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Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi
Acta Tropica 97 (2006) 140–145
Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi N´estor A˜nez-Rojas, Pablo Garc´ıa-Lugo, Gladys Crisante, Agustina Rojas, N´estor A˜nez ∗ Universidad de Los Andes, Facultad de Ciencias, Departamento de Biolog´ıa, M´erida 5101, Venezuela Received 13 May 2005; received in revised form 8 September 2005; accepted 22 September 2005 Available online 24 October 2005
Abstract GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed. © 2005 Elsevier B.V. All rights reserved. Keywords: GPI; Membrane proteins; Trypanosoma cruzi; Trypanosoma rangeli; Species differentiation
1. Introduction Chagas disease, caused by Trypanosoma (Schyzotrypanum) cruzi, Chagas 1909, is one of the major public health problems in Central and South America. The fact that nearly 20 million people are affected by chronic disease and about 100 million are under risk appears to corroborate the above statement (TDR/WHO, 2005). Trypanosoma (Tejeraia) rangeli Tejera 1920, known as the second American Trypanosoma infecting man, in contrast to T. cruzi is harmless to the vertebrate host but it is pathogenic to the triatomine-bugs acting as vectors
∗
Corresponding author. Tel.: +58 274 2401285; fax: +58 274 2401285. E-mail address: [email protected] (N. A˜nez). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.09.005
of the parasite (A˜nez and East, 1984). Mixed infections caused by both parasites are not uncommon due to the fact that the parasites share the same vertebrate hosts, vectors and have similar geographical distribution (A˜nez, 1982). The co-existence of these parasites in the same natural habits evidenced by the common demonstration of mixed infection and the detection of similar antigenic components, which have been interpreted as the cause of interference for serological tests in epidemiological studies in areas where Chagas disease is endemic, urge for a right diagnosis. On this aspect, several authors, using serological methods, have previously demonstrated antigen cross-reactions between T. rangeli and T. cruzi (Afchain et al., 1979; O’Daly et al., 1994; Gr¨ogl and Kuhn, 1984). However, others have reported several T. cruzi-specific proteins most of them useful in serological tests (Morgado et al., 1989). In addi-
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tion, Salda˜na and Sousa (1996) reported the presence of several common epitopes among T. rangeli antigens (Ag) and between T. rangeli and T. cruzi polypeptides. The authors also suggested that purification of these T. rangeli Ags, which cross-reacted with T. cruzi components, could be important on the diagnosis of Chagas disease. More recently, Salda˜na et al. (1998) described a specific 48 kDa Ag from T. rangeli, which was not detected in T. cruzi epimastigotes. The authors suggest that this Ag may be a useful marker for the identification and characterization of T. rangeli isolates. The above circumstances appear to justify the production of a new reliable methodology capable of differentiating both parasites when present in the same host, which at the same time, result in a fast, specific and cheap diagnostic test. Considering, on one hand, that the major antigenic difference between T. cruzi and T. rangeli appears to be found in the membrane proteins (Bronzina et al., 1980) and on the other hand, that membrane proteins anchored by glycosyl-phosphatidyl-inositol (GPI) have been characterized in different morphological forms of T. cruzi (Ferguson, 1997) in the present paper, we propose the detection and characterization of GPI-anchored membrane proteins to discriminate between T. rangeli and T. cruzi. 2. Materials and methods 2.1. Parasites Four T. cruzi isolates obtained by hemoculture from acute chagasic patients at the Center for Parasitological Research, Faculty of Sciences, University of Los Andes, M´erida, Venezuela, were used. The patients from whom T. cruzi isolates were characterized in this study were from four localities of Barinas state, western Venezuela, where Chagas disease is endemic. Details on the origin, maintenance of the isolates and the clinical features of each patient have been given in a previous publication (A˜nez et al., 2004). Prior to the study, the four T. cruzi isolates were genetically typed as lineage I as previously indicated (A˜nez et al., 2004). The T. cruzi isolates were identified as follows: MHOM/Ve/91/1–91; MHOM/Ve/94/4–94; MHOM/Ve/94/9–94; MHOM/Ve/95/36–95. Similarly, four T. rangeli isolates were used. From these, three were from human active infection detected by hemoculture (MHOM/Ve/99/CH-99; MHOM/Ve/99/D-99; MHOM/Ve/02/GS-02) and one from a dog also detected by hemoculture (MCAN/Ve/00/LOBITA-00). T. rangeli isolates were also obtained from samples taken at the Barinas state, Venezuela. Both T. cruzi and T. rangeli
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isolates were kept in NNN culture medium at 25 ◦ C, until they grew to obtain masses of parasites. When the culture of the parasites reached the exponential phase, they were collected and centrifuged at 3000 × g at 4 ◦ C for 15 min and washed three times in phosphate buffer saline (PBS), pH 7.4 to collect a pellet, which had an approximated concentration of 13 × 106 flagellates/ml, for each of the isolates. 2.2. Isolation and purification of T. rangeli and T. cruzi GPI-anchored membrane proteins GPI-anchored membrane proteins for both T. rangeli and T. cruzi isolates were obtained using the partition Triton X-114 method previously described by Ko and Thompson (1995). Briefly, the collected pellet was resuspended in a 10 mM Tris–HCl, 150 mM NaCl, 2% Triton X-114, pH 7.4 buffer containing a pool of protease inhibitors (Roche), with shaking during 1 h at 0 ◦ C. The suspension was spun at 8000 × g at 0 ◦ C for 10 min. The obtained pellet was then washed with a 10 mM Tris–HCl, 150 mM NaCl, 0.06% Triton X-114, pH 7.4 buffer and kept at −20 ◦ C. The supernatant was kept at −20 ◦ C during 24 h and then it was slowly unfrozen at room temperature and later placed during 12 min at 32 ◦ C to obtain the first partition (P1). P1 was then spun at 3000 × g for 3 min, obtaining two phases. The aqueous phase containing hydrophilic proteins (HP) was separated and kept at −20 ◦ C. The detergent phase was re-suspended in 3 V of the above indicated buffer and kept at 0 ◦ C for 10 min. The P1 process was repeated and the aqueous phase containing the rest of HP was discharged. The detergent phase was then mixed with 3 V of the same buffer, kept at 0 ◦ C for 10 min and spun at 18,000 × g at 0 ◦ C for 10 min. The pellets containing hydrophobic proteins, not GPI-anchored protein (hp) were kept at −20 ◦ C. The supernatant was then submitted to a second partition process (P2). The proteins contained at the detergent phase were precipitated with 3 V of cold acetone and spun at 3000 × g, to obtain the GPI-anchored proteins. 2.3. Protein measurement Concentration of protein fractions (HP, hp and GPI) were measured following the method reported by Lowry et al. (1951) using bovine serum albumin as standard. 2.4. Gel electrophoresis Protein gel electrophoresis was carried out on 12% sodium dodecyl sulphate polyacrylamide (SDS-PAGE)
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on a 0.75 mm thick minigel and stained with Comassie blue R-250 as described by Laemmli (1970). Samples of proteins were precipitated with 3 V of cold acetone and re-suspended in sampling buffer (0.2% SDS, 10% glycerol, 50 mM dl-dithiothreitol, 168 mM Tris–HCl, pH 6.8). The molecular weight markers were from Biorad® . 2.5. Rabbit immunization with T. rangeli and T. cruzi GPI-anchored proteins Concentration of GPI-anchored proteins from epimastigotes of both species of parasites were quantified following the method of Lowry et al. (1951). For both T. rangeli and T. cruzi, the amount of protein to be used was re-suspended in 500 l PBS and mixed 1:1 with Freund’s adjuvant to 1 ml final volume. Immunizations with proteins of each of the parasites were carried out in rabbits following the schedule of three challenges, 15 days each. The first immunization was done with 100 g protein, followed by a second and third challenge with 50 g each. 2.6. Indirect immunofluorescence to detect surface GPI-anchored proteins in cultured specimens of T. rangeli and T. cruzi Cultured T. rangeli and T. cruzi epimastigotes were placed on glass slides, dried at room temperature and fixed with 100% acetone. The slides were then incubated in wet chamber during 1 h with sera anti-T. rangeli and T. cruzi GPI-anchored membrane proteins from immunized rabbits, using dilutions from 1:64 to 1:2048. Later, the slides were washed three times in PBS and incubated at dark with 1:80 diluted antirabbit IgG FIT conjugate (Sigma) for 1 h, washed three times in PBS and then mounted in buffered glycerin to be observed under a fluorescent microscope at 400×. 2.7. Western blot analysis Following SDS-PAGE, the proteins were electrotransferred (100 V constant for 1 h at 4 ◦ C) to PVDF membranes (Immobilon P). The anti-T. rangeli and antiT. cruzi GPI-anchored proteins antibodies (Abs) diluted 1:300, were incubated overnight with the membranes and after a thorough washing, a 1:15,000 rabbit serum antiIgG linked peroxidase was added and incubated for 3 h. Reaction was developed using 3,3 -diaminobenzidine as a substrate.
2.8. Evidence for GPI-linked proteins by phosphatidyl-inositol specific phospholipase C (PIPLC) treatment A 60 g of delipidated GPI-anchored proteins was re-suspended in 300 l of 100 mM Tris–HCl, pH 7.5 containing 0.1% sodium deoxycholate and two units of Bacillus cereus PIPLC. The suspension was incubated 4 h at 37 ◦ C following Ko and Thompson (1995). Proteins of reaction mixture were precipitated with cold acetone and then re-solubilized in 2% Triton X-114, 150 mM NaCl and 10 mM Tris–HCl, pH 7.4. After partitioning, the proteins were precipitated both from the aqueous phase and the Triton X-114 phase with cold acetone and then re-suspended in sampling buffer. 2.9. Biotinylation of parasite surface proteins Parasites were washed three times with cold PBS pH 8.0 and re-suspended at a concentration of 2 × 107 epimastigotes/ml. Later, 0.5 mg/ml sulpho-NHS-LC-biotin was added and incubated 30 min at room temperature and then washed three times in the same solution as recommended by Altin and Pagler (1995). After purification of biotynilated parasites, a SDS-PAGE was performed with the separated protein fractions (HP, hp and GPI), which were electro-transferred to PVDF membrane (Immobilon P) and located by Western blot using ExtrAvidin® peroxidase conjugate and developed with 3,3 -diaminobenzidine as substrate. 3. Results 3.1. Trypanosoma rangeli and Trypanosoma cruzi GPI-anchored proteins: isolation and analysis In both T. rangeli and T. cruzi, the presence of a wellstabilized and reproducible protein pattern was detected. The differences observed during the purification process in the protein composition of the obtained fractions (HP, hp and GPI) indicate the efficiency of the separation method and its reproducibility to obtain specific protein patterns. In general, the GPI-anchored proteins fraction represents about 1% of the solubilized proteins in both species of parasites. A similar migration pattern was observed in SDS-PAGE for the four T. rangeli isolates, which resulted different from the four T. cruzi isolates used in the present study (Fig. 1). The GPI-anchored proteins pattern showed a strong band of 90 kDa molecular mass for the T. rangeli isolates, while those of the T. cruzi fractions revealed a major band at 30 kDa, showing a different migration pattern between the two species
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Fig. 1. GPI-anchored proteins from Trypanosoma rangeli and Trypanosoma cruzi cultured epimastigotes. SDS polyacrylamide gel stained with Coomassie blue. Lanes 1–4: Trypanosma rangeli isolates—(1) MHOM/Ve/99/CH-99; (2) MHOM/Ve/99/D-99; (3) MHOM/Ve/02/GS-02; (4) MCAN/Ve/00/Lobita-00. Lane 5: molecular mass marker. Lanes 6–9: Trypanosoma cruzi isolates; (6) MHOM/Ve/91/1–91; (7) MHOM/Ve/94/4–94; (8) MHOM/Ve/ 94/9–94; (9) MHOM/Ve/95/36–95. Lane 10: molecular mass marker. In all cases, 25 g GPI-anchored proteins were placed in each well.
of parasites. In addition, a strong band of 85 kDa, and a faint-stained band of 56 kDa were detected in the isolate MHOM/Ve/99/CH-99 of T. rangeli (Lane 1) absent in the T. cruzi isolates. The use of biotinylated parasites also showed differences between the biotin marked GPI-anchored membrane proteins of the two species. In fact, while T. rangeli showed a major band at 90 kDa, T. cruzi revealed strong bands at 30 and 35 kDa (not shown). The treatment of GPI-anchored proteins with PIPLC produced a change on the protein solubility, being found about 40% of them in the aqueous phase after treatment. PIPLC-treated proteins showed a delayed migration in SDS-PAGE as compared to the migration pattern of the proteins obtained with detergent treatment (results no shown). 3.2. Immunological characterization of T. rangeli and T. cruzi GPI-anchored proteins Western blots revealed the response to rabbit immunesera generated by both T. rangeli and T. cruzi GPIanchored proteins. Fig. 2 gives details on the observed differences among the more antigenic proteins detected in the GPI fractions of the two compared species of Trypanosoma, as well as on the similarity observed among the isolates belonging to the same species. The use of sera samples containing T. rangeli GPI Abs against GPIT. rangeli fraction revealed an identical pattern for the four isolates, strongly identifying proteins of 90, 85 and 56 kDa and revealing a very weak band of 30 kDa when T. cruzi fractions were used (Fig. 2A). On the contrary, the use of T. cruzi GPI Abs against GPI-T. cruzi frac-
Fig. 2. GPI-anchored proteins from T. rangeli and T. cruzi epimastigotes. Western blot from SDS polyacrylamide gel. (A) Developed with T. rangeli anti-GPI Abs (1:300). Lanes 1–4 and 6–9 contain, respectively, T. rangeli and T. cruzi GPI-anchored proteins from the isolates indicated in Fig. 1 conserving the same order. (B) Developed with T. cruzi anti-GPI Abs (1:300). Lanes 1–4 and 6–9 as indicated above. Lanes 5 and 10: molecular mass marker. Reaction was carried out with 25 g proteins in each well.
tions, revealed proteins of 30, 38, 70 and 100 kDa, and a 28 kDa weak band when GPI-T. rangeli fractions were used (Fig. 2B). 3.3. Immnunofluorescence for location of membrane GPI-anchored proteins in T. rangeli and T. cruzi Specific anti-T. rangeli and anti-T. cruzi GPIanchored proteins Abs from sera of immunized rabbits were only able to recognize specific antigenic GPI-anchored proteins present at the membrane of the correspondent parasite when immunofluorescense was detected (Fig. 3). A regular and uniform specific fluorescence on the parasite membrane was observed when anti-GPI Abs diluted from 1:64 to 1:2048 was used. Fig. 3 shows the results of the fluorescence as observed in both T. rangeli and T. cruzi epimastigotes indicating negative results for cross-reactions.
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Fig. 3. Immunofluorescence detected in Trypanosoma rangeli and Trypanosoma cruzi epimastigotes challenged against anti-T. rangeli anti-T. cruzi GPI-anchored proteins Abs from immunized rabbits: (A) T. cruzi–T. cruzi GPI Abs; (B) T. cruzi–T. rangeli GPI Abs; (C) T. rangeli–T. rangeli GPI Abs; (D) T. rangeli–T. cruzi GPI Abs.
4. Discussion Previous studies have demonstrated that GPIanchored proteins are very immunogenic for a wide range of models (Low, 1989). In the present work, GPIanchored membrane proteins obtained from T. rangeli and T. cruzi cultured epimastigotes were able to differentiate the two parasites and recognize their antigenic capability. Confirmation of the presence and location of these molecules in our system was carried out: (i) by using biotinylated parasites to obtain biotin marked GPI-anchored proteins; (ii) by performing immunofluorescent specific protein detection; (iii) by producing changes in protein solubility using PIPLC. The obtained results undoubtedly indicated the presence of GPIanchored protein fractions and their location in the plasma membrane of the specimens of T. rangeli and T. cruzi used during the experiments. The present results allow us to state that despite the great similarity reported for these two trypanosomes, there are enough differences in the GPI-anchored protein patterns found in the plasma membrane to discriminate
between both species. This assumption is strongly supported by the fact that some proteins detected in T. rangeli were absent in T. cruzi. Indeed, the presence of the major GPI-anchored specific proteins of 90, 85 and 56 kDa in the surface of T. rangeli epimastigotes were not detected in T. cruzi when developed by Western blot. Conversely, the presence of the major GPI-anchored membrane proteins detected at 30, 70 and 100 kDa in the surface of T. cruzi epimastigotes were also absent in T. rangeli. Undoubtedly, these differences give the system a potential diagnostic value. This fact, together with the proven specific antigenicity observed in our system, lead us to consider these proteins as a biochemical marker to identify and differentiate T. rangeli from T. cruzi. Our results appear to corroborate previous findings by Salda˜na et al. (1998) who described the identification and purification of a specific 48 kDa T. rangeli antigen, suggesting it as a marker to identify and characterize T. rangeli isolates. In addition, in a very preliminary assay, the consistence and usefulness of the GPI-anchored proteins in the diagnosis of T. rangeli and T. cruzi specific infection was tested with sera from
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infected people revealing the same differences showed in the experimental model (data no shown). All the above suggests the potential of these biochemical markers as diagnostic tools to be used in areas where Chagas disease is endemic and where both T. cruzi and T. rangeli co-exist. The circumstance of having the system to separate the specific GPI-anchored membrane proteins from both parasites, allowed us to create a simple device as a tool for diagnosis in situ, which at the same time, is reliable, specific, cheap and easy to use (this information will be soon published elsewhere). Acknowledgements We are indebted to Dr. G. Fermin for carefully reviewing of the manuscript. The financial support of CDCHTULA-C-1209-03-03-A and FONACIT-G-2005000370 (NA) is gratefully acknowledged. References Afchain, D., Le Ray, D., Fruit, J., Capron, A., 1979. Antigenic makeup of Trypanosoma cruzi culture forms identification of a specific component. J. Parasitol. 65, 507–514. Altin, J.G., Pagler, E.B., 1995. A one-step procedure for biotinylation and chemical crosss-linking of lymphocyte surface and intracellular membrane-associated molecules. Anal. Biochem. 224, 382–389. A˜nez, N., 1982. Studies on Trypanosoma rangeli TEJERA, 1920. Part IV: a re-consideration of its systematic position. Mem. Inst. Oswaldo Cruz. 77 (4), 405–415. A˜nez, N., East, Y., 1984. Studies on Trypanosoma rangeli TEJERA, 1920. Part II: its effect on feeding behaviour of triatomid-bugs. Acta Trop. 41, 93–95. A˜nez, N., Crisante, G., Maia da Silva, F., Rojas, A., Carrasco, H., Umezawa, E., Stolf, A.M., Ramirez, J.L., Teixeira, M.M.G., 2004.
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Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas disease. Trop. Med. Intern. Health 9 (12), 1319–1326. Bronzina, A., D’Alessandro, A., Segura, E., 1980. Diferencias y similitudes antig´enicas entre Trypanosoma rangeli y Trypanosoma cruzi. Medicina 40, 45–49. Ferguson, M.A.J., 1997. The surface glyconjugates of trypanosomatid parasites. Philos. Trans. R. Soc. Lond. B 352, 1295–1302. Gr¨ogl, M., Kuhn, R.E., 1984. Identification of antigens of culture forms of Trypanosoma cruzi and Trypanosoma rangeli recognized by sera from patients with chronic Chagas disease. J. Parasitol. 70, 822–824. Ko, Y., Thompson, G., 1995. Purification of glycosylphosphatidylinositol anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis. Anal. Biochem. 224, 166–172. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. Low, M.G., 1989. The glycosylphosphatidylinositol anchor of membrane proteins. Biochem. Biophys. Acta 988, 427–454. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Morgado, M.G., Ivo-Dos Santos, J., Pinho, R.T., Arg¨uelles, E., Rezende, J.M., Galv˜ao-Castro, B., 1989. Trypanosoma cruzi: identification of specific epimastigote antigens by human sera. Mem. Inst. Oswaldo Cruz. 84, 309–314. O’Daly, J.A., Carrasco, H., Fernandez, V., Rodriguez, M.B., 1994. Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli. Acta Trop. 56, 265–287. Salda˜na, A., Sousa, O.E., 1996. Trypanosoma rangeli and Trypanosoma cruzi: cross-reaction among their immunogenic components. Mem. Inst. Oswaldo Cruz. 91, 81–82. ¨ Salda˜na, A., Harris, R.A., Orn, A., Sousa, O.E., 1998. Trypanosoma rangeli: identification and purification of a 48 kDa-specific antigen. J. Parasitol. 84, 67–73. TDR/WHO, 2005. Scientific Working Group on Chagas disease, Buenos Aires, Argentina.
Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi N´estor A˜nez-Rojas, Pablo Garc´ıa-Lugo, Gladys Crisante, Agustina Rojas, N´estor A˜nez ∗ Universidad de Los Andes, Facultad de Ciencias, Departamento de Biolog´ıa, M´erida 5101, Venezuela Received 13 May 2005; received in revised form 8 September 2005; accepted 22 September 2005 Available online 24 October 2005
Abstract GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed. © 2005 Elsevier B.V. All rights reserved. Keywords: GPI; Membrane proteins; Trypanosoma cruzi; Trypanosoma rangeli; Species differentiation
1. Introduction Chagas disease, caused by Trypanosoma (Schyzotrypanum) cruzi, Chagas 1909, is one of the major public health problems in Central and South America. The fact that nearly 20 million people are affected by chronic disease and about 100 million are under risk appears to corroborate the above statement (TDR/WHO, 2005). Trypanosoma (Tejeraia) rangeli Tejera 1920, known as the second American Trypanosoma infecting man, in contrast to T. cruzi is harmless to the vertebrate host but it is pathogenic to the triatomine-bugs acting as vectors
∗
Corresponding author. Tel.: +58 274 2401285; fax: +58 274 2401285. E-mail address: [email protected] (N. A˜nez). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.09.005
of the parasite (A˜nez and East, 1984). Mixed infections caused by both parasites are not uncommon due to the fact that the parasites share the same vertebrate hosts, vectors and have similar geographical distribution (A˜nez, 1982). The co-existence of these parasites in the same natural habits evidenced by the common demonstration of mixed infection and the detection of similar antigenic components, which have been interpreted as the cause of interference for serological tests in epidemiological studies in areas where Chagas disease is endemic, urge for a right diagnosis. On this aspect, several authors, using serological methods, have previously demonstrated antigen cross-reactions between T. rangeli and T. cruzi (Afchain et al., 1979; O’Daly et al., 1994; Gr¨ogl and Kuhn, 1984). However, others have reported several T. cruzi-specific proteins most of them useful in serological tests (Morgado et al., 1989). In addi-
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tion, Salda˜na and Sousa (1996) reported the presence of several common epitopes among T. rangeli antigens (Ag) and between T. rangeli and T. cruzi polypeptides. The authors also suggested that purification of these T. rangeli Ags, which cross-reacted with T. cruzi components, could be important on the diagnosis of Chagas disease. More recently, Salda˜na et al. (1998) described a specific 48 kDa Ag from T. rangeli, which was not detected in T. cruzi epimastigotes. The authors suggest that this Ag may be a useful marker for the identification and characterization of T. rangeli isolates. The above circumstances appear to justify the production of a new reliable methodology capable of differentiating both parasites when present in the same host, which at the same time, result in a fast, specific and cheap diagnostic test. Considering, on one hand, that the major antigenic difference between T. cruzi and T. rangeli appears to be found in the membrane proteins (Bronzina et al., 1980) and on the other hand, that membrane proteins anchored by glycosyl-phosphatidyl-inositol (GPI) have been characterized in different morphological forms of T. cruzi (Ferguson, 1997) in the present paper, we propose the detection and characterization of GPI-anchored membrane proteins to discriminate between T. rangeli and T. cruzi. 2. Materials and methods 2.1. Parasites Four T. cruzi isolates obtained by hemoculture from acute chagasic patients at the Center for Parasitological Research, Faculty of Sciences, University of Los Andes, M´erida, Venezuela, were used. The patients from whom T. cruzi isolates were characterized in this study were from four localities of Barinas state, western Venezuela, where Chagas disease is endemic. Details on the origin, maintenance of the isolates and the clinical features of each patient have been given in a previous publication (A˜nez et al., 2004). Prior to the study, the four T. cruzi isolates were genetically typed as lineage I as previously indicated (A˜nez et al., 2004). The T. cruzi isolates were identified as follows: MHOM/Ve/91/1–91; MHOM/Ve/94/4–94; MHOM/Ve/94/9–94; MHOM/Ve/95/36–95. Similarly, four T. rangeli isolates were used. From these, three were from human active infection detected by hemoculture (MHOM/Ve/99/CH-99; MHOM/Ve/99/D-99; MHOM/Ve/02/GS-02) and one from a dog also detected by hemoculture (MCAN/Ve/00/LOBITA-00). T. rangeli isolates were also obtained from samples taken at the Barinas state, Venezuela. Both T. cruzi and T. rangeli
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isolates were kept in NNN culture medium at 25 ◦ C, until they grew to obtain masses of parasites. When the culture of the parasites reached the exponential phase, they were collected and centrifuged at 3000 × g at 4 ◦ C for 15 min and washed three times in phosphate buffer saline (PBS), pH 7.4 to collect a pellet, which had an approximated concentration of 13 × 106 flagellates/ml, for each of the isolates. 2.2. Isolation and purification of T. rangeli and T. cruzi GPI-anchored membrane proteins GPI-anchored membrane proteins for both T. rangeli and T. cruzi isolates were obtained using the partition Triton X-114 method previously described by Ko and Thompson (1995). Briefly, the collected pellet was resuspended in a 10 mM Tris–HCl, 150 mM NaCl, 2% Triton X-114, pH 7.4 buffer containing a pool of protease inhibitors (Roche), with shaking during 1 h at 0 ◦ C. The suspension was spun at 8000 × g at 0 ◦ C for 10 min. The obtained pellet was then washed with a 10 mM Tris–HCl, 150 mM NaCl, 0.06% Triton X-114, pH 7.4 buffer and kept at −20 ◦ C. The supernatant was kept at −20 ◦ C during 24 h and then it was slowly unfrozen at room temperature and later placed during 12 min at 32 ◦ C to obtain the first partition (P1). P1 was then spun at 3000 × g for 3 min, obtaining two phases. The aqueous phase containing hydrophilic proteins (HP) was separated and kept at −20 ◦ C. The detergent phase was re-suspended in 3 V of the above indicated buffer and kept at 0 ◦ C for 10 min. The P1 process was repeated and the aqueous phase containing the rest of HP was discharged. The detergent phase was then mixed with 3 V of the same buffer, kept at 0 ◦ C for 10 min and spun at 18,000 × g at 0 ◦ C for 10 min. The pellets containing hydrophobic proteins, not GPI-anchored protein (hp) were kept at −20 ◦ C. The supernatant was then submitted to a second partition process (P2). The proteins contained at the detergent phase were precipitated with 3 V of cold acetone and spun at 3000 × g, to obtain the GPI-anchored proteins. 2.3. Protein measurement Concentration of protein fractions (HP, hp and GPI) were measured following the method reported by Lowry et al. (1951) using bovine serum albumin as standard. 2.4. Gel electrophoresis Protein gel electrophoresis was carried out on 12% sodium dodecyl sulphate polyacrylamide (SDS-PAGE)
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on a 0.75 mm thick minigel and stained with Comassie blue R-250 as described by Laemmli (1970). Samples of proteins were precipitated with 3 V of cold acetone and re-suspended in sampling buffer (0.2% SDS, 10% glycerol, 50 mM dl-dithiothreitol, 168 mM Tris–HCl, pH 6.8). The molecular weight markers were from Biorad® . 2.5. Rabbit immunization with T. rangeli and T. cruzi GPI-anchored proteins Concentration of GPI-anchored proteins from epimastigotes of both species of parasites were quantified following the method of Lowry et al. (1951). For both T. rangeli and T. cruzi, the amount of protein to be used was re-suspended in 500 l PBS and mixed 1:1 with Freund’s adjuvant to 1 ml final volume. Immunizations with proteins of each of the parasites were carried out in rabbits following the schedule of three challenges, 15 days each. The first immunization was done with 100 g protein, followed by a second and third challenge with 50 g each. 2.6. Indirect immunofluorescence to detect surface GPI-anchored proteins in cultured specimens of T. rangeli and T. cruzi Cultured T. rangeli and T. cruzi epimastigotes were placed on glass slides, dried at room temperature and fixed with 100% acetone. The slides were then incubated in wet chamber during 1 h with sera anti-T. rangeli and T. cruzi GPI-anchored membrane proteins from immunized rabbits, using dilutions from 1:64 to 1:2048. Later, the slides were washed three times in PBS and incubated at dark with 1:80 diluted antirabbit IgG FIT conjugate (Sigma) for 1 h, washed three times in PBS and then mounted in buffered glycerin to be observed under a fluorescent microscope at 400×. 2.7. Western blot analysis Following SDS-PAGE, the proteins were electrotransferred (100 V constant for 1 h at 4 ◦ C) to PVDF membranes (Immobilon P). The anti-T. rangeli and antiT. cruzi GPI-anchored proteins antibodies (Abs) diluted 1:300, were incubated overnight with the membranes and after a thorough washing, a 1:15,000 rabbit serum antiIgG linked peroxidase was added and incubated for 3 h. Reaction was developed using 3,3 -diaminobenzidine as a substrate.
2.8. Evidence for GPI-linked proteins by phosphatidyl-inositol specific phospholipase C (PIPLC) treatment A 60 g of delipidated GPI-anchored proteins was re-suspended in 300 l of 100 mM Tris–HCl, pH 7.5 containing 0.1% sodium deoxycholate and two units of Bacillus cereus PIPLC. The suspension was incubated 4 h at 37 ◦ C following Ko and Thompson (1995). Proteins of reaction mixture were precipitated with cold acetone and then re-solubilized in 2% Triton X-114, 150 mM NaCl and 10 mM Tris–HCl, pH 7.4. After partitioning, the proteins were precipitated both from the aqueous phase and the Triton X-114 phase with cold acetone and then re-suspended in sampling buffer. 2.9. Biotinylation of parasite surface proteins Parasites were washed three times with cold PBS pH 8.0 and re-suspended at a concentration of 2 × 107 epimastigotes/ml. Later, 0.5 mg/ml sulpho-NHS-LC-biotin was added and incubated 30 min at room temperature and then washed three times in the same solution as recommended by Altin and Pagler (1995). After purification of biotynilated parasites, a SDS-PAGE was performed with the separated protein fractions (HP, hp and GPI), which were electro-transferred to PVDF membrane (Immobilon P) and located by Western blot using ExtrAvidin® peroxidase conjugate and developed with 3,3 -diaminobenzidine as substrate. 3. Results 3.1. Trypanosoma rangeli and Trypanosoma cruzi GPI-anchored proteins: isolation and analysis In both T. rangeli and T. cruzi, the presence of a wellstabilized and reproducible protein pattern was detected. The differences observed during the purification process in the protein composition of the obtained fractions (HP, hp and GPI) indicate the efficiency of the separation method and its reproducibility to obtain specific protein patterns. In general, the GPI-anchored proteins fraction represents about 1% of the solubilized proteins in both species of parasites. A similar migration pattern was observed in SDS-PAGE for the four T. rangeli isolates, which resulted different from the four T. cruzi isolates used in the present study (Fig. 1). The GPI-anchored proteins pattern showed a strong band of 90 kDa molecular mass for the T. rangeli isolates, while those of the T. cruzi fractions revealed a major band at 30 kDa, showing a different migration pattern between the two species
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Fig. 1. GPI-anchored proteins from Trypanosoma rangeli and Trypanosoma cruzi cultured epimastigotes. SDS polyacrylamide gel stained with Coomassie blue. Lanes 1–4: Trypanosma rangeli isolates—(1) MHOM/Ve/99/CH-99; (2) MHOM/Ve/99/D-99; (3) MHOM/Ve/02/GS-02; (4) MCAN/Ve/00/Lobita-00. Lane 5: molecular mass marker. Lanes 6–9: Trypanosoma cruzi isolates; (6) MHOM/Ve/91/1–91; (7) MHOM/Ve/94/4–94; (8) MHOM/Ve/ 94/9–94; (9) MHOM/Ve/95/36–95. Lane 10: molecular mass marker. In all cases, 25 g GPI-anchored proteins were placed in each well.
of parasites. In addition, a strong band of 85 kDa, and a faint-stained band of 56 kDa were detected in the isolate MHOM/Ve/99/CH-99 of T. rangeli (Lane 1) absent in the T. cruzi isolates. The use of biotinylated parasites also showed differences between the biotin marked GPI-anchored membrane proteins of the two species. In fact, while T. rangeli showed a major band at 90 kDa, T. cruzi revealed strong bands at 30 and 35 kDa (not shown). The treatment of GPI-anchored proteins with PIPLC produced a change on the protein solubility, being found about 40% of them in the aqueous phase after treatment. PIPLC-treated proteins showed a delayed migration in SDS-PAGE as compared to the migration pattern of the proteins obtained with detergent treatment (results no shown). 3.2. Immunological characterization of T. rangeli and T. cruzi GPI-anchored proteins Western blots revealed the response to rabbit immunesera generated by both T. rangeli and T. cruzi GPIanchored proteins. Fig. 2 gives details on the observed differences among the more antigenic proteins detected in the GPI fractions of the two compared species of Trypanosoma, as well as on the similarity observed among the isolates belonging to the same species. The use of sera samples containing T. rangeli GPI Abs against GPIT. rangeli fraction revealed an identical pattern for the four isolates, strongly identifying proteins of 90, 85 and 56 kDa and revealing a very weak band of 30 kDa when T. cruzi fractions were used (Fig. 2A). On the contrary, the use of T. cruzi GPI Abs against GPI-T. cruzi frac-
Fig. 2. GPI-anchored proteins from T. rangeli and T. cruzi epimastigotes. Western blot from SDS polyacrylamide gel. (A) Developed with T. rangeli anti-GPI Abs (1:300). Lanes 1–4 and 6–9 contain, respectively, T. rangeli and T. cruzi GPI-anchored proteins from the isolates indicated in Fig. 1 conserving the same order. (B) Developed with T. cruzi anti-GPI Abs (1:300). Lanes 1–4 and 6–9 as indicated above. Lanes 5 and 10: molecular mass marker. Reaction was carried out with 25 g proteins in each well.
tions, revealed proteins of 30, 38, 70 and 100 kDa, and a 28 kDa weak band when GPI-T. rangeli fractions were used (Fig. 2B). 3.3. Immnunofluorescence for location of membrane GPI-anchored proteins in T. rangeli and T. cruzi Specific anti-T. rangeli and anti-T. cruzi GPIanchored proteins Abs from sera of immunized rabbits were only able to recognize specific antigenic GPI-anchored proteins present at the membrane of the correspondent parasite when immunofluorescense was detected (Fig. 3). A regular and uniform specific fluorescence on the parasite membrane was observed when anti-GPI Abs diluted from 1:64 to 1:2048 was used. Fig. 3 shows the results of the fluorescence as observed in both T. rangeli and T. cruzi epimastigotes indicating negative results for cross-reactions.
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Fig. 3. Immunofluorescence detected in Trypanosoma rangeli and Trypanosoma cruzi epimastigotes challenged against anti-T. rangeli anti-T. cruzi GPI-anchored proteins Abs from immunized rabbits: (A) T. cruzi–T. cruzi GPI Abs; (B) T. cruzi–T. rangeli GPI Abs; (C) T. rangeli–T. rangeli GPI Abs; (D) T. rangeli–T. cruzi GPI Abs.
4. Discussion Previous studies have demonstrated that GPIanchored proteins are very immunogenic for a wide range of models (Low, 1989). In the present work, GPIanchored membrane proteins obtained from T. rangeli and T. cruzi cultured epimastigotes were able to differentiate the two parasites and recognize their antigenic capability. Confirmation of the presence and location of these molecules in our system was carried out: (i) by using biotinylated parasites to obtain biotin marked GPI-anchored proteins; (ii) by performing immunofluorescent specific protein detection; (iii) by producing changes in protein solubility using PIPLC. The obtained results undoubtedly indicated the presence of GPIanchored protein fractions and their location in the plasma membrane of the specimens of T. rangeli and T. cruzi used during the experiments. The present results allow us to state that despite the great similarity reported for these two trypanosomes, there are enough differences in the GPI-anchored protein patterns found in the plasma membrane to discriminate
between both species. This assumption is strongly supported by the fact that some proteins detected in T. rangeli were absent in T. cruzi. Indeed, the presence of the major GPI-anchored specific proteins of 90, 85 and 56 kDa in the surface of T. rangeli epimastigotes were not detected in T. cruzi when developed by Western blot. Conversely, the presence of the major GPI-anchored membrane proteins detected at 30, 70 and 100 kDa in the surface of T. cruzi epimastigotes were also absent in T. rangeli. Undoubtedly, these differences give the system a potential diagnostic value. This fact, together with the proven specific antigenicity observed in our system, lead us to consider these proteins as a biochemical marker to identify and differentiate T. rangeli from T. cruzi. Our results appear to corroborate previous findings by Salda˜na et al. (1998) who described the identification and purification of a specific 48 kDa T. rangeli antigen, suggesting it as a marker to identify and characterize T. rangeli isolates. In addition, in a very preliminary assay, the consistence and usefulness of the GPI-anchored proteins in the diagnosis of T. rangeli and T. cruzi specific infection was tested with sera from
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infected people revealing the same differences showed in the experimental model (data no shown). All the above suggests the potential of these biochemical markers as diagnostic tools to be used in areas where Chagas disease is endemic and where both T. cruzi and T. rangeli co-exist. The circumstance of having the system to separate the specific GPI-anchored membrane proteins from both parasites, allowed us to create a simple device as a tool for diagnosis in situ, which at the same time, is reliable, specific, cheap and easy to use (this information will be soon published elsewhere). Acknowledgements We are indebted to Dr. G. Fermin for carefully reviewing of the manuscript. The financial support of CDCHTULA-C-1209-03-03-A and FONACIT-G-2005000370 (NA) is gratefully acknowledged. References Afchain, D., Le Ray, D., Fruit, J., Capron, A., 1979. Antigenic makeup of Trypanosoma cruzi culture forms identification of a specific component. J. Parasitol. 65, 507–514. Altin, J.G., Pagler, E.B., 1995. A one-step procedure for biotinylation and chemical crosss-linking of lymphocyte surface and intracellular membrane-associated molecules. Anal. Biochem. 224, 382–389. A˜nez, N., 1982. Studies on Trypanosoma rangeli TEJERA, 1920. Part IV: a re-consideration of its systematic position. Mem. Inst. Oswaldo Cruz. 77 (4), 405–415. A˜nez, N., East, Y., 1984. Studies on Trypanosoma rangeli TEJERA, 1920. Part II: its effect on feeding behaviour of triatomid-bugs. Acta Trop. 41, 93–95. A˜nez, N., Crisante, G., Maia da Silva, F., Rojas, A., Carrasco, H., Umezawa, E., Stolf, A.M., Ramirez, J.L., Teixeira, M.M.G., 2004.
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