1 Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
2 Departamento de Medicamentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
3 Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
4 Disciplina de Biologia Celular, Universidade Federal de São Paulo, São Paulo, SP 04023-062, Brazil
Correspondence
José R. Meyer-Fernandes
meyer{at}bioqmed.ufrj.br
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ABSTRACT |
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INTRODUCTION |
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A number of fundamental processes in fungi such as the cell cycle, gene transcription and mating have been shown to require protein phosphorylation (Madhani & Fink, 1998; Dickman & Yarden, 1999
; Zhan et al., 2000
). Levels of cellular phosphorylation are controlled by the coordinated actions of protein kinases and protein phosphatases (Hunter, 1995
). Acid phosphatases (EC 3.1.3.2) have been described in many yeasts (Vogel & Hinnen, 1990
; Vasileva-Tonkova et al., 1996
). These enzymes may exist as soluble or secreted forms (Jolivet et al., 1998
), or remain attached to the outer surface of the inner membrane (Arnold et al., 1988
) or cell wall (González et al., 1993
; Bernard et al., 2002
; Kneipp et al., 2003
).
The presence of surface-located acid phosphatases, called ecto- or extracytoplasmic phosphatases, has been reported in many micro-organisms (Fernandes et al., 1997; Dutra et al., 1998
; Meyer-Fernandes et al., 1999
; Braibant & Content, 2001
), including the fungi Saccharomyces cerevisiae (Mildner et al., 1975
), Candida parapsilosis (Fernando et al., 1999
), Sporothrix schenckii (Arnold et al., 1986
) and Aspergillus fumigatus (Bernard et al., 2002
). The specific functions of these enzymes are not fully known, but they probably participate in cell wall biosynthesis in yeast cells (Novick et al., 1981
). They may also have a role as safeguard enzymes to protect the cells from acidic conditions, by buffering the periplasmic space with phosphate released from polyphosphates (Touati et al., 1987
). These enzymes may also provide fungal cells with a source of orthophosphate by hydrolysing organic phosphates (Toh-e, 1989
). In C. parapsilosis, a surface phosphatase activity was correlated with fungal adhesion to mammalian cells (Fernando et al., 1999
), indicating that ectophosphatases can also influence the interaction of fungal cells with the host.
We demonstrated recently that mycelial forms of F. pedrosoi express a Zn2+-activated acid ectophosphatase in their walls, and studied it biochemically and by ultrastructural examination (Kneipp et al., 2003). In the present work we characterized an ectophosphatase activity in conidial forms of F. pedrosoi. The enzyme activity was shown to be negatively modulated by exogenous phosphate (Pi). Conidial cells expressing high ectophosphatase activity were significantly more capable of adhering to epithelial cells than fungi expressing basal levels of enzyme activity, indicating that surface phosphatases may have a role in the interaction of F. pedrosoi with host cells, an essential step in the infectious process.
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METHODS |
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Mammalian cells.
MA 104 epithelial cell line, from monkey's kidney, and 3T3-L1 mouse fibroblasts were purchased from a Rio de Janeiro cell culture collection (BCRJ, registration numbers CR053 and CR089, respectively). The cells were grown at 37 °C in 25 cm2 culture flasks containing DMEM medium (Gibco), supplemented with 10 % fetal bovine serum (FBS). The pH was maintained at 7·2 by the addition of HEPES (3 g l1) and NaHCO3 (0·2 g l1) to the medium (Freshney, 1994). The initial inoculum was 5x104 cells ml1; cultures were subcultured every 2 days and the cells maintained in exponential-phase growth as described by Freshney (1994)
.
Measurement of phosphatase activity.
Phosphatase activity was determined by measuring the rate of p-nitrophenol (p-NP) production from the hydrolysis of p-nitrophenyl phosphate (p-NPP). Intact cells (107 conidia) of F. pedrosoi were incubated at room temperature for 60 min in a reaction mixture (0·5 ml) containing 15 mM MES/HEPES buffer at pH 5·5 and 5 mM p-NPP as substrate. For determining the concentration of p-NP formed through phosphatase activity, the tubes were centrifuged at 1500 g for 10 min (4 °C). The reaction was terminated by taking 0·2 ml of the supernatant and mixing it with 0·4 ml 1 M NaOH. The addition of NaOH after centrifugation was done to avoid extraction of melanin present in F. pedrosoi (Alviano et al., 1991), which interferes with the colorimetric method. This mixture was measured spectrophotometrically at 425 nm, using p-NP as standard (Fernandes et al., 1997
). The phosphatase activity was calculated by subtracting the nonspecific p-NPP hydrolysis measured in the absence of cells. As detailed in Results and figure legends, several experiments were performed in the presence of different cations, substrates or phosphatase inhibitors, at standardized concentrations (Kneipp et al., 2003
). Cell viability was assessed before and after incubations by the Trypan blue method. The viability of the cells was not affected by the conditions used in this work.
Cytochemical detection of acid phosphatase.
The cytochemical assay was carried out as previously described (Kneipp et al., 2003). Briefly, the conidial forms were fixed for 30 min at 4 °C with 1 % glutaraldehyde in 5 % sucrose, 0·1 M cacodylate buffer at pH 7·2, washed in the same buffer, followed by 0·1 M Tris/maleate buffer pH 5·5, and incubated for 2 h at room temperature in 2 mM cerium chloride, 5 % sucrose, 0·1 M Tris/maleate buffer pH 5·5, with 2 mM sodium
-glycerophosphate as substrate. The cells were then washed in Tris/maleate and cacodylate buffers, fixed again with 4 % paraformaldehyde, 2·5 % glutaraldehyde, 5 mM CaCl2, postfixed in 1 % osmium tetroxide, dehydrated in a graded acetone series, and embedded in Epon. As a control, cells were incubated in the absence of substrate. Ultrathin sections were observed unstained in a transmission electron microscope (Zeiss 900 EM; Carl Zeiss) operated at 80 kV.
Adhesion of F. pedrosoi to host cells.
Animal cells were plated onto 24-well multidishes at a density of 105 cells per well. They were then incubated at 37 °C for 24 h in the presence of DMEM medium supplemented with 10 % FBS. Before interaction with animal cells, conidia of F. pedrosoi (106) were incubated for 30 min at room temperature in 0·9 % NaCl (control cells) or in the same solution containing 1 mM sodium orthovanadate. Conidia were then washed twice with 0·9 % saline and finally rinsed in DMEM. Fungal cells were suspended in the same medium to a ratio of 10 F. pedrosoi conida per animal cell on monolayers. After the addition of F. pedrosoi, the cells were incubated at 37 °C for 2 h, washed three times in PBS to remove nonadherent conidia, fixed in Bouin's solution and stained with Giemsa. Adhesion indices were determined with a microscope at a magnification of 1000 (Zeiss Axioplan 2, Germany). The adhesion index is the ratio of attached and internalized conidia to the number of host cells per field. For each experiment, 400 animal cells were counted.
Statistical analysis.
All experiments were performed in triplicate, with similar results obtained from at least three separate cell suspensions. Data were analysed statistically using Student's t test. The maximal velocity (Vmax) and Michaelis constant (Km) for p-NPP were calculated using a computerized nonlinear regression program (Sigma Plot 4.0; Jandel Scientific Software).
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RESULTS |
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Conidial cells expressing different levels of phosphatase activity were also compared in their sensitivity to phosphatase inhibitors (Fig. 2, white bars). Different profiles were observed; for instance, conidia cultivated in Pi-depleted medium had a surface phosphatase activity significantly more susceptible to inhibition by sodium orthovanadate, sodium fluoride and sodium molybdate than those cultivated in the complete medium. In addition, the ectophosphatase from conidia grown in Pi-depleted medium was partially inhibited by levamizole.
Conidia from both culture conditions had their surface phosphatase activity strongly inhibited by Pi (Fig. 2). The inhibition of the surface phosphatase activity in F. pedrosoi conidia by Pi followed a dose-dependent pattern, as demonstrated by Ki determination. Enzyme activity was measured in the absence of Pi (control) or in a reaction medium containing increasing concentrations of KH2PO4. Phosphatase activity was almost completely inhibited at 10 mM KH2PO4; a Ki corresponding to 2·05±0·28 mM was estimated.
Potential substrates for the ectophosphatase activity such as -glycerophosphate and
-naphthyl phosphate seemed to compete with p-NPP, inhibiting its hydrolysis (Table 1
). However,
-naphthyl phosphate and
-glycerophosphate were more efficient in inhibiting the activity of conidia cultivated in Pi-depleted medium. The possible existence of phosphotyrosin phosphatases, initially suggested by the decrease of p-NPP hydrolysis by orthovanadate (Gordon, 1991
), was confirmed by the fact that P-Tyr, but not P-Ser and P-Thr, inhibited the hydrolysis of p-NPP by conidial cells cultivated in both complete and Pi-depleted medium (Table 1
). This observation was further confirmed by the fact that intact F. pedrosoi conidial cells preferentially liberated Pi from P-Tyr (data not shown).
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Expression of ectophosphatase influences the adhesion of F. pedrosoi to animal epithelial cells and fibroblasts
The differential expression of ectophosphatase activity in fungal cells cultivated in the absence or presence of exogenous phosphate provided the basis for new experiments to investigate the possible participation of fungal ectophosphatases in the interaction of F. pedrosoi with animal cells. Conidia with greater ectophosphatase activity had indices of adhesion to host cells higher than those of fungi cultivated in the presence of Pi (i.e. expressing low ectophosphatase activity) (Fig. 4). When the epithelial cell lineage MA 104 was used, conidia from Pi-depleted medium were 7-fold more adherent than fungi from the complete medium. Conidia expressing higher indices of ectophosphatases were also more efficient in attaching to mouse fibroblasts; they were 3·7-fold more adherent than fungal cells cultivated in the complete medium. Surface phosphatases seemed to be involved in adhesion of fungal cells cultivated in Pi-depleted medium, since the enhanced conidial attachment to both cell lines was reversed by pre-treating fungi with sodium orthovanadate.
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DISCUSSION |
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Phosphatases are fundamental components in cellular events regulated by phosphorylation-dephosphorylation systems, which are coordinately controlled through the action of protein kinases and phosphoprotein phosphatases (Hunter, 1995). The ectophosphatase activity in mycelial cells of F. pedrosoi (Kneipp et al., 2003
), and that shown in F. pedrosoi conidia in the present study, are strongly inhibited by exogenously added Pi, suggesting that this reaction product may regulate the fungal ectophosphatase activity.
The presence of phosphatases regulated by phosphate in the culture medium was reported in several prokaryotic organisms (Torriani-Gorini et al., 1994; Hulett, 1996
) and also in fungi, including Neurospora crassa (Jacob et al., 1971
), Aspergillus niger (MacRae et al., 1988
), Yarrowia lipolytica (González et al., 1993
), Pichia pastoris (Payne et al., 1995
) and S. cerevisiae (Ogawa et al., 2000
). In S. cerevisiae, transcription of genes encoding acid and alkaline phosphatases and the Pi transporter are coordinately repressed and derepressed depending on the Pi concentration in the culture medium (Oshima et al., 1996
). The regulation of this adaptive response is very complex, involving several genes that signal Pi starvation (Ogawa et al., 2000
). Most of the phosphatases synthesized under Pi-limiting conditions are either located extracellularly or are associated with the plasma membrane or cell wall (Metzenberg, 1979
).
In the present study, the presence of cell wall phosphatases in conidial forms of F. pedrosoi was demonstrated by transmission electron microscopy. The phosphatase activity was strongly inhibited by Pi, and cultivation of F. pedrosoi conidia in the absence of exogenous Pi resulted in the generation of fungal cells expressing an ectophosphatase activity 130-fold higher than that expressed by fungi cultivated in the presence of Pi, which agreed with our previous biochemical observations (Kneipp et al., 2003). The depletion of phosphate from the culture medium apparently induced the expression of a different ectophosphatase, as suggested by the differences in the affinity for the artificial substrate p-NPP and by the fact that Fe3+ did not enhance the ectophosphatase activity from conidia cultivated in Pi-depleted medium. On the other hand, the expression of other surface structures was apparently not affected by exogenous phosphate; for instance, the ecto-ATPase activities of F. pedrosoi conidia after growth in the two different media described above were quite similar (data not shown).
The generation of conidial cells with clearly distinct levels of surface phosphatase activity allowed the design of experiments to investigate the putative participation of the ectophosphatase activity in the interaction of F. pedrosoi conidia with host cells. Conidia expressing higher levels of surface phosphatase activity showed 7- and 3·7-fold greater adhesion to epithelial cells and fibroblasts, respectively. To evaluate whether phosphatases were in fact involved in adhesion, experiments were performed in the presence of enzyme inhibitors. Pi, the natural inhibitor of the F. pedrosoi ectophosphatases, produces a reversible pattern of inhibition. Therefore, experimental models in which fungal cells are pre-incubated with Pi were impaired, since it is efficiently removed by washing. When Pi is added as a phosphatase inhibitor during the interaction of F. pedrosoi with host cells, surface structures of fibroblasts and epithelial cells seemed to be modified also (data not shown), making the interpretation of data very difficult. Sodium orthovanadate (irreversible inhibition) was therefore chosen as the most suitable inhibitor to control the influence of ectophosphatases in fungal adherence. The pre-treatment of fungal cells with sodium orthovanadate inhibited the adhesive ability of conidia, confirming the involvement of ectophosphatases in the adhesion of F. pedrosoi to mammalian cells.
Our results indicate that ectophosphatases, besides their possible functions in the biology of fungal cells, may play a role in the interaction of F. pedrosoi with host tissues. The reasons for increased adherence of fungal propagules with enhanced ectophosphatase activity remain unclear, but the removal of phosphate groups from surface structures of host cells could result in conformational changes resulting in the exposure of additional sites for interaction with infectious agents. Alternatively, ectophosphatases may contain adhesive domains that could directly promote the attachment of fungal cells to their hosts, therefore functioning similarly to the well-characterized microbial adhesins. Finally, they could regulate the functional activation of surface adhesins, which would be the key structures mediating fungal attachment.
The knowledge of the functions of ectophosphatases in fungal pathogens is still very preliminary. We currently report on the involvement of these enzymes in fungal adhesion, but their cell wall distribution suggests that they could also play important roles in other biological processes occurring at the fungal surface. Ectophosphatases could, for instance, regulate the functions of chitin synthase, which is activated by calmodulin-mediated phosphorylation as in N. crassa (Suresh & Subramanyam, 1997). Alternatively, ectophosphatases may regulate morphological transitions in fungal pathogens. Recent results from our laboratory demonstrated that the addition of propranolol or platelet-activating factor, which are inducers of differentiation in F. pedrosoi, stimulates the ectophosphatase activity concomitantly with morphogenetic transition (Alviano et al., 2003
). These observations are in agreement with our previous results demonstrating that the three morphological stages of F. pedrosoi, i.e. sclerotic, mycelial and conidial cells, show different levels of ectophosphatase activity (Kneipp et al., 2003
). In the same study, we demonstrated that a fungal strain recently isolated from a human case of CBM had an ectophosphatase activity significantly higher than that of a laboratory-adapted strain. In addition, sclerotic cells, which are the parasitic forms of F. pedrosoi, had a surface phosphatase activity higher than that of mycelia and conidia. Taken together, these observations suggest a link between ectophosphatase activity and fungal parasitism. Our current results indicate that the activity of surface phosphatases indeed influences the interaction of F. pedrosoi with host cells in vitro. If the same occurs in vivo, ectophosphatases could contribute to the establishment of CBM, possibly acting in the early steps of fungal adhesion.
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ACKNOWLEDGEMENTS |
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Received 16 June 2004;
accepted 6 July 2004.
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