Antibody delivery into viable epimastigotes of Trypanosoma cruzi as a tool to study the parasite biology

Abstract

American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, hence the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal (Trypano-soma cruzi). T. cruzi is an RNAi-negative parasite, therefore the silencing genes strategies by RNAi is not possible; for that reason, antibodies may be taken as a tool for studying the parasite proteins function by blocking these molecules with specific antibodies. The aim of this work was to establish a methodology for antibody delivery (antibody transfection) into viable parasites. We used anti-cyclin-A antibody (human origin) in western blot assay with epimastigote of T. cruzi proteins and this recognized a ~55 kDa polypeptide. Several methods for antibody transfection (electroporation, saponin permeabilization and a lipid-based formulation) were tested. The first two methods were unsuccessful. In electroporation was impossible to visualize the antibody inside parasites and with saponin permeabilization, antibodies were successfully introduced, but with loss of parasites viability. The lipid-based formulation method forms noncovalent complexes with antibodies. These complexes are internalized by cells and antibodies are released into the cytoplasm. With this method, a successful antibody delivery was achieved. Anti-cyclin antibodies were visualized in the cytoplasm from fixed transfected parasites (immunofluorescence assays). At 24 h post-transfection, parasites maintained their viability (90%) and were able to arrest the cell cycle in G0/G1-phase of cultured epimastigotes (cell population increased in G0/G1-phase from 50.5% to 66.2% and decreased in S-phase from 47.2% to 26%). It was also observed that anti-cyclin-A antibodies inhibit the parasite population doubling (p < 0.05, 95% CI). This is the first report of antibody-delivery into viable epimastigote forms of T. cruzi, with a simple and cheap technique, which will allows carrying out further studies of this protozoan.

Share and Cite:

Acosta-Viana, K. , Julio, H. , Matilde, J. , Eugenia, G. and Rosales-Encina, J. (2013) Antibody delivery into viable epimastigotes of Trypanosoma cruzi as a tool to study the parasite biology. Advances in Bioscience and Biotechnology, 4, 719-726. doi: 10.4236/abb.2013.46095.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] World Health Organization (2013) http://www.who.int/neglected_diseases/diseases/en/
[2] World Health Organization (2012) http://www.who.int/mediacentre/factsheets/fs340/en/index.html
[3] Cancado, J.R. (2002) Long term evaluation of etiological treatment of chagas disease with benznidazole. Revista do Instituto de Medicina Tropical de Sao Paulo, 44, 29-37. doi:10.1590/S0036-46652002000100006
[4] Guedes, P.M., Silva, G.K., Gutierrez, F.R. and Silva, J.S. (2011) Current status of Chagas disease chemotherapy. Expert Review of Anti-Infective Therapy, 9, 609-620. doi:10.1586/eri.11.31
[5] Tetaud, E., Lecuix, I., Sheldrake, T., Baltz, T. and Fairlamb, A.H. (2002) A new expression vector for Crithidia fasciculata and Leishmania. Molecular and Biochemical Parasitology, 120, 195-204. doi:10.1016/S0166-6851(02)00002-6
[6] Schimanski, B., Nguyen, T.N. and Gunzl, A. (2005) Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Eukaryotic Cell, 4, 1942-1950. doi:10.1128/EC.4.11.1942-1950.2005
[7] Martinez-Calvillo, S., Lopez, I. and Hernandez, R. (1997) pRIBOTEX expression vector: A pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene, 199, 71-76. doi:10.1016/S0378-1119(97)00348-X
[8] Xu, D., Brandan, C.P., Basombrio, M.A. and Tarleton, R.L. (2009) Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi. BMC Microbiology, 9, 90. doi:10.1186/1471-2180-9-90
[9] Taylor, M.C., Huang, H. and Kelly, J.M. (2011) Advances in Parasitology. Academic Press, Waltham.
[10] Wirtz, E., Leal, S., Ochatt, C. and Cross, G.A. (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Molecular and Biochemical Parasitology, 99, 89-101. doi:10.1016/S0166-6851(99)00002-X
[11] Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806-811. doi:10.1038/35888
[12] Kolev, N.G., Tschudi, C. and Ullu, E. (2011) RNA interference in protozoan parasites: achievements and challenges. Eukaryotic Cell, 10, 1156-1163. doi:10.1128/EC.05114-11
[13] DaRocha, W.D., Otsu, K., Teixeira, S.M. and Donelson, J.E. (2004) Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi. Molecular and Biochemical Parasitology, 133, 175-186. doi:10.1016/j.molbiopara.2003.10.005
[14] Barnes, R.L., Shi, H., Kolev, N.G., Tschudi, C. and Ullu, E. (2012) Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery. PLoS Pathogens, 8, e1002678. doi:10.1371/journal.ppat.1002678
[15] Bright, G.R., Kuo, N.-T., Chow, D., Burden, S., Dowe, C. and Przybylski, R.J. (1996) Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies. Cytometry, 24, 226-233. doi:10.1002/(SICI)1097-0320(19960701)24:3<226::AID-CYTO5>3.3.CO;2-Y
[16] Douglas, J.N., Gardner, L.A., Lee, S., Shin, Y., Groover, C.J. and Levin, M.C. (2012) Antibody transfection into neurons as a tool to study disease pathogenesis. Journal of Visualized Experiments, 67, e4154.
[17] Chakrabarti, R., Wylie, D.E. and Schuster, S.M. (1989) Transfer of monoclonal antibodies into mammalian cells by electroporation. Journal of Biological Chemistry, 264, 15494-15500.
[18] Berglund, D.L. and Starkey, J.R. (1989) Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation. Journal of Immunological Methods, 125, 79-87. doi:10.1016/0022-1759(89)90080-X
[19] Berglund, D.L. and Starkey, J.R. (1991) Introduction of antibody into viable cells using electroporation. Cytometry, 12, 64-67. doi:10.1002/cyto.990120109
[20] Wilcox, R.A. (1999) Methods in molecular biology. Humana Press Inc., Totowa.
[21] Wassler, M., Jonasson, I., Persson, R. and Fries, E. (1987) Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins. Biochemical Journal, 247, 407-415.
[22] Johnson, J.A., Gray, M.O., Karliner, J.S., Chen, C.H. and Mochly-Rosen, D. (1996) An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research. Circulation Research, 79, 1086-1099. doi:10.1161/01.RES.79.6.1086
[23] Swift, L.M. and Sarvazyan, N. (2000) Localization of dichlorofluorescin in cardiac myocytes: Implications for assessment of oxidative stress. American Journal of Physiology—Heart and Circulatory Physiology, 278, H982H990.
[24] Beckers, C.J., Dubremetz, J.F., Mercereau-Puijalon, O. and Joiner, K.A. (1994) The toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. The Journal of Cell Biology, 127, 947-961. doi:10.1083/jcb.127.4.947
[25] Kass, G., Arad, G., Rosenbluh, J., Gafni, Y., Graessmann, A., Rojas, M.R., Gilbertson, R.L. and Loyter, A. (2006) Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions. Journal of General Virology, 87, 2709-2720. doi:10.1099/vir.0.82021-0
[26] Friedler, A., Zakai, N., Karni, O., Broder, Y.C., Baraz, L., Kotler, M., Loyter, A. and Gilon, C. (1998) Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells. Biochemistry, 37, 5616-5622. doi:10.1021/bi972878h
[27] Armon-Omer, A., Graessmann, A. and Loyter, A. (2004) A synthetic peptide bearing the HIV-1 integrase 161 173 amino acid residues mediates active nuclear import and binding to importin α: Characterization of a functional nuclear localization signal. Journal of Molecular Biology, 336, 1117-1128. doi:10.1016/j.jmb.2003.11.057
[28] Todorova, R. (2008) Methods of protein delivery into mammalian cells for gene therapy and genetic studies. Acta Medica Bulgarica, 35, 3-11.
[29] Camargo, E.P. (1964) Growth and differentiation in Trypanosoma cruzi. Origin of metacyclic trypanosomes in liquid media. Revista do Instituto de Medicina Tropical de Sao Paulo, 6, 93-100.
[30] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0
[31] Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76, 4350-4354.
[32] Norris, K.A. (1998) Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance. Infection and Immununity, 66, 2460-2465.
[33] Vercesi, A.E., Bernardes, C.F., Hoffmann, M.E., Gadelha, F.R. and Docampo, R. (1991) Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in Situ. The Journal of Biological Chemistry, 266, 14431-14434.
[34] Kawase, O., Nishikawa, Y., Bannai, H., Igarashi, M., Matsuo, T. and Xuan, X. (2010) Characterization of a novel thrombospondin-related protein in Toxoplasma gondii. Parasitology International, 59, 211-216. doi:10.1016/j.parint.2010.02.001
[35] Guinet, F., Louise, A., Jouin, H., Antoine, J.-C. and Roth, C.W. (2000) Accurate quantitation of Leishmania infection in cultured cells by flow cytometry. Cytometry, 39, 235-240. doi:10.1002/(SICI)1097-0320(20000301)39:3<235::AIDCYTO10>3.3.CO;2-C
[36] Tu, X. and Wang, C.C. (2005) Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism. Eukaryotic Cell, 4, 755-764. doi:10.1128/EC.4.4.755-764.2005
[37] Diaz-Gonzalez, R., Kuhlmann, F.M., Galan-Rodriguez, C., da Silva, L.M., Saldivia, M., Karver, C.E., Rodriguez, A., Beverley, S.M., Navarro, M. and Pollastri, M.P. (2011) The susceptibility of trypanosomatid pathogens to PI3/ mTOR kinase inhibitors affords a new opportunity for drug repurposing. PLoS Neglected Tropical Diseases, 5, e1297. doi:10.1371/journal.pntd.0001297
[38] Marschall, A.L.J., Frenzel, A., Schirrmann, T., Schüngel, M. and Dubel, S. (2011) Targeting antibodies to the cytoplasm. mAbs, 3, 3-16. doi:10.4161/mabs.3.1.14110
[39] Gomez, E.B., Santori, M.I., Laria, S., Engel, J.C., Swindle, J., Eisen, H., Szankasi, P. and Tellez-Inon, M.T. (2001) Characterization of the Trypanosoma cruzi Cdc2prelated protein kinase 1 and identification of three novel associating cyclins. Molecular Biochemical Parasitology, 113, 97-108. doi:10.1016/S0166-6851(00)00382-0
[40] Santori, M.I., Laria, S., Gomez, E.B., Espinosa, I., Galanti, N. and Tellez-Inon, M.T. (2002) Evidence for CRK3 participation in the cell division cycle of Trypanosoma cruzi. Molecular and Biochemical Parasitology, 121, 225-232. doi:10.1016/S0166-6851(02)00039-7
[41] Potenza, M., Schenkman, S., Laverrière, M. and TellezIñón, M.T. (2012) Functional characterization of TcCYC2 cyclin from Trypanosoma cruzi. Experimental Parasitology, 132, 537-545. doi:10.1016/j.exppara.2012.09.002

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.