Figure 1.
MutT heterologous expression did not alter T. cruzi growth but enhanced survival against H2O2.
(A) Amplification of a 112-bp fragment of MutT from transfected parasite total RNA (MutT +) and positive controls (E. coli total DNA). pROCK +: total RNA from parasites transfected with the pROCK empty vector. MutT - and pROCK -: RT-PCR negative control without reverse transcriptase; - control: PCR control without template DNA. (B) WT and MutT parasites were grown in LIT medium and followed for 6 days until the stationary phase. Alternatively, WT and MutT were grown in LIT medium containing different concentrations of H2O2 for 3 days and then counted (C). Experiments were performed in triplicate. The survival percentage was measured in relation to untreated cells, and the bars represent the SD (*** P<0.001, * P<0.05, unpaired t test).
Figure 2.
Analysis of pROCK parasite DNA lesions compared with MutT parasites through a QPCR assay.
Specific primers were used to amplify large and small fragments of the nuclear and mitochondrial DNA of untreated cells. Normalized amplification fluorescence values are in Table 1. This values were used to estimate the relative amplification of pROCK cells compared to MutT. The pROCK average number of lesions per 10 kb of the genome in relation to MutT parasites was then estimated by the relative amplification calculated (*** P<0.0001, unpaired t test).
Figure 3.
MutT expression enhances in vitro intracellular growth.
Murine fibroblasts were exposed to trypomastigotes (MOI of 50) for 30 minutes. Monolayers were washed to remove extracellular parasites and either fixed (PFA 4%) or incubated with fresh medium without parasites for different times. Slides were stained by immunofluorescence and analyzed in fluorescence microscope. (A) Parasites infectivity was determined by counting the number of internalized trypomastigotes per 100 cells right after cell exposure to parasites. (B) Parasitophorous vacuole escape kinetics were determined by analyzing the number of parasites co-localizing with LAMP, a lysosomal marker, at different time points for 16 hours after invasion. (C) Number of intracellular parasites per infected cell for WT, pROCK and MutT infected cultures from 24 up to 96 hours post-infection (*** P<0.001, * P<0.05, two-way ANOVA test with Bonferroni post-test). (D) Representative images of murine fibroblasts infected either with WT or MutT at an MOI of 50, cells and parasites DNA were labeled with DAPI.
Figure 4.
H2O2 treatment does not affect MutT parasite infectivity and replication.
(A) Parasite infectivity after H2O2 treatment was determined by pre-treating parasites with 50 µM H2O2 before the invasion assay and counting the internalized trypomastigotes per 100 cells right after cell exposure to parasites. (B) Number of intracellular parasites related to time for cultures infected with WT, pROCK or MutT parasites pre-treated or not with 50 µM H2O2. The asterisk symbol (*) refers to significant differences of MutT parasites compared with controls, and the hash mark (#) indicates a significant difference of parasites in the H2O2-treated group and the untreated parasites (*** P<0.001, ** P<0.01, * P<0.05, ### P<0.001, # P<0.05, two-way ANOVA test with Bonferroni post-test).
Figure 5.
TcCPx and TcMPx expression in pROCK or MutT parasites.
T. cruzi epimastigotes lysates were prepared with exponential phase cultures after 20 min incubation or not with 50 µM H2O2. Protein extracts were quantified by Bradford assay and resolved by SDS-PAGE (A) (30 µg protein/lane). Western blot analysis of TcCPx and TcMPx from non-treated pROCK (1) and MutT (2), or 50 µM H2O2-treated pROCK (3) and MutT (4) parasites. β-tubulin was used as loading control. (B) Coomassie blue staining of the protein extracts. The best representative of three independent experiments is shown. The signal intensity obtained for each studied enzyme from pROCK untreated control was set to 100%, and the enzyme levels from the others parasites were evaluated as a percentage of the control. The results were expressed in graphs for each enzyme: TcCPx (C) and TcMPx. (D) The asterisk symbol (*) refers to significant differences from the pROCK non-treated control (*** P<0.001, one-way ANOVA test with Bonferroni post-test).
Figure 6.
Parasitemia from animals infected with MutT or WT parasites.
Three-week-old female Swiss mice were infected via intraperitoneal route with 5000 TCTs. The parasitemia was evaluated for up to 28 days post-infection by counting bloodstream form of parasites in tail vein blood. Parasitemia levels are expressed as the arithmetic mean of five mice per group (representative of three independent experiments). Significant differences between the two curves are represented in the graph (* P<0.05, unpaired t test).
Table 1.
Heterologous complementation assay with BH600 (mutT-) bacteria.
Figure 7.
T. cruzi MTH overexpressors survival after H2O2 treatment.
2O2 doses, and after 3 days, the parasites were counted. Survival percentage was measured in relation to untreated cells (** P<0.01, * P<0.05, unpaired t test).
Figure 8.
Macrophage infection experiment.
Inflammatory macrophages obtained from peritoneal cavity 3 days after injection of thioglycolate were subjected to infection with WT, pROCK, MutT or TcMTH TCTs (MOI 5). The cells were washed to remove extracellular parasites and either fixed or reincubated with medium for 48 and 72 hours. The slides were stained with Giemsa and counted to determine the infection index (percentage of infected macrophages multiplied by the average number of amastigotes per macrophage) for each parasite population (*** P<0.001, ** P<0.01, one-way ANOVA with Bonferroni post-test).