Knockout of the dhfr-ts gene in Trypanosoma cruzi generates attenuated parasites able to confer protection against a virulent challenge.
ABSTRACT Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes.
By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts(+/-) mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8(+) T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection.
This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.
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ABSTRACT: Treatment of susceptible C3H(He) mice with 10(7) live Corpus Christi strain culture-derived Trypanosoma cruzi provided protection against a subsequent Brazil strain challenge. This protection was indicated by a greater than 10-fold decrease in parasitemia and an increase in longevity (including survival) in many groups. The Corpus Christi organisms were unable to establish an apparent infection, but viability is an important element in the treatment in that freeze-thawed, non-viable preparations of the Corpus Christi strain were unable to provide protection. Adoptive transfer of resistance was achieved with spleen cells from Corpus Christi-treated, Brazil-infected mice which had recovered from the acute phase of infection. The T cell-depleted population of these spleen cells was able to transfer resistance whereas the T cell-enriched population was not protective. Passive transfer of serum from Corpus Christi-treated and Brazil-infected mice provided a temporary decrease in parasitemia in infected mice. The results presented herein suggest that Corpus Christi-induced protection to virulent T. cruzi challenge is mediated by antibody mechanisms.Journal of Parasitology 11/1984; 70(5):760-6. · 1.32 Impact Factor
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ABSTRACT: Immunity against lethal, bloodstream forms of Trypanosoma cruzi was achieved in mice by preinoculation of approximately equal to 10(5) culture epimastigotes of an attenuated T. cruzi strain (TCC). The risks of TCC inoculation in terms of pathogenicity or eventual increase in virulence of TCC progeny were evaluated. No pathogenic parasites could be selected from TCC progeny by either mouse, triatome, or culture passages. Immunizing doses of live TCC did not induce in adult mice alterations resembling chronic Chagas' disease, as judged by patterns of mortality, tissue damage, autoantibodies, or parasite recovery. On the basis of the same criteria, However, a remarkable similarity could be established between the disease caused in mice by inoculation of low numbers (10(2)) of pathogenic trypomastigotes and human chronic Chagas' disease. Although patent parasitemias were never revealed in fresh blood mounts obtained from TCC-inoculated mice, a few hemocultures and xenodiagnoses gave positive results, particularly soon after inoculations at birth. The parasites recovered by either method remained in the attenuated, epimastigote stage. In rabbits, no local lesions, fever, weight loss, or histopathological alterations were detected after subcutaneous inoculation of 10(7) TCC organisms, although one fifth of the animals yielded positive hemocultures of epimastigotes. The contrasting host response to cultured epimastigotes as compared with blood trypomastigotes indicates that, in experimental Chagas' disease, immunoprotection is not necessarily associated with immunopathology.Infection and Immunity 05/1982; 36(1):342-50. · 4.07 Impact Factor
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ABSTRACT: The possibility of preventing chronic infection by a battery of 17 wild isolates of Trypanosoma cruzi was studied in Swiss mice preimmunized with culture forms of an attenuated strain (TCC). Mice were challenged intradermally with low numbers of wild trypomastigotes obtained from naturally infected insect vectors captured within a 57,000 km2 subtropical area in northern Argentina and which had not undergone any laboratory propagation. A significant degree of protection was observed in all cases, according to one or more parameters. Immunization reduced the level of parasitemia (P < 0.05) in infections caused by 4 out of 13 isolates, as evaluated by microscope counts performed on fresh blood mounts and in 10 out of 15 isolates as evaluated by xenodiagnosis. A lesser degree of histopathology (P < 0.05) was detected in the heart (7 out of 17 isolates), urinary bladder (10 out of 17 isolates) and skeletal muscle (10 out of 17 isolates). None of these parameters reflected infection or pathology in TCC-immunized, non-challenged mice. While antigenic variation frustrates vaccination against African trypanosomes, the effective protection shown here against 17 T. cruzi primary isolates indicates lack of antigenic variation and thus the possibility of effective vaccination in Chagas' disease.International Journal for Parasitology 09/1986; 16(4):375-80. · 3.64 Impact Factor
Knockout of the dhfr-ts Gene in Trypanosoma cruzi
Generates Attenuated Parasites Able to Confer
Protection against a Virulent Challenge
Cecilia Perez Brandan1*, Angel M. Padilla2, Dan Xu2, Rick L. Tarleton2, Miguel A. Basombrio1
1Instituto de Patologia Experimental - CONICET, Universidad Nacional de Salta, Salta, Argentina, 2Center for Tropical and Emerging Global Diseases, University of
Georgia, Athens, Georgia, United States of America
Background: Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing
countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side
effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines;
however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the
molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for
parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may
protect against such reversion to virulent genotypes.
Methods and Findings: By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T.
cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in
growth of dhfr-ts+/2mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower
number of T. cruzi-specific CD8+T cells, in comparison to those induced by wild type parasites, was detected in mice
infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC
mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant
parasites still displayed a significant control over the secondary infection.
Conclusion: This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer
protection against further T. cruzi infections.
Citation: Perez Brandan C, Padilla AM, Xu D, Tarleton RL, Basombrio MA (2011) Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites
Able to Confer Protection against a Virulent Challenge. PLoS Negl Trop Dis 5(12): e1418. doi:10.1371/journal.pntd.0001418
Editor: Ana Rodriguez, New York University School of Medicine, United States of America
Received June 30, 2011; Accepted October 21, 2011; Published December 13, 2011
Copyright: ? 2011 Perez Brandan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by grant PICT 2005 32739 to MAB and NIH Grant PO1 AI0449790 to RLT. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Chagas disease is one of the major health problems in Latin and
Central America, where an estimated of 7.7 million people are
infected . This disease is the consequence of the infection by the
protozoan parasite Trypanosoma cruzi. This flagellate is transmitted
to mammalian hosts, including humans, by the feces of infected
triatomine insects. Infection is also possible via mother to fetus
during pregnancy and by contaminated blood transfusion. So far
there is no effective vaccine against Chagas disease and the current
available drugs have considerable side effects.
Animals surviving infection by T. cruzi become resistant to
subsequent homologous infections. This resistance exceeds, both
in strength and duration, the protection achieved with various
experimental T. cruzi vaccines. Several naturally attenuated strains
have been used in immunization-infection assays in experimental
models [2,3]. TCC is a naturally attenuated strain of T. cruzi that
was thought to be unable to persistently infect immunocompetent
mice ; however, recent experiments demonstrated that this
strain does persist in experimental animals (Padilla AM,
unpublished data). The results of immunization with this
attenuated strain were promising since inoculation of live TCC
epimastigotes provided protection against infection with the
virulent Tulahuen strain and against each of 17 wild isolates
obtained from an endemic area for Chagas in Argentina . The
protective capacity of this naturally attenuated strain was also
evaluated in field trials against natural vector-derived infection; the
TCC strain was not naturally transmitted in either guinea pigs or
dogs and these TCC inoculated animals were protected against
secondary natural infections [6–8]. Unfortunately, the potential of
reversion of the TCC strain to a virulent phenotype or persistence
in immunocompromised hosts cannot be foretold, rendering this
method not completely safe for broad application in domestic
Gene targeting methods have provided a better understanding
of trypanosomatid genetics, allowing the introduction or removal
of specific genes from the genome of these organisms. The
generation of attenuated parasites unable to sustain infection and
cause pathology through removal of virulence or metabolic factors
is now a reasonable possibility. A range of genetically altered
www.plosntds.org1December 2011 | Volume 5 | Issue 12 | e1418
parasites has been used as experimental vaccines [9,10] but
according to the literature, only four T. cruzi knockout lines have
been evaluated as experimental immunogens. In one approach, a
monoallelic mutant clone for the calmodulin-ubiquitin gene was
obtained from the virulent Tulahuen strain of T. cruzi. Mice
inoculated with different doses of mutant epimastigotes and later
challenged with virulent wild type Tulahuen trypomastigotes were
strongly protected, as shown by a reduction in parasite burden
. The second approach involved a T. cruzi line (L16) carrying a
targeted biallelic deletion of the lyt-1 gene. Also in this case, long-
term protection against a virulent challenge was observed in mice
pre-inoculated with L16 parasites as shown by a reduction in
parasite load in blood . In the third study, a biallelic knockout
of the gp72 gene in Y T. cruzi strain was shown to be highly
attenuated and able to induce long lasting protection against a
subsequent infection by virulent T. cruzi . Recently, T. cruzi
parasites lacking enoyl co-A hydratase genes (ech1+/2ech22/2)
were used for oral route immunization assays, showing that
vaccination with genetically modified T. cruzi parasites confers
protection against a further virulent challenge .
In the case of other parasitic protozoa, like Plasmodium sp or
Leishmania sp, the generation of genetically attenuated parasites for
use as protective vaccines has been more frequently reported
[10,15–18]. One particular approach was the generation of
Leishmania major dhfr-ts null mutants. In trypanosomatids dhfr-ts is
a single copy gene which codes for the bifunctional enzyme
dihydrofolate reductase-thymidylate synthase (DHFR-TS) [19,20].
This enzyme catalyzes sequential reactions in the biosynthesis of
dTMP. Therefore inhibition of this enzyme results in thymidine-
less death. Leishmania major parasites completely lacking the dhfr-ts
gene were generated through gene targeted deletion by homolo-
gous recombination . As expected, these mutant parasites were
auxotrophic and their safety and protective potential as experi-
mental vaccines were evaluated . dhfr-ts2/2parasites were able
to persist in mice for up to 2 months; however, they were
incapable of causing disease in both susceptible and immunode-
ficient mouse models. A substantial resistance to challenge with
virulent L. major parasites was detected . Moreover, heterol-
ogous protection against challenges with different Leishmania
species was also observed .
Here we studied the biological effect of introducing a mutation
in the dhfr-ts gene of the naturally attenuated TCC strain of T. cruzi
as a safety device to avoid the potential reversion to virulent
variants. Moreover, the effect of the same mutation was evaluated
in dhfr-ts+/2mutant clones of the virulent Tulahuen strain. We
also investigated the persistence of these parasites and their
capacity to induce an immune response in infected hosts and
protect against a subsequent infection.
All animal protocols adhered to the National Institutes of
Health (NIH) ‘‘Guide for the care and use of laboratory animals’’
and were approved by the School of Health Sciences, National
University of Salta and the University of Georgia Institutional
Animal Care and Use Committee.
Parasites and culture procedures
Wild type forms of the naturally attenuated TCC and the virulent
Tulahuen strains of T. cruzi were used, as well as two mutant clones
derived from the Tulahuen strain carrying a targeted mutation of one
dhfr-ts allele . Epimastigote forms were grown at 28uC in liver
digested neutralized tryptose medium (LDNT), supplemented with
10%fetal bovine serum (FBS).Metacyclictrypomastigoteswere either
obtained fromstationaryphase epimastigoteculturesorbyadding1%
triatomine gut homogenate  to epimastigote cultures and
harvesting the parasites after 7 to 10 days. In both cases, complement
resistant forms were purified using normal non decomplemented
serum, quantified in a hemocytometer and further used to inoculate
experimental animals. For the challenge experiments, fluorescent CL-
tdTomato  as well as Tulahuen and CL wild type trypomastigotes
were used. These trypomastigote forms were obtained either from
Vero cell monolayers cultures or from infected mice. Infected Vero
cells were cultured in RPMI 1640 medium with 10% FBS in a humid
atmosphere containing 5% CO2at 37uC.
Generation of Trypanosoma cruzi mutant parasites
To generate parasites of the TCC strain of T. cruzi with a
disruption of the dhfr-ts gene, the plasmid pBSdh1f8Neo was used.
This plasmid contains the coding sequence of the dhfr-ts gene
interrupted by the coding sequence of the neomycin phospho-
transferase gene and it has been previously used for the generation
of single knockout parasites, by homologous recombination, of the
Tulahuen strain of T. cruzi . Transgenic parasites were
generated as previously described . A total of 107early-log
epimastigotes were centrifuged at 1,620 g for 10 min and
suspended in 100 ml Human T Cell NucleofectorTM Solution
(Lonza, Cologne) at room temperature. The resuspended parasites
were then mixed with 10 mg DNA in a total volume of 10 ml and
electroporated using the program ‘‘U-33’’ in an AMAXA
Nucleofector Device (Lonza). The electroporated parasites were
then cultured in 25 cm2 culture flasks with 10 ml LDNT medium
and 300 mg/ml of G418 were added at 24 h post-transfection.
Individual clones were obtained by single cell sorting into a 96-well
plate using MoFlow cell sorter (Dako-Cytomation-Denmark).
Molecular characterization of mutant parasites
For Southern blot analysis, genomic DNA from a selected TCC
clone and from TCC wild type parasites was purified using the
Phenol-Chloroform method. The DNA was then digested,
separated by 0.7% agarose gel electrophoresis and the gels were
blotted onto nylon membranes (Hybond-N 0.45-mm-pore-size
filters; Amersham Life Science) using standard methods . For
probes generation, a 795 bp DNA segment corresponding to
Neomycin Phosphotransferase gene was amplified from plasmid
Chagas disease is the clinical manifestation of the infection
produced by the flagellate parasite Trypanosoma cruzi and
currently there is no vaccine to prevent this disease.
Therefore, different approaches or alternatives are urgently
needed. Vaccination with live attenuated parasites has
been used effectively in mice to reduce parasitemia and
histological damage. However, the use of live parasites as
inmunogens is controversial due to the risk of reversion to
a virulent phenotype. In this work we genetically
manipulated a naturally attenuated strain of T. cruzi in
order to produce parasites with impaired replication and
infectivity, using the mutation as a safety device against
reversion to virulence. We show that genetically modified
parasites display a lower proliferation rate in vitro and
induced almost undetectable levels of T. cruzi specific
CD8+T cells when injected in mice. Furthermore, the
immune response induced by these live mutant parasites
confers protection against a subsequent virulent infection
even a year after the original immunization.
T. cruzi dhfr-ts KO Vaccine
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pBSSK-neo1f8  using primers Neo_for (59 ATGATTGAA-
CAAGATGGATT 39) and Neo_rev (59 AGAACTCGTCAA-
GAAGGCGA 39) while dhfr-ts gene was amplified from genomic
DNA of TCC wild type parasites using primers DH5_f (59
TGTCGCTGTTTAAGATCCGC 39) and DH6_r (59 CCAT-
GAAGATGGCGGTTTAG 39). Labeling of the probes and DNA
hybridization were performed according to the protocol supplied
with the PCR-DIG DNA-labeling and detection kit (Roche Applied
PCR analyses were carried out using as template DNA from
TCC wild type as well as TCC dhfr-ts+/2parasites. The primers
used for PCR analysis were specific for the upstream gene of the
dhfr-ts gene (PG1 59 CTTCGAGGAGCTTTGCTGTT 39 and
PG2 59 GATCCAACCAACTGGAGGAA 39 ) in combination
with a primer specific for the neomycin phosphotransferase gene
(N 59 GATCTCCTGTCATCTCACCT 39).
Epimastigote growth assays
26105epimastigotes from mutant and wild type parasites were
grown in 6-wells plates containing 5 ml of LDNT medium per
well. Samples were done by triplicate and the number of growing
parasites was quantified daily in a hemocytometer.
Infectivity assays in mice
In order to evaluate the infectivity of dhfr-ts+/2mutant parasites,
different mouse strains were used. C57BL/6J (B6) mice were
purchased from The Jackson Laboratory. IFNc2/2, Balb/c, Swiss
and nude (nu/nu) mice (1 to 2 months old) were bred and
maintained in our animal facility under specific pathogen-free
conditions. Animals were inoculated by intraperitoneal (i.p.) route
with metacyclic or trypomastigote forms of mutant and wild type
parasites as specified.
To test the immunological protection induced by mutant clones,
mice were first inoculated with 56105dhfr-ts+/2metacyclic
parasites and later challenged, at different time points, with 104
blood trypomastigotes of the Tulahuen wild type strain or with
2.56105culture trypomastigotes derived forms of the fluorescent
CL-tdTomato strain  or CL wild type.
Blood (10 ml) was drawn from the tail tip of mice under slight
anesthesia, and the number of parasites per 100 fields (parasitemia)
was recorded from fresh blood mounts under microscope (6400).
For in vivo fluorescence detection, footpads of mice subcutaneously
infected with CL-tdTomato parasites were imaged every other day
using the Maestro2 In Vivo Imaging System (CRi, Woburn, MA)
with the green filter set (acquisition settings: 560 to 750 in 10 nm
steps; exposure time 88.18 ms and 262 binning). Collected images
were unmixed and analyzed with the Maestro software v2.8.0A.
Hemocultures were performed by seeding, under sterile conditions,
200 ml of heparinized blood into 2 ml of LIT medium (Liver
Infusion Tryptose) supplemented with 10% FBS. The cultures were
incubated at 28uC and analyzed at day 15, 30, 45, and 60. For PCR
detection of T. cruzi, 700 ml of blood from inoculated animals was
processed following strict PCR decontamination procedures.
Sample storage, DNA extraction, and amplification using primers
121 and 122 were performed as previously described .
Total immunoglobulin G antibodies against T. cruzi were measured
by the enzyme-linked immunosorbent assay (ELISA) using T. cruzi
epimastigote homogenate asantigen.The antibodyconcentration was
expressed as the optical density at a 492-nm wavelength.
Trypanosoma cruzi specific CD8+T cells determination
T. cruzi-infected mice were bled and whole blood was stained
with a MHC class I tetramer containing the T. cruzi specific
peptide TSKB20 (TSKB20/Kb-PE tetramer) as previously
described . Cells were stained with anti-CD8–allophycocya-
nin, anti-CD11b–Cy5-PE, anti-CD4–Cy5-PE and anti-B220–
Cy5-PE (all from Caltag, Burlingame, CA). CD8+T cells were
gated in the CD42CD11b2B2202lymphocyte population. Flow
cytometry was carried out on a FACSCalibur flow cytometer
(Becton Dickinson, San Diego, CA), and data were analyzed with
FlowJo software (Tree Star, Inc., Ashland, OR).
Continuous variables, such as antibody titers and parasite
concentrations in blood samples, were analyzed with the two-tailed
Wilcoxon signed-rank test for time course plots and with the
Mann-Whitney or Kruskal-Wallis test for single-day measure-
ments. Values are expressed as mean 6 standard errors of the
mean from at least three separate experiments. Differences
between two groups were considered significant at p,0.05.
Generation of TCC dhfr-ts mutant parasites
Using constructs targeted for the interruption of the dhfr-ts gene,
single-allele knockout parasites (dhfr-ts+/2) for the TCC strain of T.
cruzi could easily be achieved by electroporation with the plasmid
pBSdh1f8Neo and selection in 300 mg/ml of G418, as it was
previously shown for the Tulahuen strain of this parasite . The
genome locus of dhfr-ts gene is shown in Figure 1A. Southern Blot
analysis of a TCC dhfr-ts+/2clone confirmed the correct insertion
of the neomycin phosphotransferase gene interrupting the coding
sequence of dhfr-ts in the parasite genome (Figure 1B). By using a
combination of the enzymes SalI and EcoRI, which cut outside the
recombination DNA fragment electroporated and by using
neomycin phosphotransferase sequence as a probe, we could
confirm the correct interruption of the target gene, since a 3 kb
band was obtained as expected. When hybridizing with the dhfr-ts
probe, bands of 2 kb and 3 kb were obtained, indicating successful
interruption of one dhfr-ts allele. PCR analyses using specifically
designed primers upstream of the dhfr-ts gene in combination with
primers for the neomycin phophotransferase gene also revealed
the correct insertion of the antibiotic marker interrupting the
target gene (Figure 1C). However, repeated attempts to interrupt
the second copy of this gene and create null mutant parasites did
not succeed, either for TCC dhfr-ts+/2or Tulahuen dhfr-ts+/2
parasites. Moreover, thymidine addition to the culture media did
not help in obtaining null parasites, suggesting that the dhfr-ts gene
may be essential for T. cruzi survival in vitro. Only in one occasion
and after several attempts, we were able to obtain resistance to
both, neomycin and hygromycin, but these selected parasites still
retained a copy of the dhfr-ts gene (data not shown).
Cell growth in vitro is significantly affected in dhfr-ts+/2
To determine if the interruption of one allele of the dhfr-ts gene
affects the ability of T. cruzi to replicate in culture, dhfr-ts+/2
epimastigotes from the TCC and the Tulahuen strains, were
seeded in 6-well plates in LDNT medium without selecting
antibiotic pressure and parasites were counted daily until
stationary phase was reached. After day 5, significant impairment
T. cruzi dhfr-ts KO Vaccine
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in TCC dhfr-ts+/2epimastigote growth was detected when
compared to TCC wild type (Figure 2A). However, these
differences were less evident in Tulahuen mutant parasites when
compared to Tulahuen wild type epimastigotes (Figure 2B).
Addition of thymidine (100 mg/ml) did not improve mutant
parasite growth (data not shown).
In vivo infectivity of Tulahuen dhfr-ts+/2metacyclic
Infectivity of Tulahuen dhfr-ts+/2metacyclic trypomastigotes
was determined by quantifying parasite load in blood from
animals independently inoculated with either individual mutant
clones or Tulahuen wild type parasites as a control. Nude mice as
well as IFNc2/2mice infected with 56104Tulahuen dhfr-ts+/2
parasites succumbed after 20–25 days of infection even though the
parasite load in these infected mice was significantly lower than
with Tulahuen wild type parasites (Figure 3A–B). In a Balb/c
mouse model differences in parasite load between mice receiving
Tulahuen wild type (26104metacyclic trypomastigotes/mouse)
and mutant lines (26105metacyclic trypomastigotes/mouse) were
evident, despite the fact that 10-fold fewer wild type parasites were
used to initiate these infections (p,0.05) (Figure 3C). In summary,
Figure 1. Disruption of one allele of the dhfr-ts gene in the TCC strain of T. cruzi. (A) Diagram of the expected genomic loci of dhfr-ts in
single knockout parasites. (B) Southern Blot analysis of genomic DNA of wild type and a dhfr-ts+/2TCC clone digested by a combination of SalI/EcoRI
enzymes and hybridized with a DNA probe complementary to the neomycin phosphotransferase gene or the dhfr-ts gene. (C) PCR analysis using a
combination of specific primers complementary to the coding sequence of the upstream gene and the neomycin resistance gene.
Figure 2. In vitro growth for dhfr-ts+/2and wild type epimastigotes. (A) Growth curve of TCC wild type versus TCC dhfr-ts+/2clone and (B)
growth curve of Tulahuen wild type versus Tulahuen dhfr-ts+/2clone. These results are representative of 3 independent experiments.
T. cruzi dhfr-ts KO Vaccine
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results from three independent experiments with three different
mouse strains led to the conclusion that the parasite load in mice
receiving Tulahuen dhfr-ts+/2parasites was significantly lower than
in mice receiving wild type parasites.
In vivo infectivity of TCC dhfr-ts+/2metacyclic
To determine if the naturally attenuated TCC strain could be
rendered even less infective via mutation of the dhfr-ts gene, we
evaluated the infectivity of wild type and dhfr-ts mutant TCC lines
in different mouse strains. Since TCC parasites naturally display
undetectable levels by direct blood examination in immunocom-
petent infected mice, the establishment of infection by TCC
mutant parasites was determined by PCR and hemoculture in
blood samples taken at day 15 post inoculation. No positive
hemocultures were obtained from immunocompetent Balb/c or
Swiss mice injected with 56105TCC dhfr-ts+/2metacyclic
parasites (Table 1). However, using nude mice infected with 105
TCC dhfr-ts+/2metacyclic parasites, parasite recovery by hemo-
culture was demonstrated in 3/3 animals infected with TCC wild
type and in 4/5 animals infected with TCC dhfr-ts+/2parasites.
Lower proportions of infected animals were detected by PCR in
immunocompetent Balb/c and Swiss mice inoculated with the
mutant as compared to wild type TCC (Table 1). No mortality was
observed in animals infected with mutant or wild type TCC
parasites. Thus, the natural attenuation of TCC leaves a narrow
range to measure further attenuation in the mutants. Nevertheless,
in every measurable case the rates of infection obtained with TCC
dhfr-ts+/2were lower than those of TCC wild type. These results
led us to conclude that mutation of one allele of the dhfr-ts gene is
sufficient to render mutant parasites less virulent than their
parental line. We then wondered if these parasites were capable of
surviving for long periods of time in the infected hosts; therefore
we evaluated the persistence of TCC dhfr-ts+/2parasites after 60
and 120 days post infection. Day 120 samples were obtained after
immunosupression with cyclophosphamide (5 doses of 250 mg/kg
of cyclophosphamide per mouse and samples taken 10 days after
the last dose). On day 60, all immunocompetent animals were
negative by both, PCR and hemoculture, whereas 80% (4/5) nude
mice still remained positive. On day 120, 3 surviving immuno-
competent animals (2 Balb/c and 1 Swiss) were negative by PCR
and hemoculture. Parallel determinations in TCC wild type
infected animals did not differ from TCC dhfr-ts+/2in immuno-
competent mice, except for the fact that in 1 out of 3 animals, a
positive PCR signal was obtained. These results show that
parasites are maintained below detectable levels of our most
Figure 3. In vivo infectivity of Tulahuen dhfr-ts+/2and Tulahuen wild type metacyclic trypomatigotes. (A) Parasitemia curves of IFNc2/2
mice inoculated with 56104metacyclic trypomastigotes of Tulahuen wild type and dhfr-ts+/2parasites. (B) Parasite load of nude mice inoculated with
56104metacyclic trypomastigotes of Tulahuen wild type and dhfr-ts+/2parasites at day 20 post-infection. (C) Parasitemia curves of Balb/c mice
inoculated with 26104metacyclic trypomastigotes of Tulahuen wild type and 26105metacyclic trypomastigotes of Tulahuen dhfr-ts+/2metacyclic
trypomastigotes. Values are given as means; error bars indicate standard errors of the means.
T. cruzi dhfr-ts KO Vaccine
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stringent techniques, opening the possibility that in some cases
might even be completely clear although total parasite elimination
is difficult to assess.
TCC dhfr-ts+/2parasites inoculation induces a low level of
specific CD8+T cells
Parasite–specific CD8+T cells have been shown to be crucial in
the immunity against T. cruzi . It has been shown that the wild
type TCC strain, despite being naturally attenuated, is able to
induce parasite-specific CD8+T cells in infected mice (Padilla AM,
unpublished data). Therefore, and by staining with the MHC class
I tetramer containing the T. cruzi specific epitope TSKB20 ,
we were able to determine the generation of specific CD8+T cells
in peripheral blood of mice inoculated with TCC dhfr-ts+/2and
wild type parasites. For this purpose C57BL/6J (B6) mice were
injected with 56105TCC mutant parasites. Blood samples were
analyzed at day 15 post inoculation. As shown in Figure 4A, CD8+
T cells specific response was normal in mice infected with TCC
wild type parasites, while in mutant infected mice, the level of
CD8+T cells positive for the staining with the MHC class I
tetramer containing the TSKB20 tetramer was not significantly
different from naı ¨ve mice (Figure 4A). Since the attenuation of
TCC dhfr-ts+/2parasites seems to be stronger than wild type
parasites we analyzed the CD8+response in a more sensible mouse
model. For this purpose IFNc2/2mice were inoculated with
56104metacyclic trypomastigotes of mutant and wild type TCC
parasites. In this case, we also detected differences in the T. cruzi
specific CD8+T cell profile displayed 22 days after infection. The
percentage of parasite-specific CD8+T cell was significant lower in
mice infected with TCC dhfr-ts+/2parasites when compared to
TCC wild type infected ones (Figure 4B). Only one mouse infected
with the dhfr-ts+/2displayed a defined MHC class I tetramer
positive population different from the naive background levels and
more similar to the TCC wild type infected ones. These results
reinforce the previous one, demonstrating the high attenuation of
the TCC dhfr-ts+/2parasites.
Protective immunity acquired by infection with TCC wild
type and dhfr-ts+/2parasites
The TCC strain of T. cruzi has been extensively used by our
group as a live vaccine [7,8,32]. Since TCC dhfr-ts+/2mutant
parasites displayed in several experiments a lower infectivity
than TCC wild type, we tested whether this attenuation would
affect the protective effect of TCC against a virulent challenge.
For this purpose we carried out two independent short term
immunization assays. In one experiment, groups of 4, 30-day-
old C57BL/6 female mice were inoculated with either 56105
metacyclic trypomastigotes of TCC wild type, similar forms of
TCC dhfr-ts+/2parasites or PBS as a control group. At day 15
post first inoculation, the animals were boosted with the same
dose of parasites. To determine if this immunization regimen
induced a cellular immune response, blood samples were taken
during the immunization phase. In the protection assays mice
immunized with the TCC dhfr-ts+/2parasites reached levels of
CD8+T cells specific for the TSKB20 epitope different from the
naive background only after a second boost (Figure 5A). Fifteen
days after the boost, the animals were challenged with 104
metacyclic forms of the virulent CL strain of T. cruzi.
Parasitemia was measured in fresh blood mounts twice a week
in all animals. Mice previously inoculated with either TCC wild
type or TCC dhfr-ts+/2showed a lower parasite load than
challenged naı ¨ve mice (Figure 5B). Despite the lower number of
specific CD8+T cells detected in mice immunized with mutant
parasites, no differences were found between the protection
conferred by wild type and dhfr-ts+/2TCC, suggesting that the
interruption of one dhfr-ts allele did not affect their vaccine-
induced protection. Similar results were obtained in another
short term immunization assay with Balb/c male mice
immunized with the same doses and regimen as above but
Table 1. Infectivity of TCC dhfr-ts+/2and TCC wild type
parasites in different mouse strains.
Nude3/3 4/5 ND*ND*
Swiss 0/40/5 2/40/5
*ND: not done.
Figure 4. T. cruzi CD8+specific response in mice infected with TCC dhfr-ts+/2and wild type parasites. Frequency of TSKB20-specific CD8+
T cells in (A) B6 mice infected with 56105metacyclic parasites of mutant and wild type TCC parasites (n=4) and (B) IFNc2/2mice infected with 56104
metacyclic trypomastigotes of mutant and wild type TCC parasites (n=6 and n=3 respectively). Bars represent the mean frequencies of CD8+
tetramer-positive lymphocytes per group; error bars represent standard errors of the mean.
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challenged with 56103blood trypomastigotes of the virulent
Tulahuen wild type strain. Specific anti-T. cruzi antibody levels
in sera were undetectable for mice immunized with TCC wild
type parasites or TCC dhfr-ts+/2(Figure 5C) and clearly
different from the level for mice infected with Tulahuen wild
type parasites, as determined by ELISA at 14 days post-boost.
This was expected since previous results from our group showed
that the TCC strain per se is not a good inducer of a humoral
response . However, Balb/c mice pre-infected with TCC
wild type or TCC dhfr-ts+/2metacyclic trypomastigotes showed
reduced numbers of circulating parasites in the peripheral blood
(Figure 5D). Mortality on immunized and challenged mice
was null in this animal model. Again, in this experiment no
differences were detected in the protective capacity of dhfr-ts+/2
versus wild type TCC parasites.
dhfr-ts+/2mutant parasites are able to confer long lasting
protection against a subsequent T. cruzi virulent infection
To determine the duration of the protection observed in short
term immunization-challenge experiments, we carried out a long
term immunization assay. For this purpose, B6 mice immunized
with TCC dhfr-ts+/2or TCC wild type parasites were challenge
370 days post infection with virulent parasites. In this case, we
employed an approach of challenging with CL-tdTomato parasites
expressing the fluorescent protein td-tomato  which can be
tracked in vivo at the site of the infection. This technology allows us
a more quantitative determination of the parasite control at the
site of infection during the days following the challenge. This early
determination is important since one desirable characteristic of a
vaccine is to confer a rapid response and control of the parasites at
the entry location, limiting their proliferation and spread through
other organs. Groups of 3, 30-day-old C57BL/6 female mice were
inoculated with either 56105
trypomastigotes, TCC dhfr-ts+/2parasites or PBS as a control
group. Blood samples were taken at day 300 post infection in order
to establish the percentage of T. cruzi specific CD8+T cells. At 300
days post infection, only one mouse inoculated with TCC dhfr-ts+/2
have a detectable population of CD8+T cells specific for the
TSKB20 epitope. However, TCC wild type infected mice displayed
a consistent TSKB20 specific population (Figure 6A). At day 370
post-infection, these mice were challenged in the footpad with
TCC wild type metacyclic
Figure 5. Short term protection in immunocompetent mice infected with TCC mutant parasites. (A) Lymphocytes were recovered from
blood of B6 mice immunized with TCC wild type (grey bar) and TCC dhfr-ts+/2(white bar) 14 days after the boost and were stained with the TSKB20
MHC I tetramer. Bars represent the mean frequencies of CD8+tetramer-positive lymphocytes for four mice per group; error bars represent standard
errors of the mean. (B) Parasitemia curve of B6 mice infected with 56105TCC dhfr-ts+/2metacyclic trypomastigotes, TCC wild type metacyclic
trypomastigotes and PBS and challenge with 104virulent CL parasites. (C) Dispersion diagrams of antibody levels in either naive animals (non
immunized) and those immunized with 56105metacyclic trypomastigotes of TCC dhfr-ts+/2or TCC wild type. The results are expressed as the ratio of
the absorbance of each serum sample at a 490-nm optical density (OD) to the cutoff value. Dotted lines indicate the cutoff adopted for positivity,
calculated as the mean of the values determined for the naive controls plus three times the standard deviation. Positive controls were infected with
Tulahuen wild type parasites. (D) Parasitemia curve of Balb/c mice infected with TCC dhfr-ts+/2metacyclic trypomastigotes, TCC wild type metacyclic
trypomastigotes or PBS and challenge with 56103virulent Tulahuen blood trypomastigotes. Values are given as means; error bars indicate standard
errors of the mean.
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2.56105metacyclic trypomastigotes of the fluorescent CL-
tdTomato strain. Fluorescence at the site of infection was
measured for 13 consecutive days as a surrogate measurement
of parasite load. Figure 6B depicts the evolution of parasite load
during 13 days. Mice previously infected with TCC wild type
metacyclic trypomastigotes a year before were still considerably
protected against the virulent challenge. Despite displaying a
more attenuated behavior, TCC dhfr-ts+/2infection produced a
similar protective effect compared to TCC wild type parasites.
Overall, these observations indicate that both, wild type and
dhfr-ts+/2TCC primo infection conferred a long-lasting protec-
tion against secondary infections.
Targeted gene deletion has been one of the most important
tools for the study of gene functions, mainly in those organisms
where the current techniques for gene silencing by RNA
interference has failed . The first T. cruzi mutant line carrying
a targeted deletion of a metabolic gene was generated over 18
years ago . Unfortunately, the list of genes that have been
altered for reverse genetic studies in T. cruzi has so far not
increased considerably [24,35–49]. In our limited experience, the
complete deletion of an identified gene through homologous
recombination is not an easy task. The mutants at the dhfr-ts locus
obtained in this work attempting to delete both copies of the dhfr-ts
gene support this notion. Despite the correct replacement of the
endogenous dhfr-ts gene by different antibiotic resistance genes, the
presence of an extra copy in the genome may suggest an evasion
strategy by the parasite to avoid the loss of this essential gene.
Apparently, duplications of the target gene or the whole
chromosome may be taking place. Similar events showing target
locus amplification were observed when trying to obtained null
mutant T. cruzi parasites for the enoyl-CoA hydratase (ech) and
UDP-Glcp 49-epimerase (TcGALE) genes [24,38]. Identifying the
frequency at which duplication events take place could be
important for targeted deletion protocols and for probing the
plasticity of the genome of this intriguing parasite. Possibly,
trisomy and polyploidy are more frequent events than expected.
Overall, our attempts to create a null mutant of the DHFR-TS
enzyme strongly suggest that the dhfr-ts gene is essential in T. cruzi
epimastigotes, even when exogenous thymidine is provided.
The enzyme dihydrofolate reductase thymidylate synthase of T.
cruzi is involved in a number of different vital processes, essential
for parasite survival. The impairment in dhfr-ts+/2epimastigote
growth is in agreement with depletion of one allele, since the
enzyme product of this gene is involved in the synthesis of
thymidine monophosphate, needed for DNA assembly and
therefore, for cellular replication. The significant loss of the ability
of Tulahuen dhfr-ts+/2parasites to develop blood parasitism in
immunocompetent mice suggests that this gene may be considered
as a virulence factor of T. cruzi. A reduction in the virulence of
knockout parasites in animal models has been previously observed
in other T. cruzi lines. Such is the case for the T. cruzi Ynull line,
carrying a biallelic targeted deletion of the gp72 gene. This
mutation impaired the ability of Y strain parasites to maintain a
latent infection in immunocompetent mice . Similar results
were also obtained for other T. cruzi mutants [11,12,50]. Here we
report that the disruption of one copy of the dhfr-ts gene in the
naturally attenuated TCC strain of T. cruzi results in even more
attenuated parasites than the parental strain.
In experimental infections in mice with T. cruzi virulent
parasites a strong CD8+T cell response against immunodominant
peptides encoded in trans-sialidase family genes is observed .
However, this specific CD8+T cell response against a single
epitope (TSKB20) in mice infected with TCC dhfr-ts+/2parasites
was considerably lower than in mice infected with TCC wild type.
The development of specific CD8+T cells is determined not only
by the kind but by the amount of available antigen. The lower
proportion of T. cruzi specific CD8+T cells in mice infected with
TCC dhfr-ts+/2parasites could probably be correlated with the
inherent propagation rate previously observed for these mutant
parasites. Therefore, a late antigen presentation to dendritic cells
or a lower availability of parasite antigens capable of reaching sites
of priming for the CD8+T cell response, may be taking place.
However; both TCC wild type and TCC dhfr-ts+/2parasites,
activated a protective immune response against a second virulent
infection. Despite of generating a lower proportion of TSKB20
Figure 6. Long-term protective immunization with TCC dhfr-ts+/2metacyclic trypomastigotes against virulent challenge with T. cruzi
CL-tdTomato. A) CD8+T cells positive for TSKB20 at day 300 post infection in B6 mice inoculated with 56105metacyclic trypomastigotes of mutant
and wild type TCC parasites. B) Parasite load after challenge, at day 370 post infection, with 2.56105bloodstream forms of the virulent CL-tdTomato
strain. Fluorescence levels were measured during 13 days. Values are given as means; error bars indicate standard errors of the means.
T. cruzi dhfr-ts KO Vaccine
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specific CD8+T cells, dhfr-ts+/2parasites were able to induce
protection in the immunized mice. This is in agreement with
previous work showing that TSKB20 specific CD8+T cells
contribute to an optimal control of the acute infection, but are not
crucial for the development of immune resistance . Consid-
ering that the TSKB20 specific CD8+T cells account for
approximately 30% of the total CD8+T cells in C57BL/6
infected mice at the peak of the response, it is interesting to see that
TCC dhfr-ts+/2vaccinated mice are still protected, even when they
display a lower proportion of TSKB20+CD8+T cells (compared to
TCC wild type infected mice) prior to challenge. This suggests that
other cell populations against alternative, still undefined, epitopes
may be induced by the vaccination with attenuated parasites with
an important role in the protection elicited. An alternative non
exclusive explanation is that the level of CD8+T cell response
generated and maintained by the immunization, although barely
detectable may be efficient enough to crucially curb the initial
replication of challenging parasites resulting in lower local and
systemic parasite level. The elucidation of those mechanisms will
help in defining the desired characteristics of vaccines against T.
cruzi infection and their rational development.
A point worthy of mention is that the interruption of a copy of
the dhfr-ts gene in the already naturally attenuated TCC strain
seemed to render these parasites undetectable by highly sensitive
methods after 60 days post inoculation in immunocompetent mice.
Parasite recovery in low level infections is considerably difficult;
thus, dhfr-ts+/2TCC parasites are not detected by a sensitive
technique previously used to demonstrate parasite clearance by
effective drug treatment  suggesting that these mutant
parasites may be kept at extremely low numbers without
significantly affecting their protective capacity. This result has
considerable implications since if genetically modified live
attenuated parasites are planned to be used in vaccination of
animal reservoirs, one crucial aspect is that vaccinating parasites
should be unable to be transmitted and integrated in the natural
cycle. Even if mutant parasites are not completely cleared from the
vaccinated animals, the considerable reduction in their number
and ability to develop in the insect vector  should decrease the
chances of being transmitted. Therefore this result opens the
possibility of developing a genetically modified line with increased
safety characteristics than naturally attenuated parasites without
compromising the protection induced. Although targeted deletion
of specific genes can be conceived as a potential approach to
generate attenuated lines, genetic manipulation or complete
abrogation of infectivity could lead to a loss of protective
immunity. Since the immune mechanisms of protection in T.
cruzi infection are not completely understood, it is still debatable if
in the case of live attenuated vaccines, the persistence of the
vaccinating parasites is required for maintaining the protection in
a long term. Our results support the hypothesis that a highly
controlled acute infection with genetically attenuated parasites is
enough to induce a protective response which can be maintained
for a long term under conditions of vaccinating-parasite
persistence below detection levels or even complete clearance.
We are grateful to Ruben Cimino for the serological determinations. We
thank Julie Nelson of the Center for Tropical and Emerging Global
Diseases Flow Cytometry Facility at the University of Georgia. Skillful
technical assistance was provided by Alejandro Uncos, Renato Uncos and
Conceived and designed the experiments: CPB AMP MAB RLT.
Performed the experiments: CPB AMP DX. Analyzed the data: CPB
AMP. Contributed reagents/materials/analysis tools: CPB AMP DX.
Wrote the paper: CPB AMP.
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