Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

Abstract

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-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 parasitemia 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.

Full text

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Cell culture and animal infection with distinct Trypanosoma cruzi
strains expressing red and green fluorescent proteins
S.F. Piresa, W.D. DaRochaa, J.M. Freitasa, L.A. Oliveirab, G.T. Kittenb, C.R. Machadoa,
S.D.J. Penaa, E. Chiaric, A.M. Macedoa, S.M.R. Teixeiraa,*
aDepartamento de Bioquı ´mica e Imunologia, Instituto de Cie ˆncias Biolo ´gicas – UFMG, Universidade Federal de Minas Gerais,
Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
bDepartamento de Morfologia, Universidade Federal de Minas Gerais, Brazil
cDepartamento 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.
? 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
Chagas disease, caused by the protozoan parasite Try-
panosoma cruzi, is one of the major public health problems
in Latin America where 16–18 million people are affected
(World Health Organization, http://www.who.int/ctd/cha-
gas/disease.htm). The disease usually presents an acute
phase characterized by high parasitemia and a variable
clinical course, ranging from asymptomatic to severe
chronic cardiac and/or gastrointestinal disease. The rea-
sons for such variation in both the severity and prevalence
of the diverse clinical forms of the disease are not under-
stood, but have been attributed to differences in the human
genetic background and to the highly diverse genetic profile
of the parasite population. In spite of its extreme genetic
variability, several molecular markers have defined two
highly divergent sub-groups of strains, named T. cruziI
and II, in the T. cruzi population (for a review, see Macedo
et al., 2004). Recently, the existence of a third ancestral
lineage named T. cruzi III has been proposed (Freitas
et al., 2006).
0020-7519/$30.00 ? 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2007.08.013
*Corresponding author. Tel.: +55 31 3499 2665; fax: +55 31 3499 2614.
E-mail address: santuzat@icb.ufmg.br (S.M.R. Teixeira).
www.elsevier.com/locate/ijpara
Available online at www.sciencedirect.com
International Journal for Parasitology 38 (2008) 289–297

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During development in the vertebrate host, the number
of detectable parasites rapidly decay and in most cases their
detection in infected tissues during the chronic phase can
only be monitored through sensitive immunohistochemis-
try techniques (Andrade and Andrews, 2004; Souto-
Padron et al., 2004; Correa-de-Santana et al., 2006) or by
PCR amplification of parasite DNA sequences (Andrade
et al., 1999; Vago et al., 2000; Freitas et al., 2005). PCR
amplification also allows the characterization of the genetic
profiles of different strains isolated directly from cardiac
and megaesophagus tissues of chagasic patients (Vago
et al., 2000; Freitas et al., 2005). In animal models of infec-
tion, the use of Low-Stringency Single Specific Primer
(LSSP)-PCR permitted the identification of a differential
tissue distribution when distinct cloned parasite strains
were simultaneously inoculated, thus reinforcing the
importance of T. cruzi genetic polymorphisms in determin-
ing the pathology of Chagas disease (Andrade et al., 1999).
As an approach to detect intact T. cruzi in infected ani-
mals, various groups have developed genetically modified
strains capable of expressing different markers. Using the
Escherichia coli b-galactosidase gene, Buckner et al.
(1999) generated transfected cell lines that allowed the
detection of T. cruzi in mouse tissues by histochemical
staining with 5-bromo-4-chloro-3-indolyl-beta-D-galacto-
pyranoside (X-Gal). However, besides requiring laborious
tissue fixation and X-Gal staining, this method does not
allow study of the infection dynamics of living, b-galacto-
sidase-expressing parasites. Transfection of epimastigotes
with various vectors containing the jellyfish green fluores-
cent protein (GFP) gene has resulted in GFP expression
in all three stages of the parasite. In the first report, the vec-
tor used contains the GFP gene linked to the amastin 30
untranslated region (UTR), which confers increased stabil-
ity of amastin mRNA in amastigotes, and the construct
was targeted to the tcr 27 locus (Teixeira et al., 1999).
The GFP-containing vector described by Dos Santos and
Buck (2000), pPAGFPAN, is present in stably transfected
parasites in at least three forms: single copy, head-to-tail
concatemers and integrated in multiple loci in the parasite
genome. Using the pTEX vector, which is also episomally
maintained, Ramirez et al. (2000) described the use of
GFP to improve transfection protocols in T. cruzi. More
recently, we developed the pROCKNeo vector, designed
to allow integration of a foreign gene by homologous
recombination into the tubulin locus, which contains a
large array of alternating copies of a- and b-tubulin genes.
This vector also contains sequences derived from the flank-
ing regions of the T. cruzi glycosomal glyceraldehyde 3-
phosphate dehydrogenase (gGAPDH) genes, allowing con-
stitutive expression of the foreign gene in all parasite forms
(DaRocha et al., 2004).
Using the pROCKNeo vector, we describe here the gen-
eration of different T. cruzi strains stably expressing GFP
or red fluorescent protein (RFP). When analyzed using
fluorescence or confocal microscopy, green and red fluores-
cent parasites were easily observed in single or double-
infected tissue culture cells, as well as in blood and various
tissues of infected animals. These fluorescent parasites con-
stitute a powerful new tool for in vivo studies addressing
key issues for the understanding of Chagas disease pathol-
ogy such as parasite invasion mechanisms, differential tis-
sue tropism and mechanisms of genetic exchange that
may contribute to create the large genetic variability in
the T. cruzi population.
2. Materials and methods
2.1. Parasite cultures
Epimastigote forms of different T. cruzi strains, Tula-
hue ´n and JG (both from the T. cruzi II lineage), the
Col1.7G2 clone derived from the Colombiana strain
(belonging to T. cruzi I lineage) and CL Brener (a hybrid
cloned strain) were grown in liver infusion tryptose (LIT)
medium supplemented with 10% FCS at 28 ?C as described
by Camargo (1964). Tissue culture trypomastigotes were
obtained after infecting Vero cell monolayer cultures with
stationary phase epimastigotes grown in LIT medium.
2.2. Parasite transfections
Two vectors were used to generate stable transfectants.
The first, pROCKGFPNeo, contains the GFP coding
region and sequences that allow DNA integration at the
b-tubulin loci (DaRocha et al., 2004). The second vector,
pROCKRFPNeo, was engineered to replace the GFP cod-
ing region by RFP, derived from the pDsRed vector (Clon-
tech), into the XbaI/XhoI sites of pROCKGFPNeo.
Epimastigotes growing at a density of 1–2 · 107parasites/
mL in LIT plus 10% FCS were harvested, washed once
with PBS and resuspended to a final density of 108para-
sites/mLinelectroporation
0.15 mM CaCl2, 10 mM K2HPO4, 25 mM 4-(2-hydroxy-
ethyl)-1-piperazi-neethanesulphonic acid (HEPES), 2 mM
EDTA, 5 mM MgCl2, pH 7.6). Aliquots of 0.4 mL of cell
suspension were mixed with 50 lL of DNA (50 lg) in ice-
cold 0.2 cm cuvettes and electroporated using a Bio-Rad
pulser set at 0.3 kV and 500 lF with three pulses (10 s inter-
vals between pulses). In order to generate stable transfec-
tants, parasites were electroporated with NotI linearized
DNA plasmids and 24 h after transfection, 200 lg/mL of
the aminoglycoside antibiotic G418 (Invitrogen) was added
to the medium. Drug selection was completed 30 days after
electroporation as monitored by the number of surviving
parasites in mock-transfected cultures (DaRocha et al.,
2004).
buffer(120 mMKCl,
2.3. Selection of GFP and RFP positive T. cruzi clones
After selection of recombinant T. cruzi, cloned sub-pop-
ulations were obtained by serial dilution in 96-well plates
containing LIT medium supplemented with 10% fresh
human blood and 200 lg/mL of G418. Parasite numbers
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were determined by counting cells with a Neubauer cham-
ber and then serially diluted to a final density of 0.1 and
0.2 cells/well in a total volume of 0.2 mL. Parasites in 96-
well plates were incubated at 28 ?C in humid chambers
for 3–4 weeks and analyzed with a fluorescence micro-
scope. GFP- and RFP-positive parasites were transferred
into 1 mL LIT medium supplemented with 200 lg/mL of
G418 in 24-well tissue culture plates and incubated at
28 ?C. Alternatively, transfected parasite populations were
sub-cloned in blood-agar plates prepared using 0.75% (w/
v) low melting point (LMP) agarose and 2% of defibrinated
human blood mixed with LIT-medium containing 200 lg/
mL of G418. Defibrinated blood was obtained by collect-
ing blood in a sterile glass bottle containing glass beads
that were manually rotated. After pouring into Petri dishes,
the medium was allowed to solidify and the plates were
inverted and incubated at 37 ?C for 48 h to dry (Gomes
et al., 1991). Different numbers of parasites (102, 103and
104) diluted in 0.1 mL of LIT medium were spread over
the agar surface with a sterile glass loop and incubated at
28 ?C. After 25–30 days, isolated colonies could be
observed on the agarose surface and individual colonies
were picked and transferred into 1 mL of LIT medium sup-
plemented with G418 in a 24-well tissue culture plate.
2.4. Pulse-field gel electrophoresis (PFGE), Southern and
Northern blot analyses
Epimastigotes were embedded in agarose blocks as
described by Engman et al., 1987 and pulse-field gel elec-
trophoresis (PFGE) was carried out as reported by Cano
et al., 1995 with the following modifications: the chromo-
somes were separated in 0.8% agarose gels using a program
with five phases of homogeneous pulses (N/S, E/W) for
135 h at 83 V interpolation. Phase 1 has a pulse time of
90 s (run time 30 h); phase 2, 200 s (30 h); phase 3, 350 s
(25 h); phase 4, 500 s (25 h) and phase 5, 800 s (25 h). Chro-
mosomes from Hansenula wingei (Bio-Rad) were used as
molecular mass standards. Separated chromosomes were
transferred to nylon filters and hybridized with [32P]-
labeled tubulin and RFP probes as described (DaRocha
et al., 2004). For northern blot analyses, total RNA was
extracted from 5 · 108epimastigotes using the RNeasy
Mini Kit (QIAGEN), following the procedure recom-
mended by the manufacturer. Ten micrograms of RNA
were fractionated on 1.5% denaturing agarose–formalde-
hyde gels and transferred to nitrocellulose membranes fol-
lowing standard protocols
Hybridization was performed as described above for
Southern hybridization.
(Teixeiraetal., 1994).
2.5. Cell culture infections
Approximately 105Vero cells were plated in 24-well
plates containing glass coverslips. Vero cell infection was
performed for approximately 18 h at 37 ?C at a multiplicity
of infection (MOI) of 10, using trypomastigotes from each
T. cruzi population (transfected or non-transfected con-
trols) that were collected from the supernatant of previ-
ously infected Vero cell cultures. After infection, cell
cultures were washed with PBS to eliminate non-internal-
ized parasites and re-incubated with fresh media without
parasites for 6 days before fixation with methanol at
?20 ? C. Fixed cultures were stained with Giemsa and
May Grunwald according to Giaimis et al. (1992), and
the coverslips mounted onto glass slides for microscopic
analysis. Cell infection and the number of amastigotes
per cell were evaluated by counting 300 Vero cells per cov-
erslip for each parasite population. Three independent
experiments were performed for each infection.
2.6. Infection in mice, preparation and staining of mouse
tissues
Four to 6 week old, male, IFN-c knockout (GKO) mice
were inoculated i.p. with 100,000 culture-derived trypom-
astigotes from each transfected T. cruzi population in a vol-
ume of 200 lL. Parasitism was quantified beginning on day
7 p.i. by examining 5 lL of tail blood at a magnification of
40·. Mouse infections were monitored daily. At the peak of
parasitemia, hearts, diaphragms and rectums of infected
animals were collected, washed in PBS (pH 7. 2), embedded
in Tissue-Tek O.C.T compound (Miles, Inc., Elkhart, Ind.)
and frozen at ?70 ?C until used for sectioning. Ten
micrometer thick frozen sections were prepared with a
cryostat microtome (Bright Instrument company LTDA),
mounted on glass slides and fixed with 4% paraformalde-
hyde in PBS for 10 min at 25 ?C. Slides containing tissue
slices were stained with 5 lg/mL 40,6-diamidino-2-pheny-
lindole (DAPI) (Molecular Probes) for 1 min at room tem-
perature, washed three times with PBS and mounted onto
glass slides in a mixture of 90% glycerol and 10% of
0.5 M Tris–HCl pH 9.0. Animal infection protocols were
approved by the local animal ethics committee.
2.7. Confocal microscopy analyses
For confocal microscopic analyses, coverslips contain-
ing infected Vero cells were washed twice in cold PBS sup-
plemented with 3% FCS and fixed for 10 min at 25 ?C with
4% paraformaldehyde in PBS. After fixation, cells were
washed twice in PBS supplemented with 3% FCS, stained
with DAPI for 1 min at room temperature, washed three
times with PBS and mounted onto glass slides in a mixture
of 90% glycerol and 10% of 0.5 M Tris–HCl pH 9.0. Para-
site fluorescence was detected in a Zeiss LSM 510 META
confocal microscope provided by the Electron Microscopy
Center (CEMEL-ICB/UFMG).
2.8. Flow cytometry analyses
Approximately 5 · 106parasites were washed with PBS
supplemented with 3% FCS and MFF (0.05% paraformal-
dehyde, 0.05% sodium cacodylate and 0.033% NaCl) for
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291

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30 min at 4 ?C. Fixed parasites were analyzed using a FAC-
Scan (Becton Dickinson) with 105gated events acquired for
analysis. Untransfected control parasites, which showed a
significant amount of intrinsic fluorescence, were used to
establish cut-off values for the analyzed parameters.
3. Results
3.1. Characterization of transfected T. cruzi cell lines
expressing GFP/RFP genes
Epimastigote cultures of the Tulahue ´n, JG, Colombiana
and CL Brener strains were transfected with linearized
pROCKGFPNeo or pROCKRFPNeo vectors. These vec-
tors contain GFP or RFP expression cassettes flanked by
b-tubulin sequences, which allow integration by homolo-
gous recombination into the tubulin locus of the parasite
genome (Fig. 1a). After electroporation, epimastigotes
were cultivated in media containing 200 lg/mL G418 for
4 weeks, i.e., until no parasites could be detected in
mock-transfected cultures. G418-resistant parasites were
cultivated through weekly passages in the presence of the
drug and the percentages of fluorescent parasites were
determined by fluorescent microscopy. One month after
transfection, we observed 22% and 12% of transfected par-
asites of the JG strain expressing GFP and RFP, respec-
tively. For the Tulahue ´n strain, 28% and 25% of
parasites were expressing GFP and RFP, respectively.
The CL Brener clone presented 30% and 22% GFP- and
RFP-expressing parasites,
Col1.7G2 clone presented 23% and 9% GFP- and RFP-
expressing parasites, respectively. For all transfected
strains, biological parameters including parasite mobility,
morphology (form, size and granularity) and growth rate
were comparable to untransfected, wild type (WT) T. cruzi
populations, indicating that the transfection process and
genetic recombination within b-tubulin loci had not altered
the basic features of these parasites (data not shown).
respectively, whereasthe
D
GFP GFP
GFP or RFP
GFP or RFP
NeoR
NeoR
NotI
NotI
NotI
NotI
β-Tubulin
β-Tubulin
Ribosomal
promoter promoter
HX1 HX1
gGAPDH
I/II IRI/II IR
gGAPDH
II 3’UTRII 3’UTR
β-Tubulin
β-Tubulin
pBluescript
backbone backbone
Ribosomal
gGAPDH
gGAPDH
pBluescript
1.05
1.32
1.66
1.81
2.30
Probes
Probes
EtBrEtBr
RFPRFP
tubulin tubulin
RFP1
RFP2
WT
RFP1 RFP2
WT
RFP1RFP2
WT
WT
GFP
WT
GFP
WT
GFP
ProbesProbes
Neo NeoEtBrEtBr
a
bc
Fig. 1. Integration and mRNA expression of green/red fluorescent protein (GFP/RFP) genes following parasite transfection. (a) Schematic representation
of the pROCKGFP/RFPNeo vector construct. The expression vector, which allows integration of a foreign gene in the b-tulubin locus, has the
Trypanosoma cruzi ribosomal promoter followed by the ribosomal protein TcP2b (HX1) 50intergenic region that provides the spliced leader addition site
for the GFP/RFP mRNA. It also contains the 30UTR plus intergenic sequences derived from the gGAPDH I/II genes, which provide signals for
polyadenylation of the GFP/RFP genes as well as for trans-splicing signals for the neomycin resistance gene (NeoR), used as a drug selectable marker.
(b) Expression of GFP and NeoRtranscripts. Northern blot analyses were performed with total RNA extracted from wild type parasites (WT) and
epimastigotes of the Tulahue ´n strain that had been transfected with pROCKGFPNeo (GFP). After separation in agarose gels, RNA samples were stained
with ethidium bromide (left panel), transferred to a nitrocellulose membrane and hybridized with [32P] labeled probes corresponding to the GFP and the
NeoRcoding region. (c) Southern blot analyses of transfected parasites. High molecular weight DNA, isolated from WT cells and two cloned cell lines
derived from epimastigotes of the JG strain that were transfected with pROCKRFPNeo (RFP1 and RFP2) was separated by PFGE and stained with
ethidium bromide (EtBr). After transfer to nylon membranes, DNA samples were hybridized with [32P] labeled probes corresponding to RFP and
b-tubulin coding regions. Numbers on the right correspond to the sizes of chromosomal molecular weight markers.
292
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Transfected parasite populations were cloned by serial
dilution followed by plating in blood-agar medium. Two
clones expressing GFP, TulaGFP1 and TulaGFP2, both
with 40% of the parasites presenting green fluorescence
and two clones of the Tulahue ´n strain expressing RFP,
TulaRFP1 and TulaRFP2, in which 40% and 70%, respec-
tively, of the parasites displayed red fluorescence were
selected. After another round of cloning in blood-agar
plates, we isolated two new sub-clones derived from Tula-
GFP2 and TulaRFP1 in which 95% and 84% of the para-
sites displayed green and red fluorescence, respectively.
We also obtained, after one step of serial dilution cloning,
two clones of the JG strain, named JGRFP1 and JGRFP2,
in which 88% and 70%, respectively, of the parasites
expressed RFP. As shown in Fig. 1b, expression of GFP
and the Neo resistance mRNAs in the transfected cell lines
was confirmed by Northern blot analyses. The sites of inte-
gration of the transfected DNA into the parasite genome
were also determined by PFGE analyses (Fig. 1c). DNA
from two clones derived from the JG strain transfected
with pROCKRFPNeo co-hybridizes with chromosomal
bands that are recognized by the RFP and b-tubulin
probes, thus indicating that the RFP gene was integrated
at the tubulin locus in homologous chromosomes present-
ing different sizes. Using a GFP probe, we have previously
shown that GFP-expressing Tulahue ´n and Col1.7G2
clones also presented hybridization signals compatible with
vector integration in the tubulin locus (DaRocha et al.,
2004). The stability of the fluorescent markers in the trans-
fected populations and clones, was evaluated after cultiva-
tion for 1 or 2 months in the absence or in the presence of
200 or 400 lg/mL of G418. Flow cytometry analyses
showed that, in contrast with the transfected populations,
in which a decrease of the percentages of fluorescent para-
sites occur, the percentages of fluorescent cells within the
clones were constant even after 2 months of culturing with-
out drugs. Also, increasing the G418 concentration from
200 lg/mL to 400 lg/mL did not cause significant changes
in the fluorescent protein expression ratios (see Supplemen-
tary Fig. S1).
3.2. Cell culture and animal infection with transfected
parasites
Next, fluorescent protein expression was evaluated in
the mammalian forms of the parasite life cycle by infecting
Vero cell cultures. Similar to epimastigotes, transfected
parasites exhibited intense green or red fluorescence in
the trypomastigote and amastigote stages (Fig. 2). Trans-
fected parasites were not only able to infect Vero cells,
but trypomastigotes and amastigotes maintained high lev-
els of fluorescence even after 6 months of passage through
tissue cell culturing without G418. None of the transfected
clones presents a population of cells which were 100% fluo-
rescent. However, similar to epimastigotes, non-fluorescent
trypomastigotes and amastigotes were observed in Vero
cells. To determine whether genetic modification has
altered the infection capacity of the transgenic parasites,
we added equal numbers of WT and transfected trypom-
astigotes obtained from previously infected Vero cells, to
freshly plated host cells. Six days after infection, the num-
bers of infected cells, as well as the average number of
intracellular amastigotes in each cell, in a total of 300 Vero
cells were determined (Fig. 3). We found no significant dif-
Fig. 2. Expression of green/red fluorescent proteins (GFP/RFP) in Trypanosoma cruzi. Cloned, transfected epimastigotes of the Tulahue ´n (a), JG (b), CL
Brener (c) and Colombiana (d) strains expressing RFP (a and c) or GFP (b and d) were visualized under a fluorescent microscope at 60· magnification.
Vero cells were incubated with parasites from stationary-phase cultures of two clones of transfected Tulahue ´n strain, TulaGFP2 (e and f) and TulaRFP1 (g
and h) and stained with DAPI 6 days p.i. (e and g). Infected cells containing green and red intracellular amastigotes, respectively, visualized at 40· original
magnification with a Zeiss LSM 510 META confocal microscope. (f and h) Merged fluorescence and differential interference contrast (DIC) images.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S.F. Pires et al. / International Journal for Parasitology 38 (2008) 289–297
293

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ferences in the infectivity between transfected and WT par-
asites (x2= 0.96), suggesting that, at least for tissue culture
cells, the transfection process did not compromise parasite
infectivity.
The in vivo infectivity of fluorescent parasites was also
evaluated using GKO mice which are highly susceptible
to T. cruzi infection. This was necessary since preliminary
studies using BALB/c mice, as well as other reports (Teixe-
ira et al., 1999), have shown that inoculation of 100,000
trypomastigotes of either WT or GFP-expressing parasites
from the Tulahue ´n strain resulted in extremely low levels of
parasitemia. Two groups of five mice each were infected
with WT or the TulaGFP2 clone. Twelve days after infec-
tion, we observed fluorescent T. cruzi in the blood drops
from tails of all five mice infected with transgenic parasites
(Fig. 4a and b). The intense fluorescence shown by the
transfected parasites facilitated their identification and
quantification in the animal bloodstream. When we ana-
lyzed other mouse tissues, fluorescent amastigotes were eas-
ily identified in the heart and diaphragm of animals
infected with TulaGFP2 (Fig. 4c and d), whereas no para-
sites were found in the inflammatory foci of animals
infected with WT trypomastigotes (data not shown).
Therefore, although animals infected with the Tulahue ´n
strain do not show intense parasitemia or tissue parasitism,
the use of fluorescent T. cruzi clearly helps in the detection
of parasites when using direct microscopic examination.
Finally, we incubated Vero cell cultures with TulaGFP1
and TulaRFP2 simultaneously. Four to 6 days later, we
were able to detect single cells containing large numbers
of both red and green amastigotes (Fig. 5a and b). Cells
were also co-infected with transfected and cloned popula-
tions derived from two strains belonging to distinct T. cruzi
lineages, a Colombiana strain expressing GFP (a T. cruzi I
strain) and Tulahue ´n expressing RFP (a T. cruzi II strain)
(Fig. 5c and d). The detection of green and red fluorescent
amastigotes inside the cells, although rare (occurring in less
than 1% of infected cells) clearly shows that a single host
cell can be infected by parasites belonging to two distinct
strains.
4. Discussion
Although several groups have reported the introduction
of foreign genes in T. cruzi, few reports have described the
use of integrative linear vectors containing sequences
homologous to endogenous genes other than rRNA
sequences to promote chromosomal integration of the
exogenous gene. Most papers describe the use of vectors
that replicate episomally, in the form of large concatemers,
in transfected epimastigotes. In addition, these plasmids
must be maintained in the cell under continuous drug selec-
tion. The introduction of sequences derived from the
rRNA promoter, described by several authors (Teixeira
et al., 1995; Tyler-Cross et al., 1995; Martinez-Calvillo
et al., 1997; Vazquez and Levin, 1999; Dos Santos and
Buck, 2000; Lorenzi et al., 2003), in addition to yielding
a much greater expression of the reporter gene, facilitates
the integration of the transfected vector into the nuclear
genome by homologous recombination with the rDNA
locus. Using the pTREX vector containing GFP and
RFP genes to transfect T. cruzi, we have observed a high
percentage of fluorescent cells as early as 12 h after electro-
poration. However, in contrast to other reports, the num-
ber of fluorescent parasites significantly diminishes 12
days after transfection even in the presence of drug selec-
tion (data not shown). Therefore, we designed a new vector
that targets the integration of a foreign gene to the tubulin
locus in the parasite genome, resulting in stable transfected
parasite populations that can be cultivated even in the
absence of drug selection (DaRocha et al., 2004).
Similar to the b-galactosidase-expressing T. cruzi
described by Buckner et al. (1999), which was generated
by integration of the E. coli lacZ gene into the calmodu-
lin/ubiquitin locus, we targeted the integration of fluores-
cent GFP and RFP sequences into a multicopy gene
array. After transfection, selection and cloning, the trans-
genic parasites showed high levels of green and red fluores-
cence even after 6 months of culture in the absence of
drugs. The stability of this genetic modification allowed
us to detect fluorescence not only in epimastigotes but also
in trypomastigotes and amastigotes derived from mamma-
lian cell cultures and in several tissues of infected mice. It is
however intriguing that in all transfected cloned popula-
a
0
2
4
6
8
10
12
14
Tula WTTulaRFP1TulaGFP2
% of infected cells
0
5
10
15
20
25
30
35
40
45
Tula WT TulaRFP1TulaGFP2
Nº amastigotes/cell
b
Fig. 3. Approximately, 105Vero cells were platted and infected with 106
transfected trypomastigotes (TulaRFP1 and TulaGFP2 clones) or wild
type parasites. After 6 days, cell monolayers were stained with Giemsa/
May Grunwald. The percentage of infected cells (a) and the number of
amastigotes per cell (b) were determined, with the numbers corresponding
to the mean of three independent experiments.
294
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Page 7

tions we always observed a small amount of non-fluores-
cent cells even after several months of drug selection.
GFP- and RFP-negative
observed even after one or more rounds of cloning proce-
dures using a serial dilution protocol or plating in blood-
agar media. Although we observed an increase in the per-
centage of green and red fluorescent cells after each step
of the cloning procedure, none of the cultures displayed a
population of parasites that was 100% fluorescent. As sug-
gested by preliminary data analyzing G418-resistant para-
site populations that have lost the fluorescent markers,
the most likely explanation is the occurrence of additional
recombination events that may take place after drug selec-
tion and cloning. Associated with regulatory mechanisms
acting differentially in distinct genome locations, these
recombination events may affect the expression of the
GFP or RFP genes in a subset of the transfected cells.
Southern blot analyses indicated that GFP and RFP
genes integrate at the tubulin locus in the genome of differ-
ent T. cruzi strains. Here we have shown the integration of
the RFP gene in the b-tubulin locus of the JG strain, which
belongs to the T. cruzi II main lineage. In an earlier report,
we showed that the same vector allowed integration of
GFP in b-tubulin locus of the Col1.7G2 clone, a strain that
belongs to the T. cruzi I lineage (DaRocha et al., 2004).
Because tubulin sequences are highly conserved even
parasitescontinuedto be
among distant species, we anticipate that it is possible to
generate GFP- and RFP-expressing parasites in most other
T. cruzi strains.
To our knowledge, this is the first report describing the
presence of stable, fluorescent parasites expressing GFP
and RFP in infected animal tissues. Similar to the work
described by Buckner and colleagues (1999), which used
b-galactosidase staining to visualize the transfected para-
sites in host tissues, we used targeted integration of the for-
eign gene marker. Thus, we can speculate that plasmid
integration into the parasite genome may contribute to
selection of a more homogeneous transfected population,
which facilitates the visualization of low numbers of
amastigotes present in infected animal tissues. It must how-
ever be noted that with the Tulahuen strain, we were only
able to detect GFP and RFP expressing parasites in GKO
animals. Similarly, Santos et al. (2000) used GFP-express-
ing parasites that were transfected with an episomal vector
to infect immunosuppressed mice. However, these authors
were not able to show fluorescent parasites in infected ani-
mal tissues. Using the pTREX vector, Guevara et al. (2005)
showed T. cruzi and Trypanosoma rangeli expressing GFP
and RFP in the hemolymph of experimentally infected
Rhodnius prolixus, in single and mixed infections. However,
similar to the work by Santos et al. (2000), these authors
did not show the presence of fluorescent parasites in animal
Fig. 4. Animal infection with tissue culture-derived trypomastigotes expressing green fluorescent protein (GFP). Fluorescent micrographs of mouse blood
obtained 12 days p.i. of interferon-c knockout mice containing a clone expressing GFP (TulaGFP2) at 100· magnification (a) and merged with a
transmitted light image (b). (c and d) Confocal images of mouse heart tissue infected with the same clone (original magnifications 63·). (c) Confocal image
for the GFP channel. (d) Merged differential interference contrast (DIC) image obtained with a Zeiss LSM 510 META confocal microscope.
S.F. Pires et al. / International Journal for Parasitology 38 (2008) 289–297
295

Page 8

tissues. In contrast to transfected parasites derived from
the Tulahue ´n strain, which required highly susceptible ani-
mals for infection, we were able to infect BALB/c mice
with GFP expressing clones derived from JG, Colombiana
and CL Brener strains. Although we were not able to visu-
alize fluorescent parasites in several tissues including heart,
diaphragm and rectum, probably because of the low para-
sitemia, infection of BALB/c mice with all of these strains
allowed detection of fluorescent trypomastigotes in the
bloodstream much easier than with WT parasites (not
shown).
In one of our current studies involving the GFP- and
RFP-expressing parasites, we incubated Vero cells with try-
pomastigotes expressing green and red fluorescence, to ver-
ify whether distinct parasites could replicate in the cytosol
of the same cell. We demonstrated that, even when the par-
asites belong to two different T. cruzi lineages, they are able
to infect the same cell. These fluorescent parasites are now
being used to investigate the genetic exchange process in T.
cruzi, a theme that has drawn much attention (Machado
and Ayala, 2001; Pedroso et al., 2003; Gaunt et al., 2003;
Freitas et al., 2006). In 2003, Gaunt et al. showed that
two T. cruzi clones carrying two different drug-resistance
markers and cultivated together through the entire life
cycle generated double drug-resistant hybrids. These
hybrids showed fusion of parental genotypes, loss of
alleles, homologous recombination and uniparental inheri-
tance of kinetoplast maxicircle DNA. Since hybrid clones
were only detected after passage through mammalian cells,
it has been speculated that the fusion event, which is
thought to be very rare, occurs only during the mammalian
amastigote stage. In contrast, using green and red fluores-
cent Trypanosoma brucei, (Gibson et al., 2006) have shown
that genetic exchange occurs in the insect vector. The
hybrid nature of yellow trypanosomes, present in the sali-
vary glands, was confirmed by analysis of molecular kary-
otypes and microsatellite alleles. Although we have not
detected any evidence for hybrids, the availability of T. cru-
zi expressing the two markers (GFP and RFP) will allow us
to perform more in depth studies of genetic exchange in
this organism.
Acknowledgements
We are grateful to Dr. Oscar Bruna-Romero for expert
assistance with confocal images, Dr. Luciana Andrade, Dr.
Fig. 5. Co-infection of Vero cells with parasites expressing different fluorescent proteins. Vero cell cultures were incubated with equal numbers of
transfected trypomastigotes from the TulaRFP1 and TulaGFP2 clones. Four to 6 days after infection, cells were fixed, stained with DAPI and then
intracellular fluorescent parasites were visualized by confocal microscopy using FITC and rhodamine filters to distinguish green and red amastigotes in the
same infected cell (a and b). Vero cell cultures were also incubated with equal numbers of trypomastigotes from cloned transfected cell lines derived from
the Colombiana (Col1.7G2GFP) and Tulahue ´n (Tula RFP1) strains (c and d). Cells were visualized at 80· original magnification using a Zeiss LSM 510
META confocal microscope. In b and d, merged fluorescence and DIC images are shown.
296
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Page 9

Conceic ¸a ˜o Machado and Dr. Leda Quercia Vieira for ani-
mal infections and Afonso Viana and Neuza A. Rodrigues
for expert technical assistance. This work was supported by
Fundac ¸a ˜o de Amparo a ` Pesquisa de Minas Gerais (FAP-
EMIG), Conselho Nacional de Desenvolvimento Cientı´fico
e Tecnolo ´gico (CNPq), WHO Special Program for Re-
search and Training in Tropical Diseases, and the Howard
Hughes Medical Institute.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.ijpara.
2007.08.013.
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