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Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite
International Journal for Parasitology 42 (2012) 819–827
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Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite André L.A. Dos-Santos a,b, Claudia F. Dick b,c, Michele Alves-Bezerra a,d, Thaís S. Silveira b,c, Lisvane Silva Paes e, Katia C. Gondim a,d, José R. Meyer-Fernandes a,b,⇑ a
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, 21941-590 Rio de Janeiro, Brazil Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil d Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, 21941-590 Rio de Janeiro, Brazil e Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-000 São Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 30 May 2012 Accepted 31 May 2012 Available online 26 June 2012 Keywords: Trypanosoma rangeli Epimastigotes Rhodnius prolixus Salivary glands Cell adhesion P-Tyr ecto-phosphatase activity
a b s t r a c t Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ectophosphatase of living T. rangeli epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and b-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyrresidues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Trypanosoma rangeli is a protozoan that parasitizes many eukaryotic organisms. It infects mammals including humans as well as other animals, but it is completely harmless to its mammalian hosts even though the parasitism can persist for several months or years (Dick et al., 2010; Grisard et al., 2010; Stoco et al., 2012). Trypanosoma rangeli, however, is pathogenic to triatomine vectors such as Rhodnius prolixus (Fonseca-de-Souza et al., 2008, 2009; Gurgel-Gonçalves et al., 2012). Trypanosoma rangeli has a complex life ⇑ Corresponding author at: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil. Tel.: +55 21 2562 6781; fax: +55 21 2270 8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
cycle, involving distinct morphological and functional forms within the vector, but the basis of its infectivity is still poorly understood. The interaction between the parasite and its invertebrate host begins with the ingestion of trypomastigote forms by the insect. After a blood meal, the trypomastigotes differentiate into epimastigotes in the midgut and multiply intensely. A few days after infection, they cross the intestinal barrier to reach the hemolymph, where the short epimastigotes soon disappear to be replaced by a massive colonization of long epimastigotes. The long epimastigotes survive in the hemolymph and/or inside the hemocytes and the parasites subsequently complete their cycle in the salivary glands of the insect, where metacyclogenesis takes place (Azambuja and Garcia, 2005; Gomes et al., 2006; Fonseca-de-Souza et al., 2009; Dick et al., 2010). Thus, in the T. rangeli life cycle, a critical step is the
0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.05.011
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adhesion of the epimastigotes to the R. prolixus salivary gland surface, which culminates in invasion of the organ (De Oliveira and De Souza, 2001; Azambuja and Garcia, 2005). It is interesting to note that T. rangeli epimastigotes are able to attach to the R. prolixus salivary gland surface in vitro (Basseri et al., 2002). Many studies have demonstrated that ecto-phosphatase activities are linked to cell adhesion and invasion by several microorganisms, including protozoa (Zhong et al., 1998; Anaya-Ruiz et al., 2003; Aguirre-García and Okhuysen, 2007), bacteria (Wong et al., 2011) and fungi (Kneipp et al., 2004; Collopy-Junior et al., 2006; Portela et al., 2010). In the case of T. rangeli, it has been demonstrated that living parasite epimastigotes can hydrolyze the artificial substrate, p-nitrophenyl phosphate (pNPP), and this activity is strongly inhibited by ammonium molybdate, sodium orthovanadate and zinc chloride, three well-known phosphotyrosine (PTyr) phosphatase inhibitors (Fonseca-de-Souza et al., 2009; Dick et al., 2010). The objective of this work was to investigate the role of T. rangeli ectophosphotyrosine phosphatase activity in adhesion to R. prolixus salivary glands, with the aim of elucidating the mechanisms underlying the pathogenicity of the parasite towards the insect. 2. Materials and methods 2.1. Materials All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO, USA). Nitrocellulose membranes for western blotting analyses were from GE Healthcare Lifesciences (São Paulo, Brazil). The anti-P-Tyr antibody and the secondary antibody (anti-mouse IgG alkaline conjugated antibody) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The substrate for alkaline phosphatase was purchased from Promega (São Paulo, Brazil). Distilled water was deionized using a Milli-Q system of resins (Millipore Corp., Bedford, MA, USA) and was used in the preparation of all solutions. 2.2. Parasites and growth conditions Epimastigote forms of both H14 and Macias (Mac) strains of T. rangeli were supplied by Dr. Maria Auxiliadora Sousa, Fiocruz, Rio de Janeiro, Brazil, and those of the T. rangeli Choachi (Cho) strain were supplied by Dr. Alessandra Guarneri, Centro de Pesquisa René Rachou, Belo Horizonte, Brazil. The Y and Dm28c Trypanosoma cruzi strains were also obtained from Dr. Maria Auxiliadora Sousa. The H14 and Cho strains were maintained in liver infusion tryptose medium (LIT) supplemented with 20% FCS (Cripion, Rio de Janeiro, Brazil) at 28 ± 2 °C and subcultivated for periods of 7 days, during which the parasites attained stationary growth phase in the short epimastigote form. When long epimastigote forms were used, the second period of subculture was 24–30 days (Gomes et al., 2006). The culture medium consisted of: 4.0 g/L NaCl, 0.4 g/L KCl, 7.1 g/L Na2HPO4, 2.0 g/L glucose, 5.0 g/L liver infusion broth; 5.0 g/L tryptose, 200 mg/L hemin and 30 mg/L folic acid, supplemented with 20% FCS. The pH was adjusted to 7.2 with HCl. Immediately prior to all assays, the parasites were harvested from the culture medium by centrifugation at 1,500g at 4 °C for 10 min and washed three times in cold 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0) (Dick et al., 2010) to ensure complete removal – and to avoid any interference – of the phosphate present in the culture medium. The purity (95–100%) of the short and long epimastigote forms at the corresponding period of culture was assessed by light microscopy. The Mac strain of T. rangeli and both strains of T. cruzi, which do not differentiate into short and long epimastigote forms, were subcultivated for 5 and 7 days,
respectively. The optimal concentration of FCS for the T. cruzi cultures was 10%. The cellular viability of the parasites was assessed after and before incubations using Trypan blue dye exclusion and motility. The cells were incubated in the presence of 0.01% (v/v) Trypan blue for 10 min in the buffer used for each experiment. Their viability was not affected under the conditions employed here (Dick et al., 2010). Viability of T. rangeli after treatment with different phosphatase inhibitors was also routinely assayed by Trypan blue exclusion and by observation of motility, because cell damage could compromise interaction between the parasite and the salivary gland of R. prolixus. The inhibitors used throughout this work did not affect parasite viability. 2.3. Ecto-phosphatase activity The phosphatase activity was determined using P-Tyr as a substrate, measuring the rate of inorganic phosphate (Pi) production (Amazonas et al., 2009). Epimastigotes of T. rangeli (3.0 107 cells) were incubated at 25 °C for 1 h in 0.5 mL of 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.2), 5 mM P-Tyr. Pi release was linear over time under these experimental conditions. Different phosphatase inhibitors (ammonium molybdate, sodium orthovanadate, zinc chloride and levamisole) or EDTA were used in experiments aimed at characterizing the sensitivity of the activity and to compare their influence in the adhesion of T. rangeli with the R. prolixus salivary glands. The tubes were centrifuged at 1,500g for 15 min at 4 °C and the supernatants were analyzed spectrophotometrically for Pi following the Fiske and Subbarow (1925) method. The phosphatase activity was calculated by subtracting the nonspecific P-Tyr hydrolysis measured in the absence of parasite cells from the total hydrolysis. 2.4. Insects Insects were taken from a colony of R. prolixus maintained at 28 °C and 80–90% relative humidity. Adult insects were fed on live rabbit blood at 3 week intervals, starting 15 days after molting. Experimental insects were adult males fasted for 5 weeks after the second or third meal. All animal care and experimental protocols were conducted following the guidelines of the institutional care and use committee (Committee for Evaluation of Animal Use for Research from the Federal University of Rio de Janeiro, CAUAP-UFRJ, Brazil). The protocols were approved by CAUAP-UFRJ under registry #IBQM001. 2.5. Isolation of R. prolixus salivary glands Intact salivary glands were carefully collected by cutting off the heads of the insects and immediately immersing them in PBS solution at pH 7.2. Whole salivary glands were dissected under stereoscopic vision (20). To obtain homogenates for immunodetection of phosphorylated proteins at Tyr residues, the glands (50 pairs) were first washed three times with PBS to remove the saliva. After the third centrifugation in microcentrifuge tubes at 1,500g for 10 min, the sedimented glands were macerated using a glass homogenizer, resuspended in PBS (to a final volume of 50 lL), and centrifuged at 9,700g for 1 min. The protein concentration of the final sediment to be used in western blotting analysis was determined by the method of Bradford (1976), using BSA as a standard. 2.6. Interaction between T. rangeli and R. prolixus salivary glands The protozoa were bound to the insect salivary glands following the methodology described by Fampa et al. (2003) with slight
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modifications. Briefly, long epimastigotes of T. rangeli (106, parasites in 200 lL) were added to the dissected salivary glands (three pairs per interaction assay) and incubated for 1 h at room temperature in 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0). In some experiments, the interaction was assayed in the presence of EDTA or the phosphatase inhibitors mentioned in Section 2.3. The salivary glands were then extensively washed with the same buffer, individually transferred to microcentrifuge tubes containing 50 lL of buffer and homogenized. The released trypanosomatids were counted in a Neubauer chamber.
2.7. Light microscopy The interactions of trypanosomatids with insect salivary glands were assayed as described in Section 2.6. After washing with buffer, the salivary glands were carefully transferred to slides and immediately observed under a Zeiss Axioplan 2 light microscope equipped with a Color View XS digital camera. Parasites were easily identified by differential interference contrast (DIC) microscopy due to their active movement. Images were gamma-corrected using Adobe Photoshop CS3 version 10.0 (Adobe Systems Incorporated, USA).
2.8. Immunodetection of P-Tyr-proteins in extracts of salivary glands from R. prolixus Samples of gland extracts (20 lg of total protein) were boiled for 5 min, subjected to electrophoresis (10% SDS–PAGE) and transferred to nitrocellulose membranes at 350 mA for 90 min. After transfer, the proteins were visualized using Ponceau red (10 min) to verify that the protein loading was similar in the three experiments carried out with different extract preparations and then the membranes were washed with deionized water to remove excess dye. The membranes were blocked for 1 h with 3% BSA in a solution containing 150 mM NaCl, 50 mM Tris–HCl (pH 7.5) and 0.1% Tween-20 (TBS-T). Western blot analysis was performed by incubating the membranes overnight in the presence of the antiP-Tyr antibody (1:2,000 dilution). A secondary anti-mouse IgG alkaline phosphatase conjugated antibody (1:5,000, 30 min) was used to reveal the immunocomplexes and the bands stained with the substrate for alkaline phosphatase.
2.9. Trypanosoma rangeli hemolymph inoculation Trypanosoma rangeli hemolymph was inoculated following the methodology described by Ferreira et al. (2010). Briefly, 1 lL of the trypanosome suspension (106 cells/mL) was inoculated into the insect thorax using a 10 lL Hamilton syringe connected to a fine glass needle (Mello et al., 1995; Gomes et al., 1999). One day after infection, the insects were exposed to citrate-treated human blood through an artificial feeding apparatus (Dick et al., 2010). The salivary glands of these insects were dissected and homogenized, and the number of parasites that had reached these organs was counted in a Neubauer hemocytometer under a light microscope.
2.10. Statistical analysis Data were analyzed statistically using an ANOVA followed by a Tukey’s multiple comparison post hoc test or t-test as indicated in the figure legends, using computer analysis (Graph Pad Prism 3.02). Statistical significance was considered at P < 0.05.
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3. Results 3.1. Correlation between P-Tyr ecto-phosphatase activity and T. rangeli–R. prolixus salivary gland interaction Preliminary experiments were conducted to optimize the adhesion assay conditions (incubation time, protozoan cells and number of salivary glands) that influence the binding of T. rangeli epimastigotes to R. prolixus salivary glands. In successive experiments, two parameters were kept constant and the other varied. Adhesion increased linearly with time for (more than) 1 h using three pairs of glands and 107 protozoan cells. Therefore, these were the conditions used throughout this group of experiments. Preliminary experiments were also carried out to optimize the P-Tyr ecto-phosphatase assay conditions. The hydrolysis of P-Tyr also increased linearly for (more than) 1 h using a cell density of 3 107 in 500 lL of incubation medium in all experiments, including those that involve pre-incubation and washing. Another control demonstrated that the P-Tyr phosphatase of intact T. rangeli is a surface enzyme and not a secreted one: if the parasites were incubated for 1 h in different incubation media without the phosphorylated substrate and then removed by centrifugation, the resulting supernatant was unable to hydrolyze P-Tyr. Another important preliminary set of experiments aimed to investigate the optimal age of culture for use. P-Tyr ecto-phosphatase of T. rangeli epimastigote forms slowly increases with the age of a culture maintained in the same medium. It presents a lag phase, being barely detectable at 14 days (Gomes et al., 2006); then gradually rises and stabilizes at high levels between 24 and 30 days and for this reason parasites aged 30 days were used throughout. Three different strains of T. rangeli were analyzed: H14, Cho and Mac. In Fig. 1A it can be seen that the long epimastigote forms (24– 30 days of culture) of the H14 and Cho T. rangeli strains exhibited a similar adhesion capacity, approximately 10-fold higher than that found with the corresponding short forms. The epimastigotes from the Mac T. rangeli and the Y and Dm28c T. cruzi strains barely adhered to the insect glands. This adhesion profile closely matched the P-Tyr ecto-phosphatase activity measured using intact living parasite cells (Fig. 1B): the long forms of the H14 and Cho strains presented with the highest ecto-phosphatase activity, six to eight times higher than that of the short forms. The epimastigotes of parasites that do not differentiate into long and short forms (and adhered poorly to the salivary glands) presented very low ectophosphatase activity, as previously shown for the Y strain using pNPP as substrate (Furuya et al., 1998). On the basis of these observations, the T. rangeli H14 strain was used throughout the following experiments.
3.2. In vivo and in vitro effects of P-Tyr ecto-phosphatase inhibitors on T. rangeli–R. prolixus salivary gland interaction The parallel between adhesion capacity and P-Tyr ecto-phosphatase activity was confirmed by using different phosphatase inhibitors (Fig. 2). Over a wide concentration range of drugs that allow different degrees of inhibition to be observed, the well-known P-Tyr ecto-phosphatase inhibitors sodium orthovanadate (Na3VO4), ammonium molybdate [(NH4)2MoO4] and zinc chloride (ZnCl2; Dick et al., 2010) progressively shut down both parasite adhesion to the glands and hydrolysis of P-Tyr. This was catalyzed by the intact parasites in a similar proportion for each compound, reaching very low values in the presence of 1 mM Na3VO4 or (NH4)2MoO4, being barely detectable with the use of ZnCl2. The gradual, smooth inhibition by Na3VO4, the initially ineffective action of (NH4)2MoO4 followed by a sudden drop above 0.6 mM, and the strong inhibitory effect of ZnCl2 at low micromolar
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Fig. 1. Adhesion of epimastigotes from different Trypanosoma rangeli (H14, Macias (Mac), Choachi (Cho)) and Trypanosoma cruzi strains (Y and Dm28c) to salivary glands of Rhodnius prolixus (A) and phosphotyrosine (P-Tyr) ecto-phosphatase activity from living T. rangeli and T. cruzi epimastigote cells (B). The epimastigote forms and the strains are indicated on the abscissae. The values represent means ± S.E.s of at least five independent experiments (different cultures and different glands). Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test.
concentrations, are profiles shared by both of the phenomena under study. The insets in Fig. 2 demonstrate the tight linear correlation between the extent of adhesion and P-Tyr ecto-phosphatase activity over a wide range of concentrations of the different inhibitors. In contrast, levamisole (an alkaline phosphatase inhibitor) depressed the ecto-phosphatase activity only slightly and had no influence on adhesion (see legend to Fig. 2). The aim of the experiments depicted in Figs. 3 and 4 was to investigate whether adhesion and ecto-phosphatase activity also behave in parallel when reversible and irreversible inhibition were examined. Trypanosoma rangeli cells were pre-incubated for 1 h in the presence of three different concentrations of Na3VO4 or (NH4)2MoO4 (Fig. 3), then washed and finally assayed for adhesion to insect glands and ecto-phosphatase activity. With high concentrations of Na3VO4, which impair the ecto-phosphatase activity irreversibly, the parasites lost their capacity to interact with the insect glands. In contrast, the ecto-phosphatase activity crippled by (NH4)2MoO4 recovered fully after washing, as did the ability of parasites to interact with the salivary glands. The experiments depicted in Fig. 4 demonstrate that EDTA, which modulates P-Tyr phosphatases in opposite ways depending on the enzyme source and the substrate used (Mei and Huganir, 1991; Pallen et al., 1991; Wang and Pallen, 1991; Walton and Dixon, 1993), also inhibits to the same great extent (at 1 mM) both adhesion and PTyr ecto-phosphatase activity. When the T. rangeli epimastigotes were pre-incubated for 1 h with EDTA and then washed, they also completely recovered their ecto-phosphatase activity and their capacity for interaction with salivary glands of the insect. The dynamics of the T. rangeli–salivary gland interaction was analyzed by DIC microcopy. In all protozoan–gland pairs, two or more T. rangeli cells were usually seen attached to the same site on the salivary gland surface, with no preferential adhesion to any particular region of the organs (Fig. 5A and B), and with formation of parasite clusters in a great number of cases. The interaction was greatly inhibited or suppressed by 1 mM Na3VO4 (Fig. 5C and D) and remained impaired after pre-incubation with the drug followed by washing (Fig. 5E and F). The aim of the experiments depicted in Fig. 6 was to investigate whether T. rangeli cells treated with Na3VO4 and then washed – and therefore with their P-Tyr ecto-phosphatase activities strongly inhibited – could colonize the salivary glands when injected into R. prolixus hemolymph. The number of protozoa adhering to the surfaces of the insect glands 10 days after blood ingestion (1 day after
thoracic injection) was very much lower than observed with untreated cells (compare empty and filled bars). 3.3. P-Tyr residues of salivary gland as possible targets of T. rangeli PTyr ecto-phosphatase activity A competition assay is presented in Table 1. The rationale for this type of experiment was that exogenous P-Tyr could compete with structural P-Tyr residues in the insect salivary gland surface proteins for the ecto-phosphatase located on the T. rangeli surface. Only P-Tyr – and pNPP, an artificial substrate for P-Tyr phosphatases – was able to diminish interaction with the parasite to any great extent. P-Ser, P-Thr and b-glycerophosphate were completely ineffective in perturbing the adhesion process. The dose–response curves for the inhibition of adhesion by exogenous P-Tyr and pNPP were quite similar (Fig. 7). Finally, Fig. 8 shows immunoanalysis of P-Tyr residues in proteins present in the extracts of R. prolixus salivary glands. Western blot analysis of the salivary gland homogenate probed with anti-PTyr reveals a great amount of potential targets for P-Tyr phosphatases, including that detected in the surface of T. rangeli. 4. Discussion The key findings of the present work are a strict correlation between a surface tyrosine phosphatase activity in the long epimastigote forms of T. rangeli and the capacity of the parasite to interact with R. prolixus salivary glands. Therefore, it is likely that dephosphorylation of tyrosine residues at the external surface of the insect gland cells is crucial for the adhesion of the parasite before invasion. Trypanosoma rangeli is the only trypanosomatid able to invade the salivary glands of its different vectors (Azambuja and Garcia, 2005), even though the process occurs with different intensities and at different rates depending on the Rhodnius sp. (D’Alessandro, 1976). On the basis of the results presented here, it may be speculated that the extent of tyrosine phosphorylation at the gland surface is one factor involved in the degree and rate of invasion, which profoundly alter the capacity of the insects to feed (Garcia et al., 1994). It is of interest that only the long epimastigote forms of two different T. rangeli strains can interact with salivary glands in vitro, whereas the short forms of the same strains – and also T. cruzi
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Fig. 2. Concentration dependence of the effect of the phosphotyrosine (P-Tyr) ecto-phosphatase activity inhibitors Na3VO4, (NH4)2MoO4 and ZnCl2 on the adhesion of Trypanosoma rangeli long epimastigotes to the Rhodnius prolixus salivary gland surfaces (A, C and E) and on P-Tyr ecto-phosphatase activity of living long epimastigotes (B, D and F). The values represent means ± S.E.s of at least three independent experiments with different protozoa and glands. S.E.s were calculated from absolute values and converted to percentage values. The control value for P-Tyr ecto-phosphatase activity was 9.2 ± 1.4 nmol Pi h 1 (107 cells) 1 and for adhesion was 2,000 ± 380 protozoa/ pair of salivary glands (no additions). ⁄P < 0.05 compared with the corresponding control without inhibitors as assessed by t-test. With the use of 1 mM of each inhibitor, the remaining percentages of adhesion and P-Tyr ecto-phosphatase activity were, respectively: 13.6 ± 1.4 and 17.3 ± 10.3 (Na3VO4), 10.3 ± 6.3 ((NH4)2MoO4), 0.3 ± 0.02 and 0.2 ± 0.05 (ZnCl2). The insets show the linear correlation between percentage adhesion and percentage P-Tyr ecto-phosphatase activity (with respect to the control) at different concentrations of the three inhibitors used (r2 = 0.97 for Na3VO4; r2 = 0.95 for (NH4)2MoO4; r2 = 0.98 for ZnCl2). The percentages of adhesion and P-Tyr ectophosphatase activity in the presence of 1 mM levamisole were 105.0 ± 4.2 and 76.4 ± 19.9 (without statistical difference with respect to the controls without additions).
epimastigotes – cannot (Fig. 1A). Among this varied ensemble of strains and forms, identical profiles are seen when the extents of adhesion (Fig. 1A) and P-Tyr ecto-phosphatase activity (Fig. 1B) are compared. This is clear evidence for the tight correlation between the two processes, an inference further supported by the experiments depicted in Fig. 2. The degree of adhesion and the activity of the P-Tyr ecto-phosphatase were investigated using different inhibitors of the enzyme, and the results showed that when the activity is gradually decreased, the adhesion is compromised to a similar extent. When the long epimastigote cells of T. rangeli were first incubated with (NH4)2MoO4, a reversible inhibitor of P-Tyr ecto-phosphatases, and then washed, both adhesion and enzyme activity were restored to control levels (Fig. 3A), whereas after
irreversible enzyme inactivation with a high concentration of Na3VO4 followed by the same washing maneuver, the interaction of the parasites with salivary glands was almost completely lost (Fig. 3B). It is important to note that vanadate can alter other orthophosphate-mediated processes (Klarlund, 1985), being a potent inhibitor of cation transport P-ATPases (Cunha et al., 1992; Sodré et al., 2009). However, vanadate is not a good inhibitor of these enzymes on intact cells as the oxidation reduction reactions that take place in the cytoplasm diminish its inhibitory effect (Cantley and Aisen, 1979; Martiny et al., 1996). With respect to ZnCl2, it is noticeable that the progressive inhibition of adhesion and activity is completed at 100 lM of the divalent cation, demonstrating that the long epimastigote form of the T. rangeli H14 strain
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Fig. 3. Effect of pre-incubation of Trypanosoma rangeli with (NH4)2MoO4 (A and B) and Na3VO4 (C and D) on the parasite–salivary gland interaction (A and C) and on phosphotyrosine (P-Tyr) ecto-phosphatase activity in living parasite cells (B and D). Parasites were pre-incubated for 1 h in the presence of the concentrations of the inhibitors shown on the abscissae and washed three times with 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0). After mixing with this solution, the parasite suspensions were centrifuged at 1,500g. The values represent means ± S.E.s of at least three independent experiments that were converted to percentage values. Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test. NS, no statistical difference. CTRL, no addition.
Fig. 4. Effects of EDTA on the adhesion of Trypanosoma rangeli to Rhodnius prolixus salivary glands (A) and on phosphotyrosine (P-Tyr) ecto-phosphatase activity of the parasite (B). In one class of experiments EDTA was added to the assay media used to investigate the two phenomena. In the other, parasites were pre-incubated for 1 h in the presence of 1 mM EDTA, washed three times with 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0), recovered after centrifugation at 1,500g and used immediately. The values represent means ± S.E.s of at least three independent experiments. S.E.s were calculated from absolute values that were converted to percentage values. Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test. CTRL, no addition.
is extremely sensitive to ZnCl2. This response contrasts with that found with the Mac strain, which is partially or totally resistant to millimolar concentrations of zinc when grown at low and high phosphate, respectively (Dick et al., 2010). Overall, these manipulations of the enzyme activity give the second line of evidence for the correlation between the two processes. It also seems clear that interaction of the parasite with the gland cells depends on the P-Tyr activity itself and not on the type of epimastigote. In the case of T. cruzi, infectivity is also associated with P-Tyr ectophosphatase in the trypomastigote and amastigote forms, which
exhibit high enzyme activity, in contrast to that in the non-infective epimastigote forms, which have a low capacity to hydrolyze phosphorylated Tyr residues (Furuya et al., 1998; Meyer-Fernandes et al., 1999). The results shown in Fig. 4 demonstrate that the P-Tyr ectophosphatase activity from T. rangeli can be included in the class of those that are sensitive to the chelator using P-Tyr as substrate. Sensitivity or not towards EDTA seems to be an important clue for the biochemical characterization of P-Tyr phosphatases, since it probably reveals different mechanisms for substrate recognition
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Fig. 5. Interaction between salivary glands of Rhodnius prolixus and Trypanosoma rangeli analyzed by differential interference contrast (DIC) microscopy. Control conditions with no additions (A and B), in the presence of 1 mM Na3VO4 added to the parasite–gland suspension (C and D), and images obtained with T. rangeli epimastigotes preincubated with Na3VO4 and washed as described in the legend to Fig. 3 (E and F). Bars: 30 lm in (A and C–F), and 10 lm in (B). Arrows indicate adhering protozoa.
Table 1 Effect of different phosphatase substrates on the Trypanosoma rangeli–Rhodnius prolixus salivary gland interaction.
Fig. 6. Total Trypanosoma rangeli counts in the salivary glands of adult Rhodnius prolixus after thoracic inoculation with long epimastigotes. Living parasites were pre-incubated for 1 h at 25 °C in PBS with 1 mM Na3VO4. Then the cells were washed three times with PBS and inoculated into R. prolixus as described in Section 2.9. Parasites were counted on the days after the blood meal (DABM) indicated on the abscissa. The values represent the numbers of protozoa in the salivary glands after the blood meal (see details in Section 2.9). Empty bars correspond to untreated parasites; filled bars correspond to vanadate-treated parasites. The values represent means ± S.E.s of a least five independent experiments. ⁄P < 0.05 compared with the matched day control as assessed by t-test.
and catalysis (Mei and Huganir, 1991; Pallen et al., 1991; Wang and Pallen, 1991; Walton and Dixon, 1993).
Additions (0.5 mM)
Protozoa–gland interaction (percentage of control)
None P-Tyr pNPP P-Ser P-Thr b-Glycerophosphate
100.0 ± 19.0 21.4 ± 2.0a 39.1 ± 10.0a 95.9 ± 9.2 93.0 ± 8.5 96.8 ± 11.4
Rhodnius prolixus salivary glands were incubated with T. rangeli long epimastigotes for 1 h at 25 °C. The control interaction (no additions; 2,000 ± 380 protozoa/pair of salivary glands) was taken as 100%. Values shown are the means ± S.E.s of a least five independent experiments (different cultures). S.E.s were calculated from absolute values and converted to percentage values. P-Tyr, phosphotyrosine; pNPP, p-nitrophenyl phosphate; P-Ser, phosphoserine; PThr, phosphothreonine. a P < 0.05 compared with control.
The long T. rangeli epimastigote forms are able to survive in the insect hemolymph and reach the salivary glands (Takle, 1988). Interestingly, when long epimastigotes with their P-Tyr ecto-phosphatase activity irreversibly inhibited are injected into the hemocoel, their capacity to colonize the salivary glands is intensely depressed (Fig. 6). This in vivo observation supports the view that
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Fig. 7. Effect of exogenous phosphotyrosine (P-Tyr) (A) and p-nitrophenyl phosphate (pNPP) (B) on the interaction between Trypanosoma rangeli and salivary glands of Rhodnius prolixus. Intact parasites and salivary glands were incubated at 25 °C in the buffer solution described in the legend to Fig. 3 (final volume: 0.2 mL) in the presence of different concentrations of the substrates, as shown on the abscissae. The values represent means ± S.E.s of at least five independent experiments. ⁄P < 0.05 compared with the control with no added substrates, as assessed by t-test.
Fig. 8. Phosphotyrosine (P-Tyr) proteins in extracts of Rhodnius prolixus salivary glands. Western blotting for tyrosine phosphorylation of salivary gland extracts from R. prolixus was carried out as described in Section 2.8 using an anti-P-Tyr antibody. The figure shows a representative immunostaining of three independent experiments using different glands preparations. Left lane: protein standards; right lane: phospho-immunosignals.
a fully preserved enzyme is required for adhesion of T. rangeli to salivary gland cells, and adds support to the idea that this could be a general prerequisite for the parasite–host cell interaction, as proposed for Entamoeba histolytica and Candida parapsilosis in experiments performed in vitro (Anaya-Ruiz et al., 2003; KifferMoreira et al., 2007). At this point a question emerges: what are the role and the mechanism of the P-Tyr ecto-phosphatase activity in the T. rangeli–salivary gland interaction? To begin with, we can hypothesize that dephosphorylation of P-Tyr residues on the gland cell surfaces enhances adhesion, and this idea is supported the experiments presented in Table 1 and Fig. 7. The inhibition of adhesion to very low values with exogenous pNPP and P-Tyr, but not with other phospho-amino acids or b-glycerophosphate, indicates specific dose-dependent competition between structural P-Tyr residues at the gland cell surface and soluble substrates that are specific for P-Tyr ecto-phosphatases. Possibly, the added substrates occupy the catalytic sites of the external phosphatase molecules on the parasite, thus preventing interaction with phosphorylated residues on the gland cell surfaces. In the case of T. cruzi, the same competition profile was encountered in studies on the invasion of myoblasts by trypomastigote forms (Zhong et al., 1998), the parasite–cell interaction step that follows adhesion. Thus,
dephosphorylation of P-Tyr residues could play a central role in the binding of T. rangeli to salivary gland cells, because ectodephosphorylation of surface phosphoproteins might favor the interaction. The loss of phosphorylation of Tyr residues alters cell morphology and cell-cell interactions owing to disruption of the cytoskeleton, as demonstrated for HeLa cells (Anaya-Ruiz et al., 2003), and in the case of T. rangeli–salivary gland interactions, dephosphorylation of P-Tyr residues could facilitate the cooperative attachment of epimastigotes after disruption of the cell surface. Fig. 8 demonstrates by immunoassay the existence of P-Tyr proteins of different molecular mass in the homogenate of salivary glands from R. prolixus that could be targets for P-Tyr phosphatases. Since incubation of the salivary glands with T. rangeli does not modify the overall phosphorylation profile (data not shown), we propose that only a specific P-Tyr protein, masked within this ensemble with a large number of bands, is the target for the surface P-Tyr ectophosphatase of T. rangeli. This view is supported by preliminary observations from our laboratory, showing that pretreatment of isolated R. prolixus salivary glands with a soluble P-Tyr phosphatase, increases binding of short and long forms of the parasite (Cho strain) by 300% and 60%, as well as that of the Mac strain by 200%. Without treatment with exogenous P-Tyr phosphatase, the interaction of the Cho strain short forms – and of epimastigotes from the Mac strain – is normally low, compared with the long forms of the Cho and H14 strains that also have a very high PTyr ecto-phosphatase activity, as seen in Fig. 1. The DIC observations (Fig. 5) show that groups of epimastigotes attach to the same cell region, forming clusters in many cases. This may indicate that some parasites take advantage of the dephosphorylating action of neighboring specimens within the cluster, in a cooperative attack prior to invasion. In studies concerning the interaction of T. rangeli with R. domesticus salivary glands, punctually damaged areas follow the adhesion of epimastigotes either as single individuals or in clusters (Meirelles et al., 2005). In the present study, we provide evidence that a key molecular mechanism for disruption of the salivary gland surface relies on dephosphorylation of P-Tyr residues likely linked to cytoskeletal structures (Anaya-Ruiz et al., 2003). Thus, P-Tyr ecto-phosphatase activity can be considered an essential player in the ensemble of different enzymes that appear to be involved in the entry of the parasite into salivary gland cells. Acknowledgments This study was supported by Grants from the Brazilian National Research Council (CNPq), the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ), the São Paulo State Research
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Foundation (FAPESP), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the National Institutes of Science and Technology, Brazil. The gifts of different parasite strains from Dr. Alessandra Guarneri (Centro de Pesquisa René Rachou, Brazil) and Dr. Maria Auxiliadora de Souza (Fundação Oswaldo Cruz, Brazil) are also acknowledged. We also thank Dr. Hatisaburo Masuda for allowing access to his colony of insects, Mr. José de S. Lima Junior and Ms. Litiane M. Rodrigues for insect care, Dr. Suzete Gomes for assistance with the triatomine experiments, and Mr. Fabiano Ferreira Esteves and Ms. Rosangela Rosa de Araújo for their excellent technical assistance. Correction of the English in this manuscript was done by BioMedES (UK), which is gratefully acknowledged.
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite André L.A. Dos-Santos a,b, Claudia F. Dick b,c, Michele Alves-Bezerra a,d, Thaís S. Silveira b,c, Lisvane Silva Paes e, Katia C. Gondim a,d, José R. Meyer-Fernandes a,b,⇑ a
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, 21941-590 Rio de Janeiro, Brazil Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil d Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, 21941-590 Rio de Janeiro, Brazil e Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-000 São Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 30 May 2012 Accepted 31 May 2012 Available online 26 June 2012 Keywords: Trypanosoma rangeli Epimastigotes Rhodnius prolixus Salivary glands Cell adhesion P-Tyr ecto-phosphatase activity
a b s t r a c t Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ectophosphatase of living T. rangeli epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and b-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyrresidues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Trypanosoma rangeli is a protozoan that parasitizes many eukaryotic organisms. It infects mammals including humans as well as other animals, but it is completely harmless to its mammalian hosts even though the parasitism can persist for several months or years (Dick et al., 2010; Grisard et al., 2010; Stoco et al., 2012). Trypanosoma rangeli, however, is pathogenic to triatomine vectors such as Rhodnius prolixus (Fonseca-de-Souza et al., 2008, 2009; Gurgel-Gonçalves et al., 2012). Trypanosoma rangeli has a complex life ⇑ Corresponding author at: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil. Tel.: +55 21 2562 6781; fax: +55 21 2270 8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
cycle, involving distinct morphological and functional forms within the vector, but the basis of its infectivity is still poorly understood. The interaction between the parasite and its invertebrate host begins with the ingestion of trypomastigote forms by the insect. After a blood meal, the trypomastigotes differentiate into epimastigotes in the midgut and multiply intensely. A few days after infection, they cross the intestinal barrier to reach the hemolymph, where the short epimastigotes soon disappear to be replaced by a massive colonization of long epimastigotes. The long epimastigotes survive in the hemolymph and/or inside the hemocytes and the parasites subsequently complete their cycle in the salivary glands of the insect, where metacyclogenesis takes place (Azambuja and Garcia, 2005; Gomes et al., 2006; Fonseca-de-Souza et al., 2009; Dick et al., 2010). Thus, in the T. rangeli life cycle, a critical step is the
0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.05.011
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adhesion of the epimastigotes to the R. prolixus salivary gland surface, which culminates in invasion of the organ (De Oliveira and De Souza, 2001; Azambuja and Garcia, 2005). It is interesting to note that T. rangeli epimastigotes are able to attach to the R. prolixus salivary gland surface in vitro (Basseri et al., 2002). Many studies have demonstrated that ecto-phosphatase activities are linked to cell adhesion and invasion by several microorganisms, including protozoa (Zhong et al., 1998; Anaya-Ruiz et al., 2003; Aguirre-García and Okhuysen, 2007), bacteria (Wong et al., 2011) and fungi (Kneipp et al., 2004; Collopy-Junior et al., 2006; Portela et al., 2010). In the case of T. rangeli, it has been demonstrated that living parasite epimastigotes can hydrolyze the artificial substrate, p-nitrophenyl phosphate (pNPP), and this activity is strongly inhibited by ammonium molybdate, sodium orthovanadate and zinc chloride, three well-known phosphotyrosine (PTyr) phosphatase inhibitors (Fonseca-de-Souza et al., 2009; Dick et al., 2010). The objective of this work was to investigate the role of T. rangeli ectophosphotyrosine phosphatase activity in adhesion to R. prolixus salivary glands, with the aim of elucidating the mechanisms underlying the pathogenicity of the parasite towards the insect. 2. Materials and methods 2.1. Materials All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO, USA). Nitrocellulose membranes for western blotting analyses were from GE Healthcare Lifesciences (São Paulo, Brazil). The anti-P-Tyr antibody and the secondary antibody (anti-mouse IgG alkaline conjugated antibody) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The substrate for alkaline phosphatase was purchased from Promega (São Paulo, Brazil). Distilled water was deionized using a Milli-Q system of resins (Millipore Corp., Bedford, MA, USA) and was used in the preparation of all solutions. 2.2. Parasites and growth conditions Epimastigote forms of both H14 and Macias (Mac) strains of T. rangeli were supplied by Dr. Maria Auxiliadora Sousa, Fiocruz, Rio de Janeiro, Brazil, and those of the T. rangeli Choachi (Cho) strain were supplied by Dr. Alessandra Guarneri, Centro de Pesquisa René Rachou, Belo Horizonte, Brazil. The Y and Dm28c Trypanosoma cruzi strains were also obtained from Dr. Maria Auxiliadora Sousa. The H14 and Cho strains were maintained in liver infusion tryptose medium (LIT) supplemented with 20% FCS (Cripion, Rio de Janeiro, Brazil) at 28 ± 2 °C and subcultivated for periods of 7 days, during which the parasites attained stationary growth phase in the short epimastigote form. When long epimastigote forms were used, the second period of subculture was 24–30 days (Gomes et al., 2006). The culture medium consisted of: 4.0 g/L NaCl, 0.4 g/L KCl, 7.1 g/L Na2HPO4, 2.0 g/L glucose, 5.0 g/L liver infusion broth; 5.0 g/L tryptose, 200 mg/L hemin and 30 mg/L folic acid, supplemented with 20% FCS. The pH was adjusted to 7.2 with HCl. Immediately prior to all assays, the parasites were harvested from the culture medium by centrifugation at 1,500g at 4 °C for 10 min and washed three times in cold 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0) (Dick et al., 2010) to ensure complete removal – and to avoid any interference – of the phosphate present in the culture medium. The purity (95–100%) of the short and long epimastigote forms at the corresponding period of culture was assessed by light microscopy. The Mac strain of T. rangeli and both strains of T. cruzi, which do not differentiate into short and long epimastigote forms, were subcultivated for 5 and 7 days,
respectively. The optimal concentration of FCS for the T. cruzi cultures was 10%. The cellular viability of the parasites was assessed after and before incubations using Trypan blue dye exclusion and motility. The cells were incubated in the presence of 0.01% (v/v) Trypan blue for 10 min in the buffer used for each experiment. Their viability was not affected under the conditions employed here (Dick et al., 2010). Viability of T. rangeli after treatment with different phosphatase inhibitors was also routinely assayed by Trypan blue exclusion and by observation of motility, because cell damage could compromise interaction between the parasite and the salivary gland of R. prolixus. The inhibitors used throughout this work did not affect parasite viability. 2.3. Ecto-phosphatase activity The phosphatase activity was determined using P-Tyr as a substrate, measuring the rate of inorganic phosphate (Pi) production (Amazonas et al., 2009). Epimastigotes of T. rangeli (3.0 107 cells) were incubated at 25 °C for 1 h in 0.5 mL of 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.2), 5 mM P-Tyr. Pi release was linear over time under these experimental conditions. Different phosphatase inhibitors (ammonium molybdate, sodium orthovanadate, zinc chloride and levamisole) or EDTA were used in experiments aimed at characterizing the sensitivity of the activity and to compare their influence in the adhesion of T. rangeli with the R. prolixus salivary glands. The tubes were centrifuged at 1,500g for 15 min at 4 °C and the supernatants were analyzed spectrophotometrically for Pi following the Fiske and Subbarow (1925) method. The phosphatase activity was calculated by subtracting the nonspecific P-Tyr hydrolysis measured in the absence of parasite cells from the total hydrolysis. 2.4. Insects Insects were taken from a colony of R. prolixus maintained at 28 °C and 80–90% relative humidity. Adult insects were fed on live rabbit blood at 3 week intervals, starting 15 days after molting. Experimental insects were adult males fasted for 5 weeks after the second or third meal. All animal care and experimental protocols were conducted following the guidelines of the institutional care and use committee (Committee for Evaluation of Animal Use for Research from the Federal University of Rio de Janeiro, CAUAP-UFRJ, Brazil). The protocols were approved by CAUAP-UFRJ under registry #IBQM001. 2.5. Isolation of R. prolixus salivary glands Intact salivary glands were carefully collected by cutting off the heads of the insects and immediately immersing them in PBS solution at pH 7.2. Whole salivary glands were dissected under stereoscopic vision (20). To obtain homogenates for immunodetection of phosphorylated proteins at Tyr residues, the glands (50 pairs) were first washed three times with PBS to remove the saliva. After the third centrifugation in microcentrifuge tubes at 1,500g for 10 min, the sedimented glands were macerated using a glass homogenizer, resuspended in PBS (to a final volume of 50 lL), and centrifuged at 9,700g for 1 min. The protein concentration of the final sediment to be used in western blotting analysis was determined by the method of Bradford (1976), using BSA as a standard. 2.6. Interaction between T. rangeli and R. prolixus salivary glands The protozoa were bound to the insect salivary glands following the methodology described by Fampa et al. (2003) with slight
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modifications. Briefly, long epimastigotes of T. rangeli (106, parasites in 200 lL) were added to the dissected salivary glands (three pairs per interaction assay) and incubated for 1 h at room temperature in 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0). In some experiments, the interaction was assayed in the presence of EDTA or the phosphatase inhibitors mentioned in Section 2.3. The salivary glands were then extensively washed with the same buffer, individually transferred to microcentrifuge tubes containing 50 lL of buffer and homogenized. The released trypanosomatids were counted in a Neubauer chamber.
2.7. Light microscopy The interactions of trypanosomatids with insect salivary glands were assayed as described in Section 2.6. After washing with buffer, the salivary glands were carefully transferred to slides and immediately observed under a Zeiss Axioplan 2 light microscope equipped with a Color View XS digital camera. Parasites were easily identified by differential interference contrast (DIC) microscopy due to their active movement. Images were gamma-corrected using Adobe Photoshop CS3 version 10.0 (Adobe Systems Incorporated, USA).
2.8. Immunodetection of P-Tyr-proteins in extracts of salivary glands from R. prolixus Samples of gland extracts (20 lg of total protein) were boiled for 5 min, subjected to electrophoresis (10% SDS–PAGE) and transferred to nitrocellulose membranes at 350 mA for 90 min. After transfer, the proteins were visualized using Ponceau red (10 min) to verify that the protein loading was similar in the three experiments carried out with different extract preparations and then the membranes were washed with deionized water to remove excess dye. The membranes were blocked for 1 h with 3% BSA in a solution containing 150 mM NaCl, 50 mM Tris–HCl (pH 7.5) and 0.1% Tween-20 (TBS-T). Western blot analysis was performed by incubating the membranes overnight in the presence of the antiP-Tyr antibody (1:2,000 dilution). A secondary anti-mouse IgG alkaline phosphatase conjugated antibody (1:5,000, 30 min) was used to reveal the immunocomplexes and the bands stained with the substrate for alkaline phosphatase.
2.9. Trypanosoma rangeli hemolymph inoculation Trypanosoma rangeli hemolymph was inoculated following the methodology described by Ferreira et al. (2010). Briefly, 1 lL of the trypanosome suspension (106 cells/mL) was inoculated into the insect thorax using a 10 lL Hamilton syringe connected to a fine glass needle (Mello et al., 1995; Gomes et al., 1999). One day after infection, the insects were exposed to citrate-treated human blood through an artificial feeding apparatus (Dick et al., 2010). The salivary glands of these insects were dissected and homogenized, and the number of parasites that had reached these organs was counted in a Neubauer hemocytometer under a light microscope.
2.10. Statistical analysis Data were analyzed statistically using an ANOVA followed by a Tukey’s multiple comparison post hoc test or t-test as indicated in the figure legends, using computer analysis (Graph Pad Prism 3.02). Statistical significance was considered at P < 0.05.
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3. Results 3.1. Correlation between P-Tyr ecto-phosphatase activity and T. rangeli–R. prolixus salivary gland interaction Preliminary experiments were conducted to optimize the adhesion assay conditions (incubation time, protozoan cells and number of salivary glands) that influence the binding of T. rangeli epimastigotes to R. prolixus salivary glands. In successive experiments, two parameters were kept constant and the other varied. Adhesion increased linearly with time for (more than) 1 h using three pairs of glands and 107 protozoan cells. Therefore, these were the conditions used throughout this group of experiments. Preliminary experiments were also carried out to optimize the P-Tyr ecto-phosphatase assay conditions. The hydrolysis of P-Tyr also increased linearly for (more than) 1 h using a cell density of 3 107 in 500 lL of incubation medium in all experiments, including those that involve pre-incubation and washing. Another control demonstrated that the P-Tyr phosphatase of intact T. rangeli is a surface enzyme and not a secreted one: if the parasites were incubated for 1 h in different incubation media without the phosphorylated substrate and then removed by centrifugation, the resulting supernatant was unable to hydrolyze P-Tyr. Another important preliminary set of experiments aimed to investigate the optimal age of culture for use. P-Tyr ecto-phosphatase of T. rangeli epimastigote forms slowly increases with the age of a culture maintained in the same medium. It presents a lag phase, being barely detectable at 14 days (Gomes et al., 2006); then gradually rises and stabilizes at high levels between 24 and 30 days and for this reason parasites aged 30 days were used throughout. Three different strains of T. rangeli were analyzed: H14, Cho and Mac. In Fig. 1A it can be seen that the long epimastigote forms (24– 30 days of culture) of the H14 and Cho T. rangeli strains exhibited a similar adhesion capacity, approximately 10-fold higher than that found with the corresponding short forms. The epimastigotes from the Mac T. rangeli and the Y and Dm28c T. cruzi strains barely adhered to the insect glands. This adhesion profile closely matched the P-Tyr ecto-phosphatase activity measured using intact living parasite cells (Fig. 1B): the long forms of the H14 and Cho strains presented with the highest ecto-phosphatase activity, six to eight times higher than that of the short forms. The epimastigotes of parasites that do not differentiate into long and short forms (and adhered poorly to the salivary glands) presented very low ectophosphatase activity, as previously shown for the Y strain using pNPP as substrate (Furuya et al., 1998). On the basis of these observations, the T. rangeli H14 strain was used throughout the following experiments.
3.2. In vivo and in vitro effects of P-Tyr ecto-phosphatase inhibitors on T. rangeli–R. prolixus salivary gland interaction The parallel between adhesion capacity and P-Tyr ecto-phosphatase activity was confirmed by using different phosphatase inhibitors (Fig. 2). Over a wide concentration range of drugs that allow different degrees of inhibition to be observed, the well-known P-Tyr ecto-phosphatase inhibitors sodium orthovanadate (Na3VO4), ammonium molybdate [(NH4)2MoO4] and zinc chloride (ZnCl2; Dick et al., 2010) progressively shut down both parasite adhesion to the glands and hydrolysis of P-Tyr. This was catalyzed by the intact parasites in a similar proportion for each compound, reaching very low values in the presence of 1 mM Na3VO4 or (NH4)2MoO4, being barely detectable with the use of ZnCl2. The gradual, smooth inhibition by Na3VO4, the initially ineffective action of (NH4)2MoO4 followed by a sudden drop above 0.6 mM, and the strong inhibitory effect of ZnCl2 at low micromolar
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Fig. 1. Adhesion of epimastigotes from different Trypanosoma rangeli (H14, Macias (Mac), Choachi (Cho)) and Trypanosoma cruzi strains (Y and Dm28c) to salivary glands of Rhodnius prolixus (A) and phosphotyrosine (P-Tyr) ecto-phosphatase activity from living T. rangeli and T. cruzi epimastigote cells (B). The epimastigote forms and the strains are indicated on the abscissae. The values represent means ± S.E.s of at least five independent experiments (different cultures and different glands). Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test.
concentrations, are profiles shared by both of the phenomena under study. The insets in Fig. 2 demonstrate the tight linear correlation between the extent of adhesion and P-Tyr ecto-phosphatase activity over a wide range of concentrations of the different inhibitors. In contrast, levamisole (an alkaline phosphatase inhibitor) depressed the ecto-phosphatase activity only slightly and had no influence on adhesion (see legend to Fig. 2). The aim of the experiments depicted in Figs. 3 and 4 was to investigate whether adhesion and ecto-phosphatase activity also behave in parallel when reversible and irreversible inhibition were examined. Trypanosoma rangeli cells were pre-incubated for 1 h in the presence of three different concentrations of Na3VO4 or (NH4)2MoO4 (Fig. 3), then washed and finally assayed for adhesion to insect glands and ecto-phosphatase activity. With high concentrations of Na3VO4, which impair the ecto-phosphatase activity irreversibly, the parasites lost their capacity to interact with the insect glands. In contrast, the ecto-phosphatase activity crippled by (NH4)2MoO4 recovered fully after washing, as did the ability of parasites to interact with the salivary glands. The experiments depicted in Fig. 4 demonstrate that EDTA, which modulates P-Tyr phosphatases in opposite ways depending on the enzyme source and the substrate used (Mei and Huganir, 1991; Pallen et al., 1991; Wang and Pallen, 1991; Walton and Dixon, 1993), also inhibits to the same great extent (at 1 mM) both adhesion and PTyr ecto-phosphatase activity. When the T. rangeli epimastigotes were pre-incubated for 1 h with EDTA and then washed, they also completely recovered their ecto-phosphatase activity and their capacity for interaction with salivary glands of the insect. The dynamics of the T. rangeli–salivary gland interaction was analyzed by DIC microcopy. In all protozoan–gland pairs, two or more T. rangeli cells were usually seen attached to the same site on the salivary gland surface, with no preferential adhesion to any particular region of the organs (Fig. 5A and B), and with formation of parasite clusters in a great number of cases. The interaction was greatly inhibited or suppressed by 1 mM Na3VO4 (Fig. 5C and D) and remained impaired after pre-incubation with the drug followed by washing (Fig. 5E and F). The aim of the experiments depicted in Fig. 6 was to investigate whether T. rangeli cells treated with Na3VO4 and then washed – and therefore with their P-Tyr ecto-phosphatase activities strongly inhibited – could colonize the salivary glands when injected into R. prolixus hemolymph. The number of protozoa adhering to the surfaces of the insect glands 10 days after blood ingestion (1 day after
thoracic injection) was very much lower than observed with untreated cells (compare empty and filled bars). 3.3. P-Tyr residues of salivary gland as possible targets of T. rangeli PTyr ecto-phosphatase activity A competition assay is presented in Table 1. The rationale for this type of experiment was that exogenous P-Tyr could compete with structural P-Tyr residues in the insect salivary gland surface proteins for the ecto-phosphatase located on the T. rangeli surface. Only P-Tyr – and pNPP, an artificial substrate for P-Tyr phosphatases – was able to diminish interaction with the parasite to any great extent. P-Ser, P-Thr and b-glycerophosphate were completely ineffective in perturbing the adhesion process. The dose–response curves for the inhibition of adhesion by exogenous P-Tyr and pNPP were quite similar (Fig. 7). Finally, Fig. 8 shows immunoanalysis of P-Tyr residues in proteins present in the extracts of R. prolixus salivary glands. Western blot analysis of the salivary gland homogenate probed with anti-PTyr reveals a great amount of potential targets for P-Tyr phosphatases, including that detected in the surface of T. rangeli. 4. Discussion The key findings of the present work are a strict correlation between a surface tyrosine phosphatase activity in the long epimastigote forms of T. rangeli and the capacity of the parasite to interact with R. prolixus salivary glands. Therefore, it is likely that dephosphorylation of tyrosine residues at the external surface of the insect gland cells is crucial for the adhesion of the parasite before invasion. Trypanosoma rangeli is the only trypanosomatid able to invade the salivary glands of its different vectors (Azambuja and Garcia, 2005), even though the process occurs with different intensities and at different rates depending on the Rhodnius sp. (D’Alessandro, 1976). On the basis of the results presented here, it may be speculated that the extent of tyrosine phosphorylation at the gland surface is one factor involved in the degree and rate of invasion, which profoundly alter the capacity of the insects to feed (Garcia et al., 1994). It is of interest that only the long epimastigote forms of two different T. rangeli strains can interact with salivary glands in vitro, whereas the short forms of the same strains – and also T. cruzi
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Fig. 2. Concentration dependence of the effect of the phosphotyrosine (P-Tyr) ecto-phosphatase activity inhibitors Na3VO4, (NH4)2MoO4 and ZnCl2 on the adhesion of Trypanosoma rangeli long epimastigotes to the Rhodnius prolixus salivary gland surfaces (A, C and E) and on P-Tyr ecto-phosphatase activity of living long epimastigotes (B, D and F). The values represent means ± S.E.s of at least three independent experiments with different protozoa and glands. S.E.s were calculated from absolute values and converted to percentage values. The control value for P-Tyr ecto-phosphatase activity was 9.2 ± 1.4 nmol Pi h 1 (107 cells) 1 and for adhesion was 2,000 ± 380 protozoa/ pair of salivary glands (no additions). ⁄P < 0.05 compared with the corresponding control without inhibitors as assessed by t-test. With the use of 1 mM of each inhibitor, the remaining percentages of adhesion and P-Tyr ecto-phosphatase activity were, respectively: 13.6 ± 1.4 and 17.3 ± 10.3 (Na3VO4), 10.3 ± 6.3 ((NH4)2MoO4), 0.3 ± 0.02 and 0.2 ± 0.05 (ZnCl2). The insets show the linear correlation between percentage adhesion and percentage P-Tyr ecto-phosphatase activity (with respect to the control) at different concentrations of the three inhibitors used (r2 = 0.97 for Na3VO4; r2 = 0.95 for (NH4)2MoO4; r2 = 0.98 for ZnCl2). The percentages of adhesion and P-Tyr ectophosphatase activity in the presence of 1 mM levamisole were 105.0 ± 4.2 and 76.4 ± 19.9 (without statistical difference with respect to the controls without additions).
epimastigotes – cannot (Fig. 1A). Among this varied ensemble of strains and forms, identical profiles are seen when the extents of adhesion (Fig. 1A) and P-Tyr ecto-phosphatase activity (Fig. 1B) are compared. This is clear evidence for the tight correlation between the two processes, an inference further supported by the experiments depicted in Fig. 2. The degree of adhesion and the activity of the P-Tyr ecto-phosphatase were investigated using different inhibitors of the enzyme, and the results showed that when the activity is gradually decreased, the adhesion is compromised to a similar extent. When the long epimastigote cells of T. rangeli were first incubated with (NH4)2MoO4, a reversible inhibitor of P-Tyr ecto-phosphatases, and then washed, both adhesion and enzyme activity were restored to control levels (Fig. 3A), whereas after
irreversible enzyme inactivation with a high concentration of Na3VO4 followed by the same washing maneuver, the interaction of the parasites with salivary glands was almost completely lost (Fig. 3B). It is important to note that vanadate can alter other orthophosphate-mediated processes (Klarlund, 1985), being a potent inhibitor of cation transport P-ATPases (Cunha et al., 1992; Sodré et al., 2009). However, vanadate is not a good inhibitor of these enzymes on intact cells as the oxidation reduction reactions that take place in the cytoplasm diminish its inhibitory effect (Cantley and Aisen, 1979; Martiny et al., 1996). With respect to ZnCl2, it is noticeable that the progressive inhibition of adhesion and activity is completed at 100 lM of the divalent cation, demonstrating that the long epimastigote form of the T. rangeli H14 strain
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Fig. 3. Effect of pre-incubation of Trypanosoma rangeli with (NH4)2MoO4 (A and B) and Na3VO4 (C and D) on the parasite–salivary gland interaction (A and C) and on phosphotyrosine (P-Tyr) ecto-phosphatase activity in living parasite cells (B and D). Parasites were pre-incubated for 1 h in the presence of the concentrations of the inhibitors shown on the abscissae and washed three times with 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0). After mixing with this solution, the parasite suspensions were centrifuged at 1,500g. The values represent means ± S.E.s of at least three independent experiments that were converted to percentage values. Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test. NS, no statistical difference. CTRL, no addition.
Fig. 4. Effects of EDTA on the adhesion of Trypanosoma rangeli to Rhodnius prolixus salivary glands (A) and on phosphotyrosine (P-Tyr) ecto-phosphatase activity of the parasite (B). In one class of experiments EDTA was added to the assay media used to investigate the two phenomena. In the other, parasites were pre-incubated for 1 h in the presence of 1 mM EDTA, washed three times with 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl (pH 7.0), recovered after centrifugation at 1,500g and used immediately. The values represent means ± S.E.s of at least three independent experiments. S.E.s were calculated from absolute values that were converted to percentage values. Lowercase letters above the bars indicate statistical differences (P < 0.05) as assessed by ANOVA followed by Tukey’s multiple comparison post hoc test. CTRL, no addition.
is extremely sensitive to ZnCl2. This response contrasts with that found with the Mac strain, which is partially or totally resistant to millimolar concentrations of zinc when grown at low and high phosphate, respectively (Dick et al., 2010). Overall, these manipulations of the enzyme activity give the second line of evidence for the correlation between the two processes. It also seems clear that interaction of the parasite with the gland cells depends on the P-Tyr activity itself and not on the type of epimastigote. In the case of T. cruzi, infectivity is also associated with P-Tyr ectophosphatase in the trypomastigote and amastigote forms, which
exhibit high enzyme activity, in contrast to that in the non-infective epimastigote forms, which have a low capacity to hydrolyze phosphorylated Tyr residues (Furuya et al., 1998; Meyer-Fernandes et al., 1999). The results shown in Fig. 4 demonstrate that the P-Tyr ectophosphatase activity from T. rangeli can be included in the class of those that are sensitive to the chelator using P-Tyr as substrate. Sensitivity or not towards EDTA seems to be an important clue for the biochemical characterization of P-Tyr phosphatases, since it probably reveals different mechanisms for substrate recognition
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Fig. 5. Interaction between salivary glands of Rhodnius prolixus and Trypanosoma rangeli analyzed by differential interference contrast (DIC) microscopy. Control conditions with no additions (A and B), in the presence of 1 mM Na3VO4 added to the parasite–gland suspension (C and D), and images obtained with T. rangeli epimastigotes preincubated with Na3VO4 and washed as described in the legend to Fig. 3 (E and F). Bars: 30 lm in (A and C–F), and 10 lm in (B). Arrows indicate adhering protozoa.
Table 1 Effect of different phosphatase substrates on the Trypanosoma rangeli–Rhodnius prolixus salivary gland interaction.
Fig. 6. Total Trypanosoma rangeli counts in the salivary glands of adult Rhodnius prolixus after thoracic inoculation with long epimastigotes. Living parasites were pre-incubated for 1 h at 25 °C in PBS with 1 mM Na3VO4. Then the cells were washed three times with PBS and inoculated into R. prolixus as described in Section 2.9. Parasites were counted on the days after the blood meal (DABM) indicated on the abscissa. The values represent the numbers of protozoa in the salivary glands after the blood meal (see details in Section 2.9). Empty bars correspond to untreated parasites; filled bars correspond to vanadate-treated parasites. The values represent means ± S.E.s of a least five independent experiments. ⁄P < 0.05 compared with the matched day control as assessed by t-test.
and catalysis (Mei and Huganir, 1991; Pallen et al., 1991; Wang and Pallen, 1991; Walton and Dixon, 1993).
Additions (0.5 mM)
Protozoa–gland interaction (percentage of control)
None P-Tyr pNPP P-Ser P-Thr b-Glycerophosphate
100.0 ± 19.0 21.4 ± 2.0a 39.1 ± 10.0a 95.9 ± 9.2 93.0 ± 8.5 96.8 ± 11.4
Rhodnius prolixus salivary glands were incubated with T. rangeli long epimastigotes for 1 h at 25 °C. The control interaction (no additions; 2,000 ± 380 protozoa/pair of salivary glands) was taken as 100%. Values shown are the means ± S.E.s of a least five independent experiments (different cultures). S.E.s were calculated from absolute values and converted to percentage values. P-Tyr, phosphotyrosine; pNPP, p-nitrophenyl phosphate; P-Ser, phosphoserine; PThr, phosphothreonine. a P < 0.05 compared with control.
The long T. rangeli epimastigote forms are able to survive in the insect hemolymph and reach the salivary glands (Takle, 1988). Interestingly, when long epimastigotes with their P-Tyr ecto-phosphatase activity irreversibly inhibited are injected into the hemocoel, their capacity to colonize the salivary glands is intensely depressed (Fig. 6). This in vivo observation supports the view that
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Fig. 7. Effect of exogenous phosphotyrosine (P-Tyr) (A) and p-nitrophenyl phosphate (pNPP) (B) on the interaction between Trypanosoma rangeli and salivary glands of Rhodnius prolixus. Intact parasites and salivary glands were incubated at 25 °C in the buffer solution described in the legend to Fig. 3 (final volume: 0.2 mL) in the presence of different concentrations of the substrates, as shown on the abscissae. The values represent means ± S.E.s of at least five independent experiments. ⁄P < 0.05 compared with the control with no added substrates, as assessed by t-test.
Fig. 8. Phosphotyrosine (P-Tyr) proteins in extracts of Rhodnius prolixus salivary glands. Western blotting for tyrosine phosphorylation of salivary gland extracts from R. prolixus was carried out as described in Section 2.8 using an anti-P-Tyr antibody. The figure shows a representative immunostaining of three independent experiments using different glands preparations. Left lane: protein standards; right lane: phospho-immunosignals.
a fully preserved enzyme is required for adhesion of T. rangeli to salivary gland cells, and adds support to the idea that this could be a general prerequisite for the parasite–host cell interaction, as proposed for Entamoeba histolytica and Candida parapsilosis in experiments performed in vitro (Anaya-Ruiz et al., 2003; KifferMoreira et al., 2007). At this point a question emerges: what are the role and the mechanism of the P-Tyr ecto-phosphatase activity in the T. rangeli–salivary gland interaction? To begin with, we can hypothesize that dephosphorylation of P-Tyr residues on the gland cell surfaces enhances adhesion, and this idea is supported the experiments presented in Table 1 and Fig. 7. The inhibition of adhesion to very low values with exogenous pNPP and P-Tyr, but not with other phospho-amino acids or b-glycerophosphate, indicates specific dose-dependent competition between structural P-Tyr residues at the gland cell surface and soluble substrates that are specific for P-Tyr ecto-phosphatases. Possibly, the added substrates occupy the catalytic sites of the external phosphatase molecules on the parasite, thus preventing interaction with phosphorylated residues on the gland cell surfaces. In the case of T. cruzi, the same competition profile was encountered in studies on the invasion of myoblasts by trypomastigote forms (Zhong et al., 1998), the parasite–cell interaction step that follows adhesion. Thus,
dephosphorylation of P-Tyr residues could play a central role in the binding of T. rangeli to salivary gland cells, because ectodephosphorylation of surface phosphoproteins might favor the interaction. The loss of phosphorylation of Tyr residues alters cell morphology and cell-cell interactions owing to disruption of the cytoskeleton, as demonstrated for HeLa cells (Anaya-Ruiz et al., 2003), and in the case of T. rangeli–salivary gland interactions, dephosphorylation of P-Tyr residues could facilitate the cooperative attachment of epimastigotes after disruption of the cell surface. Fig. 8 demonstrates by immunoassay the existence of P-Tyr proteins of different molecular mass in the homogenate of salivary glands from R. prolixus that could be targets for P-Tyr phosphatases. Since incubation of the salivary glands with T. rangeli does not modify the overall phosphorylation profile (data not shown), we propose that only a specific P-Tyr protein, masked within this ensemble with a large number of bands, is the target for the surface P-Tyr ectophosphatase of T. rangeli. This view is supported by preliminary observations from our laboratory, showing that pretreatment of isolated R. prolixus salivary glands with a soluble P-Tyr phosphatase, increases binding of short and long forms of the parasite (Cho strain) by 300% and 60%, as well as that of the Mac strain by 200%. Without treatment with exogenous P-Tyr phosphatase, the interaction of the Cho strain short forms – and of epimastigotes from the Mac strain – is normally low, compared with the long forms of the Cho and H14 strains that also have a very high PTyr ecto-phosphatase activity, as seen in Fig. 1. The DIC observations (Fig. 5) show that groups of epimastigotes attach to the same cell region, forming clusters in many cases. This may indicate that some parasites take advantage of the dephosphorylating action of neighboring specimens within the cluster, in a cooperative attack prior to invasion. In studies concerning the interaction of T. rangeli with R. domesticus salivary glands, punctually damaged areas follow the adhesion of epimastigotes either as single individuals or in clusters (Meirelles et al., 2005). In the present study, we provide evidence that a key molecular mechanism for disruption of the salivary gland surface relies on dephosphorylation of P-Tyr residues likely linked to cytoskeletal structures (Anaya-Ruiz et al., 2003). Thus, P-Tyr ecto-phosphatase activity can be considered an essential player in the ensemble of different enzymes that appear to be involved in the entry of the parasite into salivary gland cells. Acknowledgments This study was supported by Grants from the Brazilian National Research Council (CNPq), the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ), the São Paulo State Research
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Foundation (FAPESP), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the National Institutes of Science and Technology, Brazil. The gifts of different parasite strains from Dr. Alessandra Guarneri (Centro de Pesquisa René Rachou, Brazil) and Dr. Maria Auxiliadora de Souza (Fundação Oswaldo Cruz, Brazil) are also acknowledged. We also thank Dr. Hatisaburo Masuda for allowing access to his colony of insects, Mr. José de S. Lima Junior and Ms. Litiane M. Rodrigues for insect care, Dr. Suzete Gomes for assistance with the triatomine experiments, and Mr. Fabiano Ferreira Esteves and Ms. Rosangela Rosa de Araújo for their excellent technical assistance. Correction of the English in this manuscript was done by BioMedES (UK), which is gratefully acknowledged.
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