Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.
References
[1]
Anonymous (2010) Chagas disease (American trypanosomiasis) fact sheet (revised in June 2010). Wkly Epidemiol Rec 85: 334–336.
[2]
Coura JR, Dias JC (2009) Epidemiology, control and surveillance of Chagas disease: 100 years after its discovery. Mem Inst Oswaldo Cruz 104 Suppl 131–40.
[3]
Tyler KM, Engman DM (2001) The life cycle of Trypanosoma cruzi revisited. Int J Parasitol 31: 472–481.
[4]
De Souza W (2002) Basic cell biology of Trypanosoma cruzi. Curr Pharm Des 8: 269–285.
[5]
Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21: 1881–1888.
[6]
Clayton C, Shapira M (2007) Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol Biochem Parasitol 156: 93–101.
[7]
Haile S, Papadopoulou B (2007) Developmental regulation of gene expression in trypanosomatid parasitic protozoa. Curr Opin Microbiol 10: 569–577.
[8]
Palenchar JB, Bellofatto V (2006) Gene transcription in trypanosomes. Mol Biochem Parasitol 146: 135–141.
[9]
Tyler-Cross RE, Short SL, Floeter-Winter LM, Buck GA (1995) Transient expression mediated by the Trypanosoma cruzi rRNA promoter. Mol Biochem Parasitol 72: 23–31.
[10]
Nunes LR, de Carvalho MR, Buck GA (1997) Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters. Mol Biochem Parasitol 86: 211–224.
[11]
Martinez-Calvillo S, Lopez I, Hernandez R (1997) pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199: 71–76.
[12]
Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, et al. (2010) A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiol 10: 259.
[13]
dos Santos WG, Buck GA (2000) Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi. J Parasitol 86: 1281–1288.
[14]
Lorenzi HA, Vazquez MP, Levin MJ (2003) Integration of expression vectors into the ribosomal locus of Trypanosoma cruzi. Gene 310: 91–99.
[15]
Vazquez MP, Levin MJ (1999) Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene 239: 217–225.
[16]
Dube A, Gupta R, Singh N (2009) Reporter genes facilitating discovery of drugs targeting protozoan parasites. Trends Parasitol 25: 432–439.
[17]
Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312: 217–224.
[18]
Ha DS, Schwarz JK, Turco SJ, Beverley SM (1996) Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 77: 57–64.
[19]
Fumarola L, Spinelli R, Brandonisio O (2004) In vitro assays for evaluation of drug activity against Leishmania spp. Res Microbiol 155: 224–230.
[20]
Bolhassani A, Taheri T, Taslimi Y, Zamanilui S, Zahedifard F, et al. (2011) Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Exp Parasitol 127: 637–645.
[21]
DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, et al. (2004) Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92: 113–120.
[22]
Guevara P, Dias M, Rojas A, Crisante G, Abreu-Blanco MT, et al. (2005) Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology. J Med Entomol 42: 48–56.
[23]
Pires SF, DaRocha WD, Freitas JM, Oliveira LA, Kitten GT, et al. (2008) Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins. Int J Parasitol 38: 289–297.
[24]
Striepen B, He CY, Matrajt M, Soldati D, Roos DS (1998) Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol Biochem Parasitol 92: 325–338.
[25]
Sultan AA, Thathy V, Nussenzweig V, Menard R (1999) Green fluorescent protein as a marker in Plasmodium berghei transformation. Infect Immun 67: 2602–2606.
[26]
VanWye JD, Haldar K (1997) Expression of green fluorescent protein in Plasmodium falciparum. Mol Biochem Parasitol 87: 225–229.
[27]
Taylor MC, Huang H, Kelly JM (2011) Genetic techniques in Trypanosoma cruzi. Adv Parasitol 75: 231–250.
[28]
Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S (1985) In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol Biochem Parasitol 16: 315–327.
[29]
Bonaldo MC, Souto-Padron T, de Souza W, Goldenberg S (1988) Cell-substrate adhesion during Trypanosoma cruzi differentiation. J Cell Biol 106: 1349–1358.
[30]
de Souza FS, Rampazzo Rde C, Manhaes L, Soares MJ, Cavalcanti DP, et al. (2010) Knockout of the gene encoding the kinetoplast-associated protein 3 (KAP3) in Trypanosoma cruzi: effect on kinetoplast organization, cell proliferation and differentiation. Mol Biochem Parasitol 172: 90–98.
[31]
Teixeira MM, Borghesan TC, Ferreira RC, Santos MA, Takata CS, et al. (2011) Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts. Protist 162: 503–524.
[32]
Sambrook J, Russell DW (2006) The condensed protocols from Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. v, 800 p. p.
[33]
Misslitz A, Mottram JC, Overath P, Aebischer T (2000) Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol 107: 251–261.
[34]
Elias MC, Marques-Porto R, Freymuller E, Schenkman S (2001) Transcription rate modulation through the Trypanosoma cruzi life cycle occurs in parallel with changes in nuclear organisation. Mol Biochem Parasitol 112: 79–90.
[35]
Moreno M, D’Avila D A, Silva MN, Galvao LM, Macedo AM, et al. (2010) Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease. Mem Inst Oswaldo Cruz 105: 918–924.
[36]
Campos MC, Salomao K, Castro-Pinto DB, Leon LL, Barbosa HS, et al. (2010) Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi. Parasitol Res 107: 1193–1204.
[37]
da Silva C, Daliry A, PB DAS, Akay S, Banerjee M, et al.. (2011) The efficacy of novel arylimidamides against Trypanosoma cruzi in vitro. Parasitology: 1–7.
[38]
Ferreira LR, Dossin Fde M, Ramos TC, Freymuller E, Schenkman S (2008) Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis. An Acad Bras Cienc 80: 157–166.
[39]
Hsu ST, Blaser G, Jackson SE (2009) The folding, stability and conformational dynamics of beta-barrel fluorescent proteins. Chem Soc Rev 38: 2951–2965.
[40]
Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, et al. (2010) In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds. PLoS Negl Trop Dis 4: e740.
[41]
Boucher N, McNicoll F, Dumas C, Papadopoulou B (2002) RNA polymerase I-mediated transcription of a reporter gene integrated into different loci of Leishmania. Mol Biochem Parasitol 119: 153–158.
[42]
Okuno T, Goto Y, Matsumoto Y, Otsuka H (2003) Applications of recombinant Leishmania amazonensis expressing egfp or the beta-galactosidase gene for drug screening and histopathological analysis. Exp Anim 52: 109–118.
[43]
Chan MM, Bulinski JC, Chang KP, Fong D (2003) A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein. Parasitol Res 89: 266–271.
[44]
Plock A, Sokolowska-Kohler W, Presber W (2001) Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp. Exp Parasitol 97: 141–153.
[45]
Dube A, Singh N, Sundar S (2005) Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persist in in vitro and in vivo experimental models. Parasitol Res 96: 216–223.
[46]
Heussler V, Doerig C (2006) In vivo imaging enters parasitology. Trends Parasitol 22: 192–195; discussion 195–196.
[47]
Mehta SR, Huang R, Yang M, Zhang XQ, Kolli B, et al. (2008) Real-time in vivo green fluorescent protein imaging of a murine leishmaniasis model as a new tool for Leishmania vaccine and drug discovery. Clin Vaccine Immunol 15: 1764–1770.
[48]
de Carvalho TU, de Souza W (1983) Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells. Z Parasitenkd 69: 571–575.
[49]
Schmatz DM, Murray PK (1981) Trypanosoma cruzi: selective isolation of pure trypomastigotes from cultured muscle cells. J Parasitol 67: 517–521.
[50]
Gamarro F, Osuna A, Castanys S, Perez-Lopez MI, Ruiz-Perez LM (1985) Isolation and purification of amastigotes of Trypanosoma cruzi from cultured Vero cells. Z Parasitenkd 71: 15–17.
