Analysis of DNA lesions in <i>T. cruzi</i> genome after treatment with H₂O₂.

<p><b>A</b>) Nuclear and mitochondrial dose-responses after exposure to increasing H₂O₂ doses. Cells were treated for 15 min. <b>B</b>) Kinetics of damage and repair of the nuclear and mitochondrial fragments after exposure to 200 µM H₂O₂. Cells were treated for 15 min and allowed to recover for the times indicated. Data are expressed as the mean of two biological experiments. Error bars represent standard error of the mean. Statistical analysis used was unpaired <i>t</i> test. Mitochondrial DNA (<b>○</b>); Nuclear DNA (Δ). ***- P value<0,001; ** - P value<0,01.</p

8-oxoguanine levels in nuclear and mitochondrial DNA of WT and <i>TcOGG1</i>-overexpressor <i>T. cruzi</i>, with or without H₂O₂ treatment.

<p>Cells were incubated with FITC-avidin, which binds to 8-oxoguanine. Slides containing stained parasites were visualized under a fluorescence microscope and fluorescence intensity was measured with ImageJ program. Graphics were plotted using the average of different experiments and statistical analyses used Mann Whitney test. NT: non-treated; kDNA: kinetoplast (mitochondrion) DNA. *** - P value<0,001.</p

Mitochondrial function after H₂O₂ treatment.

<p>Analysis of the mitochondrial activity by measuring the basal oxygen consumption rate (OCR). Cells were treated with 0 µM and 200 µM H₂O₂ for 20 min and allowed to recover for 24 hours. Measures were done on the Seahorse Extracellular Flux Analyzer XF24. Results shown are representative of the mean of two independent experiments, performed in replicates of 3–4. The basal level of OXPHOS was calculated by the difference between the mean of rates 1 to 4 and the mean of rates 14 to 16. ** - P value<0,01.</p

Analysis of DNA lesions in <i>TcOGG1</i>-overexpressor <i>T. cruzi</i>.

<p>Ratio between nuclear and mitochondrial DNA lesions from <i>TcOGG1</i>-overexpressing cells in comparison to control cells, after treatment with 200 µM H₂O₂. Both cell populations were treated for 20 min and allowed to recover for up to 24 hours. Data are expressed as the mean of two biological experiments. Error bars represent standard error of the mean. Statistical analysis used was unpaired <i>t</i> test. ** - P value<0,01; * - P value<0,1.</p

Subcellular localization of TcOgg1 in <i>T. cruzi</i>.

<p>CL Brener strain was transfected with pTREX_<i>TcOGG1</i>, resulting in the expression of TcOgg1 fused with GFP (Ogg1-GFP), which enabled the visualization of the protein under confocal microscope. DNA was stained with propidium iodide. Images obtained were analyzed with Zeiss LSM Image Browser software. N: nucleus; K: kinetoplast (mitochondrion); DIC: differential interference contrast. 5 uM scale bars is present in C.</p

MDS distribution of sequences from the MASP expression libraries.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001779#pntd-0001779-g002" target="_blank"><b>Figure 2A</b>:</a> MDS distribution of sequences from the MASP expression libraries of bloodstream forms from sequential passages and of culture trypomastigotes after the <i>in vivo</i> step. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001779#pntd-0001779-g002" target="_blank"><b>Figure 2B</b>:</a> MDS distribution of sequences from the MASP expression libraries of culture trypomastigotes derived from distinct cell types. Black bordered genes: MASP complete genes analyzed by qRT-PCR. Tryp.0.llcmk2: culture trypomastigotes prior to infection of the two cell types; Tryp.14p.llcmk2: culture trypomastigotes derived from LLC-MK2 cells after 14 passages; Tryp.14p.l6: culture trypomastigotes derived from L6 cells after 14 passages; BloodTryp.2p: bloodstream forms after 2 passages; BloodTryp.10p: bloodstream forms after 10 passages; Tryp.4p.llcmk2: culture trypomastigotes after <i>in vivo</i> step derived from LLC-MK2 cells after 4 passages; Tryp.4p.l6: culture trypomastigotes after <i>in vivo</i> step derived from L6 cells after 4 passages.</p

The MASP Family of <em>Trypanosoma cruzi</em>: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection

<div><h3>Background</h3><p><em>Trypanosoma cruzi</em> is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the <em>T. cruzi</em> genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of <em>T. cruzi</em> to infect several host cell types and/or participate in host immune evasion mechanisms.</p> <h3>Methods</h3><p>By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.</p> <h3>Findings and Conclusions</h3><p>We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute <em>T. cruzi</em> infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.</p> </div

Libraries codes of the trypomastigote samples.

<p>Libraries codes of the trypomastigote samples.</p

Experimental models.

<p><b>A</b>: Mouse groups were sequentially infected with CL Brener clone. Blood collections were performed after 14–15 days. Bloodstream parasite forms were collected after two and ten passages. Part of the trypomastigotes isolated from mouse blood was used in LLC-MK2 and L6 cell infection (bloodstream parasite forms after two passages in mice). Trypomastigotes were collected after four sequential passages in culture. <b>B</b>: The same group of culture trypomastigotes was used in the infection of LLC-MK2 (epithelial) and L6 (myoblast) cell types. Trypomastigotes were collected after 14 sequential passages in culture.</p

Alignments with the predicted <i>TcOGG1</i> products.

<p>Amino acid sequence comparison of the predicted product of the <i>OGG1</i> gene from <i>Trypanosoma cruzi</i> (<i>TcOGG1</i>_B; Tc), <i>Trypanosoma brucei</i> (Tb), <i>Leishmania major</i> (Lm), <i>Arabidopsis thaliana</i> (At), <i>Mus musculus</i> (Mm), <i>Homo sapiens</i> (Hs) and <i>Saccharomyces cerevisiae</i> (Sc). Residues shaded in black indicate identical amino acids. Residues shaded in gray are functionally similar. Residues enclosed by the box belong to the HhH-G/PD motif. Asterisks correspond to the Ogg1 catalytic lysine residue and its auxiliary aspartic acid.</p