Available online at www.sciencedirect.com International Journal for Parasitology 38 (2008) 289–297 www.elsevier.com/locate/ijpara Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins S.F. Pires a, W.D. DaRocha a, J.M. Freitas a, L.A. Oliveira b, G.T. Kitten b, C.R. Machado a, S.D.J. Pena a, E. Chiari c, A.M. Macedo a, S.M.R. Teixeira a,* a Departamento de Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas – UFMG, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil b Departamento de Morfologia, Universidade Federal de Minas Gerais, Brazil c Departamento de Parasitologia, Universidade Federal de Minas Gerais, Brazil Received 22 June 2007; received in revised form 7 August 2007; accepted 14 August 2007 Abstract Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent pro- tein (GFP) and red fluorescent protein (RFP) genes into the b-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasi- temia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Green fluorescent protein; Red fluorescent protein; Chagas disease 1. Introduction chronic cardiac and/or gastrointestinal disease. The rea- sons for such variation in both the severity and prevalence Chagas disease, caused by the protozoan parasite Try- of the diverse clinical forms of the disease are not under- panosoma cruzi, is one of the major public health problems stood, but have been attributed to differences in the human in Latin America where 16–18 million people are affected genetic background and to the highly diverse genetic profile (World Health Organization, http://www.who.int/ctd/cha- of the parasite population. In spite of its extreme genetic gas/disease.htm). The disease usually presents an acute variability, several molecular markers have defined two phase characterized by high parasitemia and a variable highly divergent sub-groups of strains, named T. cruziI clinical course, ranging from asymptomatic to severe and II, in the T. cruzi population (for a review, see Macedo et al., 2004). Recently, the existence of a third ancestral * Corresponding author. Tel.: +55 31 3499 2665; fax: +55 31 3499 2614. lineage named T. cruzi III has been proposed (Freitas E-mail address: [email protected] (S.M.R. Teixeira). et al., 2006). 0020-7519/$30.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.08.013 290 S.F. Pires et al. / International Journal for Parasitology 38 (2008) 289–297 During development in the vertebrate host, the number infected tissue culture cells, as well as in blood and various of detectable parasites rapidly decay and in most cases their tissues of infected animals. These fluorescent parasites con- detection in infected tissues during the chronic phase can stitute a powerful new tool for in vivo studies addressing only be monitored through sensitive immunohistochemis- key issues for the understanding of Chagas disease pathol- try techniques (Andrade and Andrews, 2004; Souto- ogy such as parasite invasion mechanisms, differential tis- Padron et al., 2004; Correa-de-Santana et al., 2006)orby sue tropism and mechanisms of genetic exchange that PCR amplification of parasite DNA sequences (Andrade may contribute to create the large genetic variability in et al., 1999; Vago et al., 2000; Freitas et al., 2005). PCR the T. cruzi population. amplification also allows the characterization of the genetic profiles of different strains isolated directly from cardiac 2. Materials and methods and megaesophagus tissues of chagasic patients (Vago et al., 2000; Freitas et al., 2005). In animal models of infec- 2.1. Parasite cultures tion, the use of Low-Stringency Single Specific Primer (LSSP)-PCR permitted the identification of a differential Epimastigote forms of different T. cruzi strains, Tula- tissue distribution when distinct cloned parasite strains hue´n and JG (both from the T. cruzi II lineage), the were simultaneously inoculated, thus reinforcing the Col1.7G2 clone derived from the Colombiana strain importance of T. cruzi genetic polymorphisms in determin- (belonging to T. cruzi I lineage) and CL Brener (a hybrid ing the pathology of Chagas disease (Andrade et al., 1999). cloned strain) were grown in liver infusion tryptose (LIT) As an approach to detect intact T. cruzi in infected ani- medium supplemented with 10% FCS at 28 °C as described mals, various groups have developed genetically modified by Camargo (1964). Tissue culture trypomastigotes were strains capable of expressing different markers. Using the obtained after infecting Vero cell monolayer cultures with Escherichia coli b-galactosidase gene, Buckner et al. stationary phase epimastigotes grown in LIT medium. (1999) generated transfected cell lines that allowed the detection of T. cruzi in mouse tissues by histochemical 2.2. Parasite transfections staining with 5-bromo-4-chloro-3-indolyl-beta-D-galacto- pyranoside (X-Gal). However, besides requiring laborious Two vectors were used to generate stable transfectants. tissue fixation and X-Gal staining, this method does not The first, pROCKGFPNeo, contains the GFP coding allow study of the infection dynamics of living, b-galacto- region and sequences that allow DNA integration at the sidase-expressing parasites. Transfection of epimastigotes b-tubulin loci (DaRocha et al., 2004). The second vector, with various vectors containing the jellyfish green fluores- pROCKRFPNeo, was engineered to replace the GFP cod- cent protein (GFP) gene has resulted in GFP expression ing region by RFP, derived from the pDsRed vector (Clon- in all three stages of the parasite. In the first report, the vec- tech), into the XbaI/XhoI sites of pROCKGFPNeo. tor used contains the GFP gene linked to the amastin 30 Epimastigotes growing at a density of 1–2 · 107 parasites/ untranslated region (UTR), which confers increased stabil- mL in LIT plus 10% FCS were harvested, washed once ity of amastin mRNA in amastigotes, and the construct with PBS and resuspended to a final density of 108 para- was targeted to the tcr 27 locus (Teixeira et al., 1999). sites/mL in electroporation buffer (120 mM KCl, The GFP-containing vector described by Dos Santos and 0.15 mM CaCl2,10mMK2HPO4, 25 mM 4-(2-hydroxy- Buck (2000), pPAGFPAN, is present in stably transfected ethyl)-1-piperazi-neethanesulphonic acid (HEPES), 2 mM parasites in at least three forms: single copy, head-to-tail EDTA, 5 mM MgCl2, pH 7.6). Aliquots of 0.4 mL of cell concatemers and integrated in multiple loci in the parasite suspension were mixed with 50 lL of DNA (50 lg) in ice- genome. Using the pTEX vector, which is also episomally cold 0.2 cm cuvettes and electroporated using a Bio-Rad maintained, Ramirez et al. (2000) described the use of pulser set at 0.3 kV and 500 lF with three pulses (10 s inter- GFP to improve transfection protocols in T. cruzi. More vals between pulses). In order to generate stable transfec- recently, we developed the pROCKNeo vector, designed tants, parasites were electroporated with NotI linearized to allow integration of a foreign gene by homologous DNA plasmids and 24 h after transfection, 200 lg/mL of recombination into the tubulin locus, which contains a the aminoglycoside antibiotic G418 (Invitrogen) was added large array of alternating copies of a-andb-tubulin genes. to the medium. Drug selection was completed 30 days after This vector also contains sequences derived from the flank- electroporation as monitored by the number of surviving ing regions of the T. cruzi glycosomal glyceraldehyde 3- parasites in mock-transfected cultures (DaRocha et al., phosphate dehydrogenase (gGAPDH) genes, allowing con- 2004). stitutive expression of the foreign gene in all parasite forms (DaRocha et al., 2004). 2.3. Selection of GFP and RFP positive T. cruzi clones Using the pROCKNeo vector, we describe here the gen- eration of different T. cruzi strains stably expressing GFP After selection of recombinant T. cruzi, cloned sub-pop- or red fluorescent protein (RFP). When analyzed using ulations were obtained by serial dilution in 96-well plates fluorescence or confocal microscopy, green and red fluores- containing LIT medium supplemented with 10% fresh cent parasites were easily observed in single or double- human blood and 200 lg/mL of G418. Parasite numbers S.F. Pires et al. / International Journal for Parasitology 38 (2008) 289–297 291 were determined by counting cells with a Neubauer cham- T.