Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in <i>Trypanosoma cruzi</i> Cell Viability

<div><p>The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. <i>Trypanosoma cruzi</i> needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in <i>T. cruzi</i> (TcMTH) and generated <i>T. cruzi</i> parasites heterologously expressing <i>Escherichia coli</i> MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.</p></div

H₂O₂ treatment does not affect MutT parasite infectivity and replication.

<p>(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).</p

Parasitemia from animals infected with MutT or WT parasites.

<p>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 <i>t</i> test).</p

MutT expression enhances <i>in vitro</i> intracellular growth.

<p>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.</p

MutT heterologous expression did not alter <i>T. cruzi</i> growth but enhanced survival against H₂O₂.

<p>(A) Amplification of a 112-bp fragment of MutT from transfected parasite total RNA (<b>MutT +</b>) and positive controls (<i>E. coli</i> total DNA). <b>pROCK +</b>: total RNA from parasites transfected with the pROCK empty vector. <b>MutT -</b> and <b>pROCK -</b>: RT-PCR negative control without reverse transcriptase; <b>- control:</b> 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 <i>t</i> test).</p

<i>T. cruzi</i> MTH overexpressors survival after H₂O₂ treatment.

<p>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 <i>t</i> test).</p

TcCPx and TcMPx expression in pROCK or MutT parasites.

<p><i>T. cruzi</i> 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).</p

Macrophage infection experiment.

<p>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).</p