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Keywords:

  • Trichomonas vaginalis;
  • adenosine deaminase;
  • intact trophozoites;
  • adenosine;
  • inosine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Trichomonas vaginalis is a parasite that resides in the human urogenital tract and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. Nucleoside triphosphate diphosphohydrolase (NTPDase), which hydrolyzes extracellular di- and triphosphate nucleotides, and ecto-5′-nucleotidase, which hydrolyzes AMP, have been characterized in T. vaginalis. The aim of this study was to characterize the adenosine deaminase (ADA) activity in intact trophozoites of T. vaginalis. A strong inhibition in adenosine deamination was observed in the presence of calcium and magnesium, which was prevented by EDTA. The apparent KM value for adenosine was 1.13 ± 0.07 mM. The calculated Vmax was 2.61 ± 0.054 nmol NH3 min−1 mg−1 protein. Adenosine deamination was inhibited in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. Semi-quantitative reverse transcriptase-PCR experiments were performed and both ADA-related genes ada(125) and ada(231) mRNA were expressed, although ada(231) in higher quantity when compared with the ada(125) : α-tubulin ratio. Furthermore, a phylogenetic analysis showed that the T. vaginalis sequences formed a clade with Entamoeba histolytica and Dictyostelium discoideum sequences, and it strongly suggests homologous functions in the T. vaginalis genome. The presence of ADA activity in T. vaginalis may be important to modulate the adenosine/inosine levels during infection and, consequently, to maintain the anti-inflammatory properties through different nucleoside-signalling mechanisms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Trichomonas vaginalis is a protozoan parasite that causes trichomonosis, the most prevalent nonviral sexually transmitted disease worldwide (WHO, 2001). In women, the infection is clinically characterized by vaginitis and cervicitis (Petrin et al., 1998; Lehker & Alderete, 2000). The pathogen has been associated with serious health consequences including adverse pregnancy outcomes (Klebanoff et al., 2001), infertility (Grodstein et al., 1993), predisposition to cervical cancer (Viikki et al., 2000) and pelvic inflammatory disease (Cherpes et al., 2006), and it is a cofactor in HIV transmission and acquisition (Sorvillo et al., 2001; Van Der Pol et al., 2008).

At the infection sites, tissue stress or injury takes place and intracellular ATP can be released into the extracellular environment. Extracellular nucleotides such as ATP play a role as danger-associated molecular patterns (DAMPs) or ‘alarmins’ by acting as signalling molecules that contribute to inflammation and immune responses (Hanley et al., 2004; Bours et al., 2006). The crucial factors in purinergic signalling are the stimulation of nucleotide release, their metabolism by enzymes acting in an extracellular manner and the presence of receptors that selectively bind the resulting products and mediate signal transduction (Gounaris & Selkirk, 2005). The purinergic signalling involves specific purinergic type 1 (P1) and type 2 (P2) receptors and is important in both neuronal and non-neuronal processes, including the modulation of inflammation and specific immune responses (Robson et al., 2006; Sansom et al., 2008).

The ecto-nucleoside triphosphate diphosphohydrolase family (ecto-NTPDases) is constituted by eight members (NTPDase1–8) that hydrolyze nucleoside di- and triphosphates to the monophosphate form. Nucleoside monophosphates may then be catalyzed to nucleosides such as adenosine by the action of ecto-5′-nucleotidase. Purine salvage and the regulation of blood clotting, inflammatory processes and immune reactions are among the major roles played by these enzymes to date (Sansom et al., 2008; Burnstock & Verkhratsky, 2009). The adenosinergic signalling can be controlled by adenosine uptake via bidirectional transporters, followed by intracellular phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase (ADA; EC 3.5.4.4). ADA participates in the purine metabolism, where it degrades either adenosine or 2′-deoxyadenosine, producing inosine or 2′-deoxyinosine, respectively (Franco et al., 1997). A phylogenetic study demonstrated the existence of different ADA-related members, which include ADA1, ADA2 and a similar deduced amino acid sequence named adenosine deaminase like (ADAL) (Maier et al., 2005). Despite its intracellular location, ADA1 may occur on cell surface, anchored to two proteins, CD26 and A1 receptors, acting as an ecto-ADA cleaving extracellular adenosine (Franco et al., 1997). ADA has been described in mammalian cells and tissues, blood-feeding insects, mollusks and parasites, Plasmodium lophurae, Trichinella spiralis, Fasciola gigantica and Hyalomma dromedarii (Franco et al., 1997; Gounaris, 2002; Mohamed, 2006; Ali, 2008).

The characterization and expression of S-adenosylhomocysteinase were described in T. vaginalis, which catalyzes the reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine (Minotto et al., 1998). Those authors have previously reported the absence or the poor activity of ADA. It is important to mention that T. vaginalis is dependent on salvage pathways to generate de novo nucleotides (Heyworth et al., 1982, 1984). Munagala & Wang (2003) demonstrated that adenosine is the primary precursor of the entire pool of purine nucleotides in T. vaginalis, and activities of ADA, IMP dehydrogenase and GMP synthetase were identified in trichomonads, suggesting a metabolic pathway able to convert adenine to GMP via adenosine. Our group has investigated the purinergic system in T. vaginalis throughout the extracellular nucleotide hydrolysis, and NTPDase and ecto-5′-nucleotidase activities were described (Matos et al., 2001; Tasca et al., 2003, 2005). Considering that (1) extracellular nucleotides and nucleosides, such as adenosine and inosine, act as DAMPs playing a role in cell signalling that contribute to inflammation and immune responses (Bours et al., 2006; Sansom et al., 2008), and (2) the ectonucleotidase pathway has been characterized in T. vaginalis, the aim of this study was to characterize ADA activity, an enzyme involved in nucleoside metabolism, and to evaluate the relative mRNA expression of ADA-related genes in this mucosal parasite.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Parasite culture and preparation of parasite suspensions

Trichomonas vaginalis clinical isolate TV-VP60 (Michel et al., 2006) was used throughout this enzyme characterization study. The other five isolates were TV-30236 (from the American Type Culture Collection, ATCC) and the clinical isolates TV-LACM1, TV-LACM2, TV-LACH1 and TV-LACH2 from our Clinical Laboratory surveys (Universidade Federal do Rio Grande do Sul, Brazil). Trichomonads were cultured axenically in vitro and maintained in trypticase–yeast extract–maltose (TYM) medium (Diamond, 1957), pH 6.0, supplemented with 10% (v/v) inactivated bovine serum at 37 °C. Organisms from the logarithmic phase were evaluated before and after assays based on motility and viability using trypan blue (0.2%) exclusion. The parasites were then harvested by centrifugation and washed three times with phosphate-buffered saline (PBS) added with 2.0 mM EDTA and 2.0 mM EGTA. The final pellet was resuspended and used for the subsequent assays. Trichomonas vaginalis lysates were obtained in liquid nitrogen, at 0.1 mg−1 protein−1 mL−1, in the presence of 1.0 mM protease inhibitor cocktail.

ADA assay

An aliquot from the parasite suspension was added to the reaction mixture containing 50 mM sodium phosphate buffer (pH 7.5) to maintain the protein concentration (50–150 μg mL−1) in the final volume of 200 μL. The samples were then preincubated for 10 min at 37 °C. The reaction was initiated with the addition of the substrate adenosine (3.0 mM) and stopped, after a determined time (10–40 min), by adding the samples on 500 μL of phenol-nitroprusside reagent (50.4 mg of phenol and 0.4 mg of sodium nitroprusside mL−1). Controls with the addition of the enzyme preparation after the termination of reaction were used to correct nonenzymatic deamination of the substrate. The reaction mixtures were mixed with 500 μL of alkaline-hypochlorite reagent (sodium hypochlorite to 0.125% available chlorine, in 0.6 M NaOH). Samples were incubated at 37 °C for 15 min. The colorimetric assay was carried out at 635 nm (Giusti, 1974) to measure the ammonia produced by the enzymatic reaction and the ADA activity was expressed as nmol NH3 min−1 mg−1 protein. In all assays, at least three different experiments were performed in triplicate. The protein quantification was performed in triplicate for the parasite suspensions (Bradford, 1976) using bovine serum albumin as a standard.

Characterization of ADA activity

After the standardization of incubation time and the protein concentration in order to maintain the linearity of the enzymatic reaction, assays to determine the optimum pH were performed using 50 mM sodium phosphate buffer (mixture: 0.2 M disodium phosphate and 0.2 M sodium phosphate, pH 6.5–7.5) and sodium carbonate bicarbonate buffer (mixture: 0.2 M sodium carbonate and 0.2 M sodium hydrogen carbonate, pH 8.5). The apparent KM and Vmax values for adenosine deamination were determined from Eadie–Hofstee plots using substrate concentrations from 0.40 to 3.0 mM. The substrates 2′-deoxyadenosine, guanosine and 2′-deoxyguanosine (all in 3.0 mM) were also assayed for ADA activity. The effect of the divalent cations Ca2+ and Mg2+ at 2.5 and 5.0 mM was observed by assaying in parallel a control without the cations and a control with cations and EDTA at the same concentrations. ADA activity was measured in the presence of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent inhibitor of the ADA 1 isoenzyme, in increasing concentrations (5.0–25 μM). In order to determine as to how long the EHNA inhibition effect lasts, a 20-min incubation with the inhibitor was performed and the EHNA-treated trophozoites were incubated in culture medium (TYM). After different times (1, 6 and 24 h), the ADA activity was tested. The trichomonad-culture supernatants from EHNA-treated trichomonads were also collected to test in the T. vaginalis–neutrophils interaction assay.

Analysis of gene expression by reverse transcriptase (RT)-PCR

Trophozoites were centrifuged and washed three times with PBS buffer (pH 7.2) for total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. The purity of the RNA was spectrophotometrically quantified by calculating the ratio between absorbance values at 260 and 280 nm. Afterwards, cDNA species were synthesized using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) following the supplier's instructions from 2.0 μg of total RNA. PCR reactions were performed in a volume of 20 μL using 0.1 μM of specific primers for ADA, 2.5 mM MgCl2 and 0.5 U Taq Platinum (Invitrogen) in the supplied reaction buffer. The sequences of α-tubulin primers were in accordance with previously described data (Kucknoor et al., 2005) and the PCR conditions were as reported in previous studies (Giordani et al., 2010; Rückert et al., 2010), using 0.5 M betain. All assays were carried out using 1.0 μL of cDNA template. The conditions for all PCR were as follows: initial 1-min denaturation step at 94 °C, 1-min annealing step (ada 125 and ada 231) at 57 °C, 1-min extension step at 72 °C for 35 cycles and 10 min of a postextension cycle at 72 °C. Negative controls were included for each set of PCR. PCR products were separated on a 1.0% agarose gel with GelRed 10 × (Invitrogen) and visualized with UV light. Band intensities were analyzed by densitometry using the freeware imagej 1.37 for Windows. The alpha-tubulin gene was used for normalization and all PCR products were run in a single gel. The results are representative of three different experiments.

Identification of ADA-related T. vaginalis amino acid sequences

The identification of ADA-related T. vaginalis amino acid sequences was performed using the well-known ADA1, ADAL and ADA2 from humans (NP_000013, NP_001012987 and CAG30303), mouse (NP_031424 and AAH52048), frog (Q6GP70, AAH97573 and AAX10952), chicken (NP_001006290, NP_001025718 and AAX10953) and fish (AAH76532, NP_001028916, AAL40922 and XP_687719) as queries to perform a basic local alignment search (blastp function) via the GenBank database. ADA-related sequences from Leishmania major (XP_843322), Plasmodium falciparum (XP_001347573), T. spiralis (AAT39739), Trypanosoma brucei (XP_823299), Entamoeba histolytica (XP_655082), Dictyostelium discoideum (XP_637270) and Escherichia coli (AAA16408) were also retrieved from GenBank and included in the phylogenetic analysis. The ADA protein sequences obtained were aligned with clustalx program (Thompson et al., 1997) and a phylogenetic tree was constructed with mega 4.0 program (Tamura et al., 2007) using the statistical neighbor-joining method (Saitou & Nei, 1987) with proportional (p) distance.

Isolation of human neutrophils

Human neutrophils were isolated as described previously (Boyum, 1968), with some modifications. Briefly, venous blood of healthy volunteers was collected on a heparin anticoagulant solution, centrifuged (250 g, 10 min, 24 °C) and the resulting platelet-rich plasma was discarded. Leukocytes were obtained following erythrocyte sedimentation in 2% Dextran T-500 and centrifuged (525 g, 20 min, 24 °C) through a layering on Histopaque 1077 (Sigma, St. Louis, MO). The neutrophil-enriched pellet was subjected to a 15-s hypotonic lysis to remove the remaining erythrocytes and centrifuged (1000 g, 5 min, 24 °C). The purified neutrophils were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum and 10 mM HEPES for the next experiments. The purity of neutrophils was confirmed morphologically (>95%) and examined using flow cytometry (FACSCalibur, BD Bioscience, Franklin Lakes, NJ). The phenotypic analysis as performed by cell quest bd and paint a gate pro bd softwares, after staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD45, anti-CD15, anti-CD8 antibodies and phycoerythrin-conjugated anti-CD14, anti-CD22, anti-CD3 and anti-CD4 antibodies (BD Bioscience).

Culture condition of neutrophils and T. vaginalis

Neutrophils (2.0 × 105) were co-cultured with live and lysed T. vaginalis (1.0 × 104), 1 h EHNA-treated and nontreated trophozoites, as well as trichomonad-culture supernatants from EHNA-treated trichomonads. All conditions were performed on a 96-well microplate, for 24 h, in the presence or not of 100 ng mL−1 lipopolysaccharide (used as a positive control), 100 μM adenosine and 100 μM inosine. After incubation, the cell-free culture supernatants were collected and subjected to quantification of nitrite immediately. The results are representative of at least three independent experiments.

Measurement of nitrite production

The concentration of NO in culture supernatants was determined as nitrite using Griess reagent (Sigma) in accordance with the manufacturer's instructions.

Statistical analysis

Data were expressed by mean ± SD and analyzed by one-way anova, followed by Tukey multiple-range test or Student's t-test, considering P<0.05 as significant. The analyses were performed using the spss software.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Adenosine deamination as a function of time and protein concentration

The adenosine deamination in trophozoites of T. vaginalis was evaluated as a function of time and protein concentration in order to determine the correct assay conditions. The deamination promoted by ADA activity was linear up to 40 min (Supporting Information, Fig. S1a) and in the range of 50–150 μg protein mL−1 (Fig. S1b). Therefore, we chose to use 100 μg protein mL−1 from cultures in further enzyme assays. The viability of the trophozoites was not affected by any of the conditions used in the assays. When trophozoite suspensions were incubated with their respective times and protein contents without the substrate adenosine, there was no significant production of NH3. Therefore, the involvement of other NH3 sources was negligible in the assay condition tested.

Influence of pH on ADA activity

To evaluate the influence of pH on ADA activity, the enzyme assays were carried out in a pH range of 6.5–8.5. The buffers used were sodium phosphate (used in a pH range from 6.5 to 7.5) and sodium carbonate bicarbonate buffer (assayed for pH 8.5). The results showed that the optimum pH for ADA was 7.5 (Fig. 1a); therefore, this value was chosen for the subsequent experiments.

image

Figure 1.  Influence of pH (a) and of divalent cations (b) on ADA activity in intact trophozoites of Trichomonas vaginalis. Data are means ± SD of three different experiments (parasite suspensions) performed in triplicate.

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Effect of divalent cations on ADA activity

In order to investigate a possible effect of divalent cations on ADA activity, Ca2+ and Mg2+ were used. Both cations were able to decrease (approximately 50%) the ADA activity at the lower tested concentration (2.5 mM). When tested at a higher concentration (5.0 mM), Mg2+ inhibited 80% of ADA activity and Ca2+ completely abolished the activity. This effect is specifically caused by cations because it was prevented by the addition of EDTA (Fig. 1b).

Kinetic parameters of T. vaginalis ADA

The adenosine deamination was determined at adenosine concentrations ranging from 0.4 to 3.0 mM (Fig. 2). The apparent Michaelis–Menten constant (KM app) and maximum velocity (Vmax app) were estimated from a Eadie–Hofstee plot (inset, Fig. 2). The apparent KM was 1.13 ± 0.07 mM (mean ± SD, n=4), whereas the calculated Vmax was 2.61 ± 0.054 nmol NH3 min−1 mg−1 protein (mean ± SD, n=4).

image

Figure 2.  Dependence of ADA activity on adenosine concentrations in intact trophozoites of Trichomonas vaginalis. Data represent means ± SD of four different experiments (parasite suspensions) performed in triplicate. Inset: Eadie–Hofstee plot for adenosine. V, velocity of ADA activity, expressed in nmol NH3 min−1 mg−1 protein; V [ADO]−1, velocity/adenosine concentration.

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Different nucleoside specificity

The relative substrate specificity of T. vaginalis ADA was determined (Table S1). Adenosine and 2-deoxyadenosine were substrates for ADA, presenting the activities 1.9 ± 0.6 and 2.9 ± 0.5 nmol NH3 min−1 mg−1 protein, respectively. Guanosine and 2-deoxyguanosine were not deaminated.

Inhibition of ADA activity by EHNA

We measured the adenosine deamination in T. vaginalis in the presence and in the absence of EHNA, a potent inhibitor of ADA1 activity (Iwaki-Egawa & Watanabe, 2002; Sharoyan et al., 2006; Rosemberg et al., 2008). The incubation time of 20 min for EHNA inhibition was used because this was the optimal time for all enzyme assays, ensuring the linearity of the reaction. After the EHNA treatment, trichomonads were metabolically active because they were inoculated in TYM medium for the subsequent experiments including the ADA assay and interaction with human neurophils. Moreover, the parasites presented motility and cellular integrity checked using trypan blue dye exclusion after EHNA incubation at all concentrations. The inhibition was tested using several concentrations of EHNA (5, 10, 15, 20 and 25 μM) and the results revealed a strong and dose-dependent inhibition, reaching the complete abolishment of activity at the highest concentration of EHNA (Fig. 3). Furthermore, the EHNA inhibition was long lasting, because no activity could be detected after passage in culture medium 1 and 6 h after the EHNA treatment (Table 1). The low ADA activity detected after 24 h (0.27 ± 0.05 nmol NH3 min−1 mg−1 protein) was probably due to new trophozoites grown after the incubation in the culture medium.

image

Figure 3.  Inhibition of ADA activity in intact trophozoites of Trichomonas vaginalis by EHNA. Data represent means ± SD of three different experiments (parasite suspensions) performed in triplicate. Statistical analysis by one-way anova. Statistically significant: awhen compared with the control; bwhen compared with 5 mM; cwhen compared with 10 mM; d when compared with 15 mM (P<0.05).

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Table 1.   EHNA inhibition of Trichomonas vaginalis (TV-VP60) ADA activity
ConditionADA activity (nmol NH3 min−1 mg−1 protein)
  1. ND, not detectable under the assay conditions used.

No EHNA1.80 ± 0.05
EHNA 20 minND
EHNA 1 hND
EHNA 6 hND
EHNA 24 h0.27 ± 0.05

Interaction T. vaginalis-neutrophils

We have evaluated the interaction of EHNA-treated T. vaginalis on NO production by human neutrophils stimulated with T. vaginalis. Figure 4 shows that neutrophils alone produced low levels of NO (1.98 ± 0.35 μM); however, when stimulated with lipopolysaccharide (positive control), the concentration increased 35 times (70.26 ± 14.69 μM). When the trichomonad-culture supernatants from EHNA-treated trichomonads and the T. vaginalis lysate were incubated with neutrophils, both conditions inhibited the NO production. On the other hand, and expectedly, the co-culture with intact T. vaginalis trophozoites produced a high amount of NO. However, when incubated in the presence of 1 h EHNA-treated parasites, the NO production effect was reverted. The same effect was observed with adenosine and inosine.

image

Figure 4.  Effects of lipopolysaccharide (positive control), live and lysed Trichomonas vaginalis, T. vaginalis EHNA-treated and nontreated, as well as the supernatants of the EHNA reaction, adenosine and inosine (100 μM), on NO production by neutrophils. Scale bars represent the mean ± SD of at least three independent experiments. Data were analyzed by anova, followed by Tukey test (P<0.05). Letters represent differences when compared with: aneutrophils alone (control); bneutrophils stimulated with nontreated intact T. vaginalis;cadenosine; dinosine.

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Phylogenetic analysis and gene expression pattern of ADA in T. vaginalis

In order to identify the ADA-related sequences on T. vaginalis genome, we performed a phylogenetic analysis. NCBI blast searches of GenBank yielded two complete T. vaginalis ADA-related sequences (XP_001317231 and XP_001325125). Semi-quantitative RT-PCR experiments were performed and the relative abundance of ADA-related genes ada(125) and ada(231) mRNA vs. α-tubulin was determined by densitometry. As shown in Fig. 5a and b, both genes were expressed, although ada(231) in higher quantity when compared with the ada(125) : α-tubulin ratio. The phylogenetic tree was constructed using the neighbor-joining method and proportional (p) distance (Fig. 5c). Four well-resolved terminal clades supported by high bootstrap values were identified, confirming the presence of two ADA orthologues for T. vaginalis. The first clade grouped consistently ADA1 vertebrate sequences and ADA-related sequence from T. spiralis. The second clade was formed by E. histolytica, D. discoideum and T. vaginalis sequences. The third clade grouped the ADAL sequences, whereas the fourth clade was formed by ADA2 sequences. Plasmodium falciparum and L. major ADA-related sequences were placed independently between the four clades mentioned. Trypanosoma brucei and E. coli were the most divergent sequences. The tree topology strongly suggests homologous functions on the T. vaginalis genome.

image

Figure 5.  (a) Expression pattern of ADA-related genes ada(125) and ada(231) mRNA in Trichomonas vaginalis trophozoites. The amplifications resulted in a single product. (b) The results were expressed as OD of the ADA-related genes vs. α-tubulin expression (mean ± SE) of four independent replicate RT-PCR experiments. (c) Phylogenetic analysis of ADA-related family members. The deduced amino acid sequences were aligned with clustalx program and the phylogenetic tree was constructed using the neighbor-joining method, proportional (p) distance with mega 4.0 program. The phylogenetic tree grouped consistently. Tv, T. vaginalis; Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio; Dd, Dictyostelium discoideum; Eh, Entamoeba histolytica; Lm, Leishmania major; Pf, Plasmodium falciparum; Ts, Trichinella spiralis; Tb, Trypanosoma brucei; ADA1, ADAL and ADA2 orthologous sequences.

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ADA activity in different T. vaginalis isolates

In order to screen freshly isolated clinical isolates besides TV-VP60, we have determined ADA activity in five other T. vaginalis isolates. One isolate, TV-30236, is from ATCC and long term grown; the isolates TV-LACM1 and TV-LACM2 are fresh clinical isolates, obtained from female patients, while TV-LACH1 and TV-LACH2 are fresh clinical isolates from male patients. All isolates presented ADA activity, although we could not establish a relationship between isolate source and activity (Table S2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Herein, we described the biochemical properties of an ADA activity and two ADA-related sequences present on intact trophozoites of T. vaginalis. Cellular integrity was assessed, before and after the reactions, and the viability of the trophozoites was not affected by any of the conditions used in the assays. The influence of pH on the adenosine deamination in T. vaginalis was verified and the results demonstrated that the optimal pH for ADA activity reached at 7.5. It is known that vaginal pH in noninfected women is approximately 4.3, but can vary from below 4 to pH 7.5 during the menstrual cycle (Stevens-Simon et al., 1994). In agreement, previous studies demonstrated that the optimal pH values for ADA activities from the camel tick, H. dromedarii, and from the trematode F. gigantica were also 7.5 (Mohamed, 2006; Ali, 2008).

Cation exposures (2.5 mM) were able to decrease the adenosine deamination in T. vaginalis in approximately 50%. Higher concentration of calcium (5.0 mM) completely abolished the enzyme activity and the presence of EDTA, a chelating agent, restored ADA activity. Previous data showed that zinc and other divalent cations are able to interact with other amino acid residues and induce an inhibition of the enzyme activity (Cooper et al., 1997; Mohamed, 2006; Rosemberg et al., 2008). Because zinc is toxic to T. vaginalis, we could not perform the experiments on the influence of this metal in ADA activity in intact trophozoites (Langley et al., 1987; Houang et al., 1997). Additional studies are necessary to explain the relevance of the inhibition of ADA activity by calcium and magnesium in T. vaginalis physiology, because magnesium is the most abundant divalent cation in living cells, with a total cellular concentration between 14 and 20 mM (Schmitz et al., 2007).

The substrate curve demonstrated that the apparent KM for adenosine was around 1.13 ± 0.07 mM and the estimated Vmax for adenosine deamination was 2.61 ± 0.054 NH3 min−1 mg−1 protein in T. vaginalis. The kinetic data obtained in this study are in accordance with other studies related to ADA activity, although there are some variations of KM among different ADA members. The KM value of H. dromedarii ADA2 was estimated to 0.5 mM adenosine (Mohamed, 2006), which is relatively close to several ADAs from different sources, such as rat brain (0.45 mM) (Centelles et al., 1988), bovine brain (0.4 mM) (Lupidi et al., 1992), human (0.46 mM) and chicken liver (0.33 mM) (Iwaki-Egawa & Watanabe, 2002). However, lower KM values were reported for ADA activity from mice intestine (0.023 mM) (Singh & Sharma, 2000) and from the sand fly Lutzomyia longipalpis (0.01 mM) (Charlab et al., 2000).

Additional data on biochemical characterization revealed the strong preference of the T. vaginalis enzyme for 2′-deoxyadenosine as a substrate, even higher than that for adenosine. It was already demonstrated that the preference for both adenine nucleosides may be varied and adenosine and 2′-deoxyadenosine are the classical substrates for ADA (Iwaki-Egawa & Watanabe, 2002; Iwaki-Egawa et al., 2004). In order to verify whether adenosine deamination in T. vaginalis may be altered in the presence of a classical inhibitor of ADA1, intact trophozoites were incubated in the presence and in the absence of EHNA. ADA activity from trichomonads was strongly inhibited by increasing concentrations of EHNA, reaching complete inhibition at the highest inhibitor concentration. Furthermore, the ADA inhibition by EHNA was shown to be long lasting; even after the inhibition experiment and the cultivation in TYM medium, the activity could not be detected after 6 h. After 24 h, a very low ADA activity was found, probably due to newly grown trophozoites. Importantly, EHNA-treated T. vaginalis reverted the NO production by neutrophils found in nontreated parasites, indicating the involvement of ADA in the immunomodulatory role of purinergic signalling.

Finally, to demonstrate the presence of ADA in T. vaginalis at the molecular level, the results revealed that two ADA-related gene sequences were expressed in trophozoites. In addition, the phylogenetic analysis showed four well-resolved terminal clades, confirming the presence of two ADA orthologues for T. vaginalis in the second clade with other protozoa species, E. histolytica and D. discoideum sequences.

Trichomonas vaginalis ADA could be involved in the inflammatory process generated during the infection. Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with T. vaginalis infection (Demirezen et al., 2000), and their recruitment is known to be mediated via interleukin-8 (IL-8) (Ryu et al., 2004). Extracellular ATP stimulates IL-8 release and, conversely, adenosine inhibits IL-8 secretion (Bouma et al., 1996; Kukulski et al., 2009). Our contribution differs from that of Munagala & Wang (2003), who identified the presence of ADA activity in T. vaginalis lysates, in the parasites’ cytoplasm. The present study was performed using intact trophozoites, indicating the presence of extracellular ADA activity. During the infection, it is conceivable that T. vaginalis requires the uptake of adenosine, the primary precursor of all purine nucleotides. Consequently, decreased amounts or the lack of adenosine as an anti-inflammatory agent could result in acute symptoms due to raised inflammation. To overcome this adverse situation, the parasite has ADA activity to degrade adenosine to inosine, which also presents anti-inflammatory effects (Haskóet al., 2004). In addition, both endothelial cells and neutrophils have been consistently reported to release high levels of adenosine at sites of metabolic distress, inflammation and infection. The concentrations of extracellular adenosine are below 1.0 μM in unstressed tissues, whereas adenosine levels in inflamed or ischemic tissues can be as high as 100 μM (Haskó & Cronstein, 2004). Therefore, in the microenvironment of trichomonad infection, ADA would modulate the adenosine : inosine ratio and the maintenance of related immunological properties through different nucleoside signalling mechanisms at immune cells. Further studies are necessary to better understand the physiological significance of this enzyme in T. vaginalis and the association with the mechanisms involved in specific host–parasite interactions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

M.W. is a recipient of a fellowship from BIC/PROPESQ/UFRGS; P.d.B.V. and D.B.R. from CAPES; and A.P.F., from CNPq. This study received financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, T.T., grant #477348/2008-4).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Time (a) and protein (b) concentration curves to ensure linearity on ADA activity in intact trophozoites Trichomonas vaginalis.

Table S1. Substrate specificity of ADA from Trichomonas vaginalis.

Table S2. ADA-specific activities from different Trichomonas vaginalis isolates.

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