased by about 42% with addition of Mg21or Ca21,
and the optimum pH was in the acidic range. An inhibitor of phosphatase
activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however,
as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited
the activity in a dose-dependent manner. The results indicated that the existence of
an ecto-50-nucleotidase could play a role in the control of extracellular nucleotide
concentrations.
Introduction
Candida parapsilosis is considered to be an emerging fungal
pathogen because it is associated with an increasing range of
infections, such as fungemia, vaginitis, endocarditis, en-
dophthalmitis, septic arthritis and peritonitis (Weems,
1992; Trofa et al., 2008; Nosek et al., 2009). Candida
parapsilosis is one of the only species of pathogens to show
increasing prevalence in recent years, and the association
between C. parapsilosis infections and the presence of
intravascular devices is well documented (Krcmery &
Barnes, 2002). The mechanisms by which C. parapsilosis
evades host defenses and colonizes host tissues are poorly
understood. The search for new structures representing
virulence factors, which will enhance our understanding of
the process of Candida infections, is therefore extremely
important (Kiffer-Moreira et al., 2007a).
Candida parapsilosis is the second most common yeast
isolated from bloodstream infections around the world.
Molecule studies have provided evidence of three distinct
species within the C. parapsilosis complex, namely C. para-
psilosis, Candida orthopsilosis and Candida metapsilosis (Orsi
et al., 2010). Little is known about its pathogenesis, viru-
lence factors and ability to survive in diverse hostile envir-
onments. Consequently, it is extremely important to
understand the means that enable this opportunistic patho-
gen to survive (Haynes, 2001).
Extracellular nucleotides have been recognized for over a
decade as some of the most ubiquitous intercellular signal-
ing mechanisms (Robson et al., 2006). Moreover, these
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molecules have been shown to be related to the development
of several pathologies, including disorders of the immune
system (Hask´ o & Cronstein, 2004; Schetinger et al., 2007;
Bhardwaj & Skelly, 2009). High extracellular concentrations
of ATP may occur in response to tissue or cell damage
(Bours et al., 2006; Idzko et al., 2007). Numerous works
explain that the high ATP concentration is due to a proin-
flammatory response, which involves activation and trans-
migration of monocytes and leukocytes to inflamed sites
(Bours et al., 2006; Di Virgilio, 2007; Schetinger et al., 2007).
The signaling mechanism generated by ATP can be reverted
through the action of a set of enzymes, known as ectoen-
zymes, which are involved in the control of extracellular
nucleotide and nucleoside levels. Because the active sites of
ectoenzymes face the external medium rather than the
cytoplasm, the activities of these enzymes can be measured
using living cells (Zimmermann, 1996; Meyer-Fernandes,
2002; Sissons et al., 2004; Bours et al., 2006; Matin & Khan,
2008; Amazonas et al., 2009; Cosentino-Gomes et al., 2009;
Fonseca-de-Souza et al., 2009).
The extracellular hydrolysis of ATP can be initiated by
NTPDases (ectonucleoside triphosphate diphosphohydro-
lases) and terminated by ecto-50-nucleotidases (CD73; E.C.
3.1.3.5), resulting in its respective nucleoside adenosine
(Zimmermann, 1996, 2000; Meyer-Fernandes, 2002; Robson
et al., 2006). Ecto-50-nucleotidase is the major enzyme
responsible for the formation of extracellular adenosine
from released adenine nucleotides (Zimmermann, 2000).
Adenosine, in contrast to ATP, is described as a chemotactic
inhibitor of macrophage response and monocyte response,
suppressing proinflammatory cytokines by activating P1
receptors in the host cells, thus interfering with the estab-
lishment of an immune response. (Hask´ o & Cronstein,
2004; Bours et al., 2006; de Almeida Marques-da-Silva
et al., 2008; Kumar & Sharma, 2009). In addition, this same
adenosine can be used by pathogens to avoid various acute
or chronic inflammatory conditions, decreasing the phago-
cytic activity of macrophages (Bhardwaj & Skelly, 2009;
Thammavongsa et al., 2009).
The importance of ecto-50-nucleotidase activity and ex-
tracellular adenosine production in escaping host immune
defenses has been observed in Staphylococcus aureus (Tham-
mavongsa etal., 2009) andSchistosoma mansoni, the parasite
of schistosomiasis (Bhardwaj & Skelly, 2009). Ecto-50-
nucleotidase activities were also observed in some protozoan
parasites, such as Trichomonas gallinae (Borges et al., 2007)
and Trichomonas vaginalis (Tasca et al., 2003), showing that
ecto-50-nucleotidase could play a role in salvaging purines
from the extracellular medium. Furthermore, ectoenzymes
on the cell surface of trichomonads are shown to play a
major role in cytoadhesion, host–parasite interaction, nu-
trient acquisition and protection from cytolytic effects
(Petrin et al., 1998; Tasca et al., 2003).
Recently, our group described an ecto-ATPase activity
present on the surface of C. parapsilosis (Kiffer-Moreira
et al., 2010). This enzyme participates in the interaction
between yeast and epithelial cells and can be considered a
pathogenic marker. Additionally, a sequential dephosphor-
ylation of ATP to adenosine (ATP ! ADP ! AMP !
adenosine) was observed through reverse-phase HPLC
experiments in intact C. parapsilosis cells, indicating the
participation of different ectonucleotidases activities (ecto-
ATPase, ecto-ADPase and ecto-50nucleotidase). Little infor-
mation is available about ecto-50-nucleotidase in fungi. To
further investigate the possible involvement of ecto-50-
nucleotidase activity in C. parapsilosis adenosine produc-
tion, we characterized an ecto-50-nucleotidase activity on
the surface of living, intact C. parapsilosis cells.
Materials and methods
Materials
All reagents were purchased from Merck (Darmstadt, Ger-
many) or Sigma Chemical Co. (St. Louis, MO). Water used
in the preparation of all solutions was filtered through a
four-stage Milli-Q system (Millipore Corp., Bedford, MA).
Cell culture
Candida parapsilosis strain CCT 3834 (ATCC 22019) was
obtained from the Departamento de Patologia Cl´ ınica,
Universidade Estadual de Campinas, Sa ˜o Paulo, Brazil.
Stock cultures were maintained on solid brain–heart infu-
sion at 371C. For measurements of enzyme activity, C.
parapsilosis were cultivated for 48h at room temperature
with continuous shaking (Milani et al., 2001) in a complex
medium containing glycerol (2%, v/v), peptone (2%, w/v;
Bacto peptone; Becton Dickinson Labware, NJ) and yeast
extract (1%, w/v). Yeast cells were obtained by centrifuga-
tion and washed twice in a solution containing 116mM
NaCl, 5.4mM KCl, 5.5mM D-glucose and 10mM MES–
Hepes–Tris buffer (pH 7.2). Cell growth was estimated by
counting the number of yeast cells in a Neubauer chamber.
Cellular viability was assessed, before and after incubations,
by Trypan blue dye exclusion (Kiffer-Moreira et al., 2007b).
The viability was not affected under the conditions used
here.
Ecto-50-nucleotidase activity measurements
Ecto-50-nucleotidase activity was determined by the rate
of inorganic phosphate (Pi) released. Intact cells were
incubated for 30min at 251C in 0.2mL of reaction mixture
containing, unless otherwise specified, 116mM NaCl,
5.4mM KCl, 5.5mM D-glucose, 50mM MES–Hepes–Tris
buffer (pH 4.5), 5.0mM 50-AMP as substrate and
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Candida parapsilosis ecto-50-nucleotidase activity

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2.5?109cellsmL?1. The reaction was initiated by the addi-
tion of cells and stopped by the addition of 0.4mL of ice-
cold 25% charcoal in 0.1M HCl. This charcoal suspension
was washed at least 20 times with 0.1M HCl before use to
avoid Pi contamination (Guilherme et al., 1991). Controlsin
which cells were added after interruption of the reaction
were used as blanks. After the reaction, the tubes were
centrifuged at 1500g for 15min at 41C, and 0.1mL of the
supernatant was added to 0.1mL of Fiske Subbarow reactive
mixture (Fiske & Subbarow, 1925). The absorbance of the
released Pi was measured spectrophotometrically at 660nm.
The ecto-50-nucleotidase activity was calculated by subtract-
ing the nonspecific 50-AMP hydrolysis measured in the
absence of cells. The concentration of Pi released in the
reaction was determined using a comparison with a stan-
dard curve of Pi. The AMP hydrolysis was linear with time
under the assayconditions used and was proportional to cell
number. We also measured the hydrolysis of other nucleo-
side monophosphates, using 50-CMP, 50-IMP, 50-GMP,
50-UMPor 30-AMP as substrates under the same conditions
described above. In experiments in which high concentra-
tions of Mn21, Ca21and Sr21were tested, the possible
formation of precipitates was checked as described pre-
viously (Meyer-Fernandes & Vieyra, 1988). In the reaction
media containing 50mM MES–Hepes–Tris (pH 4.5),
116mM NaCl, 5.4mM KCl, 5.5mM D-glucose and 5mM
AMP, no phosphate precipitates were observed in the
presence of these cations under the conditions used.
Ectophosphatase activity measurements
Phosphatase activity was quantified by the release of Pi
(Fonseca-de-Souza et al., 2008) after the addition of the
substrate p-nitrophenyl phosphate. The quantification of
released Pi was carried out in the same way as described
above for determining ecto-50-nucleotidase activity.
Inhibition assay
Ecto-50-nucleotidase activity in living C. parapsilosis was
analyzed with a specific inhibitor of nucleotidases (ammo-
nium molybdate). We also tested a phosphatase inhibitor,
sodium orthovanadate, to rule out the possibility of an
ectophosphatase activity on AMP hydrolysis.
Interaction of C. parapsilosis with
peritoneal macrophage
Resident peritoneal macrophages from female BALB/c mice
were collected in 0.9% saline and plated onto glass coverslips
in 24-well tissue culture plates (Falcon; Becton Dickinson
Labware). The cells were allowed to adhere for 30min at
371C in a 5% CO2atmosphere, after which the nonadhering
cells were removed and RPMI 1640 culture medium supple-
mented with 2mM L-proline, 25mM Hepes and 10% fetal
bovine serum. Adhered cells (1?105macrophages) were
then incubated overnight under the same conditions as
above before the interaction assays. Peritoneal mouse
macrophages used in this investigation were obtained fol-
lowing the guidelines for animal experimentation of the
National Institutes of Health. The experimental protocol
was approved by the Centro de Cie ˆncias da Sau ´de (Uni-
versidade Federal do Rio de Janeiro) ethical committee for
animal experimentation.
Macrophages (1?105cells) and C. parapsilosis (1?106
cells) were left in contact for 1h at 371C in a macropha-
ge–yeast ratio of 1:10 in RPMI 1640 medium pH 8.0, with
the addition or not of adenosine or 50-AMP as indicated in
the legends. Coverslips were collected after this time, rinsed
in phosphate-buffered saline, fixed in Bouin’s fixative and
stained with Giemsa. The percentages of infected macro-
phages were determined by counting 200 cells on triplicate
coverslips of each preparation; each experiment was re-
peated at least three times. The association index between
C. parapsilosis and macrophage cells was determined using a
microscope at a magnification of ?1000 (Zeiss Axioplan 2,
Germany). Representative images were taken at a magnifica-
tion of ?400. The interaction between C. parapsilosis and
macrophages was considered as the percentage of infected
macrophages, as well as the mean number of yeast cells per
macrophage.
Statistical analysis
All experiments were performed in triplicate, with similar
results obtained in at least three separate cell suspensions.
Statistical significance for enzymatic assays was determined
by a t-test. For interaction, one-way ANOVA and Tukey post-
test were applied. P-values o0.05 were considered statisti-
cally significant.
Results
Ecto-50-nucleotidase activity
The time course of ecto-50-nucleotidase activity on the C.
parapsilosis surface was linear for 1h and directly propor-
tional to the number of cells (data not shown). At a pH of
4.5, intact cells were able to hydrolyze 50-AMP at a rate of
52.44?7.01nmolPih?110?7cells.
To confirm the ectolocalization of C. parapsilosis ecto-50-
nucleotidase activity and to rule out the possibility that the
observed 50-AMP hydrolysis was the result of secreted
soluble enzymes, a reaction mixture with cells was prepared
and incubated in the absence of the substrate 50-AMP.
Subsequently, the suspension was centrifuged to remove the
cells, and the supernatant was checked for nucleotidase
activity. The rate of 50-AMP hydrolysis observed from
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T. Russo-Abraha ˜o et al.

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the supernatant was o20% of that observed in intact cells
(Fig. 1).
Substrate specificity of C. parapsilosis
ecto-50-nucleotidase
In different cells, 50-AMP is the substrate hydrolyzed by 50-
nucleotidases at the highest rates (Zimmermann, 1992;
Hunsucker et al., 2005; Str¨ ater, 2006). Intact cells of C.
parapsilosis were able to hydrolyze all substrate monopho-
sphates tested (UMP, IMP, CMP and GMP), except 30-AMP.
50-UMP and 50-IMP were hydrolyzed at similar rates to that
of AMP, whereas 50-GMPand 50-CMP presented lower rates
of hydrolysis (Fig. 2).
Influence of divalent cations and pH on ecto-50-
nucleotidase activity
Although ecto-50-nucleotidase activity is independent of
cations (Zimmermann, 1992; Hunsucker et al., 2005), it
can be modulated by the addition of Mg21, Mn21or Ca21
(Tasca et al., 2003; Borges et al., 2007). We observed that
Mn21, Sr21and the metal chelators, EGTA and EDTA, had
no effect on the ecto-50-nucleotidase activity. However, both
Mg21and Ca21increased 50-AMP hydrolysis by about 42%
(Fig. 3a). The optimum pH for C. parapsilosis ecto-50-
nucleotidase activity was in the acidic range, with its
maximum activity at a pH of 4.5. The enzyme activity
decreased with increases in pH (Fig. 3b). In the pH range
between 4.5 and 8.5, the rate of 50-AMP hydrolysis observed
from the supernatant was o15–20% of those observed in
intact cells (data not shown).
Influence of inhibitors on ecto-50-
nucleotidase activity
In addition to the existence of ecto-ATPase activity (Kiffer-
Moreira et al., 2010) on the surface of C. parapsilosis, our
group has described the presence of a membrane-bound
acid phosphatase activity (Kiffer-Moreira et al., 2007a),
which could contribute to AMP hydrolysis. To rule out
the influence of acid phosphatase on AMP hydrolysis, we
evaluated the influence of a well-known inhibitor of phos-
phatase activities, sodium orthovanadate (de Almeida-
Amaral et al., 2006; Kiffer-Moreira et al., 2007a; Amazonas
et al., 2009; Dick et al., 2010). As shown in Fig. 4a, different
concentrations of sodium orthovanadate (0.1 and 1.0mM)
inhibited ectophosphatase activity. Nevertheless, as ex-
pected, it did not have an effect on C. parapsilosis ecto-50-
nucleotidase activity (Fig. 4b). On the other hand,
ammonium molybdate, a classical 50-nucleotidase inhibitor
(Gottlieb & Dwyer, 1983; Borges et al., 2007), inhibited ecto-
50-nucleotidase in a dose-dependent manner, with maximal
inhibition at a concentration of 0.5mM (Fig. 5).
Influence of adenosine in C. parapsilosis and
macrophage interaction
Adenosine has been implicated in many aspects to contri-
bute for pathogens escaping from host immune responses
(Bhardwaj & Skelly, 2009; Thammavongsa et al., 2009). To
verify whether adenosine and 50-AMP would prevent
macrophage to phagocyte C. parapsilosis, we perform an
in vitro interaction with peritoneal macrophage and C.
parapsilosis in the presence of a low concentration of
Fig. 1. Ecto-50-nucleotidase activity in intact cells of Candida parapsilo-
sis. Intact cells were incubated for 30min at 251C in the same reaction
medium as that described in Materials and methods, without the
substrate 50-AMP. After 30min, the cells were centrifuged and an aliquot
of cells and supernatant were assayed for 50-nucleotidase activity. The
data shown indicate the mean of enzyme activity?SE of at least three
determinations, each with different cell suspensions.
Fig. 2. Substrate specificity for ectonucleotidase activity of Candida
parapsilosis. Intact cells were incubated for 30min at 251C in the same
reaction medium as that described in Materials and methods, with
5.0mM of one of the following substrates: 50-AMP, 50-CMP, 50-IMP, 50-
GMP, 50-UMP or 30-AMP. The data shown indicate the mean of enzyme
activity?SE of at least three experiments, each with different cell
suspensions.
FEMS Microbiol Lett 317 (2011) 34–42
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Candida parapsilosis ecto-50-nucleotidase activity

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adenosine and 50-AMP (100mM). As can be seen in Fig. 6a
and b, the addition of adenosine to the interaction medium
showed a significant reduction in the percentage of infected
macrophages, whereas 50-AMP at the same concentration
did nothave an effect, comparing withcontrol.Interestingly,
the addition of 50-AMP, at 1mM, caused a decrease in the
percentage of infected macrophages (Fig. 6a and b), indicat-
ing that C. parapsilosis ecto-50-nucleotidase could have a
role in generating extracellular adenosine, to further mod-
ulate the macrophage response. On the other hand, no
significant differences were observed in the mean number
of yeasts per infected macrophage among all system tested
(Fig. 6c). In this condition in the presence of 1mM AMP, C.
parapsilosis produced 1.52?0.07nmolPih?110?6cells from
AMP hydrolysis. In the same condition, macrophages
were also able to promote AMP hydrolysis producing
1.04?0.13nmolPih?110?5cells. These data confirmed
previous data from Edelson & Cohn, 1976a,b) showing the
presence of an ecto-50-nucleotidase activity on the external
surface of macrophages and indicated that during the
interaction assays, macrophages could be also responsible
for adenosine generation contributing to a reduction in the
percentage of infected macrophages.
Discussion
50-Nucleotidase activity hasbeen described in bacteria, plant
cells and in various vertebrate tissues (Zimmermann, 1992).
Little information is available about ecto-50-nucleotidase
and extracellular free adenosine in the pathogenic processes
of fungi. In this work, we identified some biochemical
properties of C. parapsilosis ecto-50-nucleotidase that could
be involved in the release of free adenosine into extracellular
medium. The detection of cell surface-located AMP
Fig. 3. Effect of different divalent cations and pH on ecto-50-nucleotidase activity of Candida parapsilosis. (a) Intact cells were incubated for 30min at
251C in the same reaction medium as that described in the Materials and methods, with 5.0mM of 50-AMP as the substrate and one of the following
divalent cations: 5.0mM MgCl2, 5.0mM CaCl2, 5.0mM MnCl2, 5.0mM SrCl2or, in the absence of any divalent cations, 1.0mM EDTA or EGTA. (b)
Intact cells were incubated in reaction medium containing 116mM NaCl, 5.4mM KCl, 5.5mM D-glucose, 5.0mM 50-AMP and 10.0mM
MES–Hepes–Tris buffer, adjusted with HCl and NaOH to obtain pH values from 4.5 to 9.0. In this pH range, cells were viable throughout the course of
the reaction. It was not possible to observe maximal cellular viability below or above this pH range. The data shown indicate the mean of enzyme
activity?SE of at least three experiments, each with different cell suspensions. Significant difference (Po0.05) after comparison with the control is
indicated by an asterisk.
Fig. 4. Effect of sodium orthovanadate on ectophosphatase and ecto-50-nucleotidase activities of Candida parapsilosis. Intact cells were incubated for
30min at 251C in the same reaction medium as that described in the Materials and methods, with 5.0mM of 50-AMP as substrate and 0.1 or 1mM of
sodium orthovanadate. Ectophosphatase activity was measuredafter the addition of 5mM p-nitrophenylphosphate. The data shownindicate the mean
of enzyme activity?SE of at least three experiments, each with different cell suspensions. Significant difference (Po0.05) after comparison with the
control is indicated by an asterisk. The absolute values for ectophosphatase and ecto-50-nucleotidase activities were considered 10.03?0.54 and
52.44?7.01nmolPih?110?7cells, respectively.
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hydrolysis was confirmed and 50-nucleotidase activity in
supernatant was o20% of that found in intact cells (Fig. 1).
In all conditions used during the incubation periods, the
cells were viable, suggesting that the low 50-AMP hydrolysis
observed in the supernatant could be attributed to secreted
enzymes. A phosphatase inhibitor, sodium orthovanadate
(de Almeida-Amaral et al., 2006; Kiffer-Moreira et al.,
2007a; Leite et al., 2007; Amazonas et al., 2009), inhibited
ectophosphatase on the surface of C. parapsilosis; however,
no inhibitory effect was seen in the ecto-50-nucleotidase
activity (Fig. 4). The optimum pH for this nucleotidase
enzyme is in the acidic range, with maximal activity at a pH
of 4.5 (Fig. 3b). Interestingly, this result is different from
that observed in T. vaginalis strains, in which the optimum
pH was in the neutral range (Tasca et al., 2003), and in
mammalian ecto-50-nucleotidase, in which maximal en-
zyme activity was obtained in the alkaline pH range of 7–8
(Zimmermann, 1992). This assay also rules out the possibi-
lity of 50-AMP hydrolysis due to the action of ecto-ATPase
because the activity of ecto-ATPase is primarily in the
alkaline pH range (Kiffer-Moreira et al., 2010).
Candida parapsilosis ecto-50-nucleotidase activity is in-
dependent of divalent cations, but it can be activated by
Ca21and Mg21(Fig. 3a). These same characteristics were
observed for 50-nucleotidase activity in T. vaginalis (Tasca
et al., 2003). The enzyme also showed a high sensitivity to
ammonium molybdate, a classical nucleotidase inhibitor
(Gottlieb & Dwyer, 1983; Borges et al., 2007), in which
concentrations above 0.5mM abolished the enzyme activity
altogether (Fig. 5).
Intact cells of C. parapsilosis were able to hydrolyze all
substrate monophosphates, except 30-AMP. As described in
other cells, 50-nucleotidases hydrolyze exclusively nucleoside
50-monophosphates, showing no activity for 30-monopho-
sphates. 50-AMP is commonly known as the most hydrolyz-
able nucleotide by 50-nucleotidase (Zimmermann, 1992,
1996; Borges et al., 2007). Nevertheless, C. parapsilosis ecto-
50-nucleotidase activity seems to exhibit no significant
difference in hydrolyzing 50-AMP, 50-UMP and 50-IMP as
substrates (Fig. 2). The presence of 50-nucleotidase activity
in plasma membranes of protoplasts from the yeast and
Fig. 5. Effect of ammonium molybdate on ecto-50-nucleotidase activity
of Candida parapsilosis. Intact cells were incubated for 30min at 251C in
the same reaction medium as that described in the Materials and
methods, with 5.0mM of 50-AMP as the substrate and increasing
concentrations of ammonium molybdate. The data shown indicate the
mean of enzyme activity?SE of at least three experiments, each with
different cell suspensions. The absolute value for ecto-50-nucleotidase
activity was considered 52.44?7.01nmolPih?110?7cells.
Fig. 6. Interaction of Candida parapsilosis with peritoneal mouse macrophages is influenced by adenosine and 50-AMP. Resident peritoneal
macrophages from female BALB/c mice were collected as described in the Materials and methods. (a) Giemsa staining of C. parapsilosis interaction
with macrophage(10:1,yeasts per macrophageratio), for 1hat 371C, in the absence or presenceof 100mM adenosine, 100mM 50-AMP or 1.0mM 50-
AMP. Magnification: ?400. (b) Percentage of infected macrophages, (c) mean number of yeast cells per macrophage. Data are mean?SE of three
determinations with different cell suspensions. Asterisks denote a significant difference (Po0.05) in relation to the control system.
FEMS Microbiol Lett 317 (2011) 34–42
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mycelia forms of Candida albicans has been reported by
Marriot (1975). A search for nucleotidase proteins in NCBI
database at Candida genus resulted for crystal structures of
50-nucleotidase from C. albicans, pyrimidine 50-nucleoti-
dases and IMP-specific 50-nucleotidases. In C. parapsilosis,
although the genome has been sequenced recently (Butler
et al., 2009) by the Wellcome Trust Sanger Institute Patho-
gen Genomics group (http://www.sanger.ac.uk/sequencing/
Candida/parapsilosis/), most of the genes have not been
completely annotated yet. Few positive results for ecto-50-
nucleotidase (CD73) sequences were found for fungi spe-
cies, most of the genes encode a hypothetical protein with a
conserved domain for CD73 enzyme. However, no signifi-
cant sequences for CD73 in Candida genome were found.
This could indicate that the enzyme was not identified in
Candida genome or it is not conserved like others CD73
enzyme.
In Saccharomyces cerevisiae, the presence of a 50-nucleo-
tidase with a preference for hydrolyzing IMP was reported.
The purified enzyme presumably participates in IMP de-
phosphorylation and the release of inosine, a precursor of
adenine and guanine nucleotides (Itoh, 1994). To our
knowledge, there is no information about IMPase or UM-
Pase activities outside of yeast cells. These surface activities
should contribute to maintain the level of intracellular
nucleotides. Adenosine released from AMP hydrolysis may
also participate in nucleoside acquisition.
Extracellular nucleotides, such as ATP, have been consid-
ered endogenous signaling molecules that contribute to
inflammation and immune responses. These nucleotides
are involved in the initiation of the oxidative burst, stimula-
tion of neutrophil adhesion to endothelial cells and degra-
nulation of both primary and secondary neutrophil
granules, which is necessary for efficient pathogen destruc-
tion (Rounds et al., 1999; Meshki et al., 2004; Bours et al.,
2006). During an immune response, ATP may contribute to
inflammatory activation of macrophages (Hanley et al.,
2004) and induce a proinflammatory cytokine profile
(Bours et al., 2006). ATP can be released in response to
tissue injury or exogenous pathogens; therefore, signaling
danger to the host and notifying the host to initiate primary
immune responses (Bours et al., 2006). In contrast, extra-
cellular adenosine at micromolar levels inhibits the adhesion
of neutrophils to vascular endothelial cells, suppresses the
phagocytic function of macrophages and decreases reactive
oxygen species generation by immunostimulated neutro-
phils (Bours et al., 2006; Kumar & Sharma, 2009). ATP can
exert a proinflammatory effect, whereas adenosine may be
related to anti-inflammatory and immunosuppressive func-
tions depending on its concentration.
In this work, we described that extracellular adenosine
could have a role in inhibiting C. parapsilosis and macro-
phage interaction, favoring the survival of the fungus
(Fig. 6a and b). It has been described that macrophage
expresses all the four types of adenosine receptors, A1, A2A,
A2Band A3receptors. Moreover, it is also well known that
the suppression of phagocytic function of macrophage
occurs by binding of adenosine to A2 receptors (Bours
et al., 2006; Hask´ o et al., 2008; Kumar & Sharma, 2009).
Both adenosine receptor types A2Aand A2Bare expressed in
neutrophils, monocytes, macrophages, dendritic cells and T
lymphocytes, and its EC50for adenosine varies at 0.56–0.95
and 16.2–64.1mM, respectively (Bours et al., 2006). Using
adenosine at the same range, at micromolar concentrations,
we observed an inhibition of 50% in the percentage of
infected macrophages (Fig. 6a and b). Although 50-AMP, at
the same concentration, did not have an effect in the
interaction, 1mM of 50-AMP presented similar results to
that observed with 100mM of adenosine. This fact could be
explained by the action of C. parapsilosis ecto-50-nucleoti-
dase activity in generating free adenosine to the medium. At
100mM on of 50-AMP, the rate of adenosine released could
not achieve the effective concentration of free adenosine
necessary to limit macrophage function, whereas at a higher
concentration of 50-AMP, the rate of extracellular adenosine
could be more expressive. However, the presence of an
ecto-50-nucleotidase activity on the external surface of
macrophages (Edelson & Cohn, 1976a,b), able to hydrolyze
50-AMP, could indicate that during the interaction assays,
macrophages could be also responsible for adenosine gen-
eration contributing to reduction in the number of infected
macrophages.
Recently, our laboratory characterized ecto-ATPase activ-
ity on C. parapsilosis. The sequential dephosphorylation of
ATP to adenosine was demonstrated by reverse-phase HPLC
experiments, suggesting the presence of different enzymatic
activities (ecto-ATPase, ecto-ADPase and ecto-50-nucleoti-
dase) on the surface of C. parapsilosis (Kiffer-Moreira et al.,
2010). Ecto-ATPase was also associated with in vitro infec-
tious processes because pretreatment with ATPase inhibitors
led to a decrease of C. parapsilosis adhesion to host cells
(Kiffer-Moreira et al., 2010). Colonization and infection
with C. parapsilosis are dependent upon the ability of the
fungus to adhere to host cells and tissues, particularly
mucosal surfaces (Trofa et al., 2008). The specific functions
of ecto-ATPases and ecto-50-nucleotidases are not fully
known, but it has been demonstrated that they participate
in many relevant biological processes (Zimmermann, 2000;
Meyer-Fernandes, 2002). In C. parapsilosis, both enzymes
play a role in the control of extracellular nucleotide concen-
trations and could have a role in limiting inflammation and
immune responses fromthe host, favoring the establishment
of infectious processes.
The involvement of ecto-50-nucleotidases and free ade-
nosines during infections has been described for several
microorganisms including protozoa (de Almeida Marques-
FEMS Microbiol Lett 317 (2011) 34–42
c ?2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
40
T. Russo-Abraha ˜o et al.

Page 8

da-Silva et al., 2008), bacteria (Thammavongsa et al., 2009)
and platyhelminthes (Bhardwaj & Skelly, 2009). In fungal
cells, there is evidence of some functions of ecto-ATPase
(Zhong et al., 2000; Junior et al., 2005; Collopy-Junior et al.,
2006; Kiffer-Moreira et al., 2010), but little information is
available about the activity of ecto-50-nucleotidase and its
product, adenosine. Identification of the physiological role
of this enzyme would contribute to understanding the
biochemical aspects of host–parasite interactions involving
C. parapsilosis.
Acknowledgements
We would like to thank Ms Fatima Regina de Vasconcelos
Goulart for preparation of fungal cultures and Mr Fabiano
Ferreira Esteves and Ms Rosangela Rosa de Arau ´jo for
excellent technical assistance. This work was supported by
grants from the Brazilian Agencies Conselho Nacional de
Desenvolvimento Cient´ ıfico e Tecnol´ ogico (CNPq), Coor-
denac ¸a ˜o de Aperfeic ¸oamento de Pessoal de N´ ıvel Superior
(CAPES) and Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado do
Rio de Janeiro (FAPERJ).
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