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Keywords:

  • Candida albicans;
  • growth;
  • germ-tube;
  • adhesion;
  • ultrastructure;
  • suramin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Candida albicans is an opportunistic pathogen that is of growing medical importance because it causes superficial, mucosal and systemic infections in susceptible individuals. Here, the effect of suramin, a polysulfonated naphthylurea derivative, on C. albicans development and virulence was evaluated. Firstly, it was demonstrated that suramin (500 μM) arrested its growth, showing a fungicidal action dependent on cell number. Suramin treatment caused profound changes in the yeast ultrastructure as shown by transmission electron microscopy. The more important changes were the enlargement of the fungi cytoplasmic vacuoles, the appearance of yeasts with an empty cytoplasm resembling ghost cells and a reduction in cell wall thickness. Suramin also blocked the transformation of yeast cells to the germ-tube and the interaction between C. albicans and epithelial cells. In order to ascertain that the action of suramin on C. albicans growth is a general feature instead of being strain-specific, the effects of suramin on 14 oral clinical strains isolated from healthy children and HIV-positive infants were analyzed. Interestingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin than strains isolated from healthy patients. Altogether, the results produced here show that suramin interfered with essential fungal processes, such as growth, differentiation and interaction with host cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Candidiasis has emerged as one of the more alarming opportunistic diseases, with a large increase in the number of patients who are immunocompromised, aged, receiving prolonged antibacterial and aggressive cancer chemotherapy or undergoing invasive surgical procedures and organ transplantation (Pfaller et al., 1998; Eggimann et al., 2003). Invasive candidal infections in such patients are associated with crude mortality rates of 40–50%, prolonged lengths of intensive care unit stay and economic impact due to high costs (Rentz et al., 1998; Olaechea et al., 2004; Ostrosky-Zeichner & Pappas, 2006). Undoubtedly, Candida albicans is considered to be the most common and most virulent pathogenic species of the genus Candida (Pfaller et al., 1998; Eggimann et al., 2003; Olaechea et al., 2004; Ostrosky-Zeichner & Pappas, 2006). The invasion of C. albicans depends upon host immune mechanisms that become impaired; however, there are intrinsic features of C. albicans that promote its ability to cause disease (Ostrosky-Zeichner & Pappas, 2006). A number of virulence attributes have been suggested, most of which fall into three categories: host recognition by fungal cell surface adhesins, morphogenetic conversion of the organism from a unicellular growth form (yeast) to a filamentous form (hyphae and pseudohyphae) and the secretion of putative invasive biomolecules such as proteases and phospholipases (reviewed by Naglik et al., 2003).

Over the last decade, there have been changes in the epidemiology of fungal infections as well as dramatic improvements in the antifungal armamentarium. However, there are limited therapeutic alternatives to combat Candida infections, mainly azole derivatives and amphotericin B. Moreover, the toxicity and emergence of resistance to these antifungal agents are potential problems and highlight the need for alternative treatment strategies (Pfaller et al., 1998; Aperis et al., 2006).

Suramin (Germin, Naganol, Morany, Bayer 205) is a colorless, polysulfonated, symmetrical naphthalene derivative of urea. Suramin is generally considered to be the drug of choice for the early stages of human African trypanosomiasis. In humans, it is also used against adult forms of the filarial parasite Onchocerca volvulus (reviewed by Barrett & Barrett, 2000). This longstanding compound has also received renewed attention for the treatment of HIV infection and cancer because of its inhibiting action on various enzyme systems (Voogd et al., 1993; Barrett & Barrett, 2000). Owing to these effects, the American AIDS lobby pushed hard for a clinical trial of suramin against AIDS in the 1980s. The drug had no impact on the progression of AIDS, although minor effects on some incidences of the AIDS-associated cancer, Kaposi's sarcoma, were noted (Stein et al., 1989). The drug was then tested against various neoplastically transformed cell lines and went into trials against a variety of cancers, where it has been of some value against hormone refractory prostate cancer (Kehinde et al., 1995). More recently, suramin is in phase I evaluation as a chemosensitizer of doxorubicin in dogs with naturally occurring cancers (Kosarek et al., 2006). The aim of the present study was to determine whether suramin exerts a direct effect on the growth, differentiation, ultrastructure and adhesive process of C. albicans.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Microorganisms

The strain of C. albicans used throughout this work (PRI strain) was isolated from the linear gingival erythema, which is a distinct fiery red band along the margin of the gingivae and probably has a candidal etiology, of a HIV-positive child (Portela et al., 2004) attended at the Hospital Pediátrico, Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG) and the Clínica Odontopediátrica at the Faculdade de Odontologia, Universidade Federal do Rio de Janeiro (UFRJ), Brazil. This study was approved by the Ethics Committee of Núcleo de Estudos de Saúde Coletiva (NESC), UFRJ, and guardians gave their informed consent. In addition, 14 oral clinical isolates (seven strains isolated from infants with a positive diagnostic of HIV infection and seven from healthy immunocompetent children) (Portela et al., 2004) were used to reveal that the suramin action is a general feature against C. albicans species, instead of being strain specific. Before the experiments, each C. albicans isolate was cultured on Sabouraud dextrose agar and CHROMagar Candida medium (CHROMagar, France) to ensure viability and purity. Additionally, the medical data of each HIV-positive patient were obtained from the hospital records and are summarized in the Table 1.

Table 1.   Characteristics of HIV-positive infants from whom Candida albicans strains were isolated
Patient*C. albicans strains (code)Age/sexPlasma RNA viral load (copies mL−1)CD4+ T-lymphocyte counts (%)Immunological stageDrugs used in therapy
  • *

    At the time of sample collection, none of the subjects demonstrated clinical signs of classical oral candidiasis (Portela et al., 2004).

  • All C. albicans strains were isolated from the subgingival plaque by using sterile paper points (Portela et al., 2004).

1PRI11/female170 0001C3Ritonavir, nelfinavir, epivir
2WVC10/male66 0003C3Ritonavir, leucovirine, epivir, didanosine
3HMS3/male620043B1Zidovudine, didanosine
4NAS7/female20020C3Ritonavir, nelfinavir, epivir
5DCB7/male31032C2Zidovudine, didanosine, nelfinavir
6PBO8/female21026A2Stavudine, lamivudine, nelfinavir
7HAO9/male21026C3Zidovudine, lamivudine, nelfinavir
8NMN3/female11 00017C3Zidovudine, lamivudine, nelfinavir

Culture conditions and evaluation of cell growth

Candida albicans strains from the Sabouraud agar plates were inoculated into 50 mL Erlenmeyer flasks containing 10 mL of brain heart infusion (BHI) medium and grown at 37°C for 48 h in an orbital incubator shaker (200 r.p.m.) (Costa et al., 2003). Cell growth was estimated by counting the yeasts in a Neubauer chamber.

Effect of suramin on fungal growth

Yeasts (1 × 108 cells) were resuspended in 1 mL BHI liquid medium, and 10 μL aliquots (equivalent to 1 × 106 cells) of this suspension were placed in the wells of a 96-well plate and then complemented with different concentrations of suramin (10, 25, 50, 100, 250 and 500 μM) (Sigma). A control was made by replacing the suramin with phosphate-buffered saline (PBS; 150 mm NaCl, 20 mm phosphate buffer, pH 7.2). Alternatively, different cellular densities (102–106 yeasts) were also treated with 500 μM suramin. These mixtures were incubated for 24 h at 37°C without agitation. The cells were then harvested by centrifugation, washed with PBS and reinoculated into solid BHI media without drugs to measure the CFU.

Effect of suramin on fungal ultrastructure

Fungal cells (PRI strain) were incubated in the absence or presence of 250 and 500 μM suramin for 24 h at 37°C. After this time, control and suramin-treated cells were washed twice in PBS, pH 7.2, and fixed in 2.5% glutaraldehyde, 4% formaldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 5 mM CaCl2 and 3.7% sucrose for 1 h at room temperature. Cells were then rinsed in PBS and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer containing 0.8% potassium ferrocyanide and 5 mM CaCl2 for 30 min at room temperature. Cells were rinsed, dehydrated in graded acetone and embedded in Polybed 812. Ultrathin sections obtained with a Reichert Ultracut S ultramicrotome were stained with uranyl acetate and lead citrate and examined in a Zeiss 900 transmission electron microscope operating at 80 kV. Morphometric evaluation of the cell wall was performed using images at a final magnification of × 200 000. Fifty cells of each system with nearly equatorial cut surfaces were measured, and results were expressed as mean±SD.

Effect of suramin on fungal morphogenesis

Yeast to germ-tube transformation was performed by incubating 1 × 106 yeasts (PRI strain) in the following differentiation inducers: 1 mL fetal bovine serum (FBS) (Hazen & Cutler, 1979), 100 μg mL−1 hemin (Casanova et al., 1997) or 5 mM N-acetyl-d-glucosamine (NAG) (Castilla et al., 1998). For each condition, two distinct systems were made: one of them was supplemented with 500 μM suramin and the other with PBS (control). At the end of the incubation period (3 h), samples were used for microscopic assessment of germ-tube production in a Zeiss microscope (Zeiss, Germany). The percentage of germination was estimated by counting 100 cells in a Neubauer chamber.

Effect of suramin on the fungal–epithelial cell interaction

For this assay, epithelial cells of monkey kidneys (Ma-104) were suspended in Dulbecco's modified Eagle's medium (DMEM), and then placed onto a glass coverslip in a 24-well tissue culture plate (1 × 105 cells well−1) and cultivated overnight at 37°C in a 5% CO2 humidified atmosphere. Control and suramin-treated (500 μM for 1 h at 37°C) C. albicans (PRI strain) were incubated with the host cells at a 10 : 1 yeast: host cell ratio in DMEM for 1 h at 37°C. After this, extracellular yeasts were removed by extensive washes, the cells were fixed with Bouin, stained with Giemsa dehydrated in acetone and xylol and the coverslips were mounted on Permount (Fisher Chemicals). In each system, the number of associated yeasts per 100 cells was estimated by counting under a light microscope.

Statistical analysis

All of the experiments were repeated at least three times, and all systems were performed in triplicate sets. Representative images of these experiments are shown. The data were analyzed statistically using Student's t-test; P values of 0.05 or less were considered to be statistically significant.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Candidiasis is the most common oral fungal infection diagnosed in humans. The presence of Candida in the oral cavities of HIV/AIDS patients predicts the subsequent development of oral candidiasis (Pfaller et al., 1998). In fact, more than 90% of HIV-positive individuals will acquire at least one episode of oropharyngeal candidiasis during progression to AIDS, while esophageal candidiasis is considered an AIDS-defining illness (Hauman et al., 1993). Owing to the emergence of pathogens resistant to conventional antifungals and the toxicity of some antimycotics, intense efforts have been made to develop more effective antifungal agents for clinical use (Aperis et al., 2006). Considering all these facts, the effect of suramin on C. albicans development in vitro was investigated. Initially, different suramin concentrations were tested on the cellular growth of C. albicans PRI strain, a clinical oral isolate resistant to fluconazole (L.A. Braga-Silva, unpublished results). The results showed a significant reduction (P<0.05) on yeast growth only with suramin at 500 μM when 106 cells were previously treated for 24 h with this drug (Fig. 1a). Then, different cellular densities (105–102 yeasts) were treated with 500 μM suramin. The treatment of 105 cells with suramin at 500 μM inhibited the growth behavior of C. albicans by c. 50% compared with untreated cells (Fig. 1b). Additionally, cellular growth was powerfully impaired when 104–102 yeasts were incubated with this suramin concentration (Fig. 1b), which shows a fungicidal action dependent on the cell number. Interestingly, the candidacidal activity of mouse peritoneal cells, which was measured as a nonspecific microbicidal activity of phagocytes after intraperitoneal injection in mice, was considerably enhanced when suramin was used as an adjuvant (Hilgers et al., 1985).

image

Figure 1.  Effect of suramin on Candida albicans growth rate. (a) After growth in BHI medium for 48 h at 37°C, yeasts were harvested by centrifugation, washed in PBS and then incubated (106 cells) with different suramin concentrations (0–500 μM). These samples were incubated for 24 h at 37°C, harvested, washed in PBS and inoculated in fresh solid medium without drugs to measure the CFU. The growth of control cells was taken as 100%. (b) Alternatively, different cellular densities (106–102) were incubated in the absence or presence of suramin at 500 μM under the same conditions as described earlier. Values represent the mean±SD of three independent experiments performed in triplicate. Asterisk in A denotes those samples treated with suramin that have a growth rate significantly different from the control (P<0.05; Student's t-test).

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Transmission electron microscopy also confirmed the fact that suramin affects C. albicans viability (Fig. 2). Nontreated cells presented a normal cellular morphology with a typical dense cytoplasm and a distinct cell wall (Fig. 2a). However, after 24 h of treatment with suramin at 250 μM, the presence of large and irregular cytoplasmic vacuoles was detected (Fig. 2b and c), some of them containing small vesicles (Fig. 2c). Similar alterations in kind but stronger in intensity were observed when the yeasts were treated with 500 μM suramin (Fig. 2d). In some cells, the enlargement of intracellular vacuoles was so intense that they occupied almost the whole cytoplasm area (Fig. 2e). Suramin has been described as a potent inhibitor of lysosomal enzymes and an inducer of lysosomal storage diseases characterized by the presence of enlarged vacuoles in different cell types (Constantopoulos et al., 1983). Because the yeast vacuole is an acidic compartment that contains a variety of hydrolytic enzymes, the effects observed support the hypothesis that suramin interferes with the metabolism of the fungus and consequently with the yeasts' growth and differentiation. Furthermore, deformed yeasts presenting low cytoplasmic electrodensity, resembling ghost cells, were also detected at the higher suramin concentration (Fig. 2f). Moreover, the morphometric analyses of yeasts after suramin treatment showed a significant reduction of the cell wall thickness (Fig. 2g–i). Control cells exhibit a uniform electrondense cell wall with a thickness of 225.7±16.0 nm. After incubation for 24 h in the presence of 250 and 500 μM suramin, cell wall thickness was reduced to about 187.2±12.5 and 155.4±14.0 nm, respectively. As in fungi in general, the cell wall of C. albicans is a dynamic and complex multilayered structure located external to the plasma membrane. It is responsible for maintaining the shape that characterizes each growth form (yeast and hyphae) of the fungus. The cell wall also plays nutritional roles and acts as a permeability barrier that protects the protoplast against physical and osmotic injuries. Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall because it is the outermost part of the cell. It is responsible for the adherence of the pathogen and establishes a cross-talk with the host. Moreover, the cell wall of C. albicans is a significant source of antigens. Indeed, several immunodominant antigens in candidiasis have been characterized as cell wall components (reviewed by Ruiz-Herrera et al., 2006). Consequently, compounds that disorganize and/or inhibit cell wall structure might be potential drugs capable of blocking some relevant biological phenomena, such as fungal differentiation and adhesion.

image

Figure 2.  Effect of suramin on Candida albicans ultrastructure. After growth in BHI medium for 48 h at 37°C, yeasts were harvested by centrifugation, washed in PBS and 106 cells were incubated for 24 h at 37°C in the absence (a) or in the presence of 250 (b, c) and 500 μM suramin (d–f). Arrows in (b), (c) and (d) show the enlarged cytoplasmic vacuole. Arrowheads in (c) indicate small vesicles inside the vacuole. A large vacuole that occupied almost the whole cytoplasm area can be observed in (e) (asterisk). Large arrows in (f) show typical ghost yeast cells. In addition, the effect of suramin on cell wall of C. albicans is shown (g–i). The representative images demonstrate the cell wall thickness of yeasts untreated (g) and treated with 250 μM suramin (h) and 500 μM suramin (i) that was determined in at least 50 yeast cells of each system in nearly equatorial sections. Scale bars: (a–e) 0.5 μm; (f–i) 1.0 μm.

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The fascinating ability of C. albicans to undergo dramatic changes in cellular morphology has invited speculation that this plasticity contributes to the virulence of the microorganism. Hyphae are able to exert a mechanical force, which facilitates the penetration of epithelial surfaces and they can also damage endothelial cells, aiding the escape of C. albicans from the host bloodstream into deeper tissues. Hyphal morphogenesis is thus an integral part of the overall virulence strategy of C. albicans (Kumamoto & Vinces, 2005). In this context, the effect of suramin on yeast to germ-tube transformation was studied, triggered in carbon-starved cells at 37°C by three distinct inducers: FBS, hemin and NAG. As expected, more than 70% of the cells formed well-defined germ tubes when they were incubated individually with each differentiation inducer (Fig. 3a). In contrast, a significant inhibition (P<0.05) on the germ-tube emergence was detected when suramin at 500 μM was added to the induction differentiation systems (Fig. 3a). In order to verify whether suramin promoted the blockage of the differentiation through loss in cell integrity, the yeast viability was assessed. The result showed that more than 98% of the suramin-treated cells were viable when assessed by the propidium iodide (PI) exclusion method. Similarly, the effects of suramin on the differentiation process in various cell systems have been widely documented, including osteoclast (Regmi et al., 2005) and neural retina (Cirillo et al., 2001).

image

Figure 3.  (a) Effect of suramin on the germ-tube formation in Candida albicans. Germination of yeast cells was performed in the absence (control) or in the presence of 500 μM suramin for 3 h at 37°C. Both systems were individually supplemented with distinct differentiation inducers: FBS, hemin and NAG as described in ‘Material and methods’. To evaluate the suramin effects on the germination process, the number of cells presenting germ-tube formation was counted in a Neubauer chamber. (b, c) Effect of suramin on the interaction between C. albicans and epithelial cells (Ma-104). Yeasts were grown in BHI medium for 48 h at 37°C, harvested by centrifugation and incubated (106 yeasts) in the absence (control) or in the presence of 500 μM suramin for 1 h (b). The interaction process was performed as described in ‘Materials and methods’. Representative images of the interaction are shown in (c). Arrows in (c) show the C. albicans adhered to the epithelial cells. In these set of experiments, the viability of the yeast cells was not affected by the suramin treatment used in this set of experiments as judged by PI staining (data not shown). Values represent the mean±SD of three independent experiments performed in triplicate. Scale bars: 50 μm.

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Adherence of yeasts to host cells and tissues represents another important virulence factor during candidiasis (Ruiz-Herrera et al., 2006). Adhesive interactions are considered to be the initial and critical step leading to the establishment of an infection. To demonstrate this, C. albicans was incubated with suramin at 500 μM for 1 h before the interaction with epithelial cells, in order to detect the effect of suramin in the cellular interaction process. The yeasts maintained their viability after treatment for 1 h with this suramin concentration as judged by the PI exclusion method (in which more than 99% of the yeasts were viable) and by measurement of CFU (data not shown). The results showed that suramin was able to diminish (P<0.001) the association index between yeasts and epithelial cells by c. 50% (Fig. 3b, c).

The wide spectrum of suramin actions has been attributed to the presence of six negative charges at physiological pH in the molecule. Different proteins and enzymes can be affected by suramin in distinct ways. The most evident effects described were the inhibition of enzymatic activities, for example: the reverse transcriptase of retroviruses (De Clerq, 1979), protein kinase C (Gschwendt et al., 1998), DNA polymerase (Voogd et al., 1993), glycolytic enzymes of trypanosomes (Trinquier et al., 1995), serine proteases (Cadene et al., 1997), protein tyrosine phosphatases (Zhang et al., 1998) and Mg2+ATPases (Meyer-Fernandes, 2002). Morphological changes related to suramin action on several cytoskeleton components have also been described (Ohmori et al., 2001).

In order to demonstrate that the action of suramin on C. albicans development is a general feature instead of being strain-specific, the effect of suramin was analyzed on 14 distinct oral clinical strains of C. albicans isolated from healthy children (n=7) and HIV-positive infants (n=7) (Fig. 4). In this set of experiments, the yeast cells grown in BHI medium for 48 h were incubated with suramin at 500 μM for 24 h and then spread on a solid medium to measure the number of CFU. Surprisingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin in comparison with the strains isolated from healthy individuals (Fig. 4, inset). There is evidence in favor of a link between the prophylactic use of antifungal agents and the selection for resistant C. albicans yeasts other than the decreased susceptibility to these agents. For C. albicans, the development of secondary resistance to fluconazole treatment has commonly been observed in HIV-infected patients who received prolonged treatment with fluconazole for oropharyngeal candidiasis (He et al., 1994). With these considerations in mind, all the HIV-infected patients, from whom the C. albicans strains were isolated and studied herein, received anti-HIV therapy (Table 1), which could justify the selection of the more resistant C. albicans strains that presented a higher resistance to suramin action (Fig. 4). Corroborating this hypothesis, in HIV-infected subjects with oral or vaginal candidiasis, C. albicans strains are selected with heightened virulence attributes and pathogenicity (reviewed by Naglik et al., 2003).

image

Figure 4.  Effect of suramin on the growth of different oral clinical strains of Candida albicans isolated from HIV-positive (HIV+) and healthy (HIV−) children. After growth in BHI medium for 48 h at 37°C, yeasts were harvested by centrifugation, washed in PBS and 106 cells were then incubated with 500 μM suramin. These systems were incubated for 24 h at 37°C and then harvested, washed in PBS and inoculated in a fresh solid medium without drugs to measure the CFU. Control cells were taken as 100%. Each bar represents the means±SD of three independent experiments performed in triplicate. The inset shows the mean±SE of the mean of each group (P<0.05; Student's t-test).

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In conclusion, the results of the present study suggest that suramin, when used under the conditions described above, altered the growth behavior of C. albicans, causing irreversible alterations at the ultrastructural level, blocked the adherence mechanisms between fungi and epithelial cells and also arrested the yeast to germ-tube transformation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

The present study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Universitária José Bonifácio (FUJB) and Programa de Apoio a Núcleos de Excelência (PRONEX). The authors thank Dr Marta H. Branquinha for the useful critical English review as well as for the valuable suggestions on the manuscript. The authors also thank Venício F. Veiga for assistance with microscopy.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
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