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

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

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

Screenings of MASP B-cell epitopes using SPOT peptide arrays.

<p>Blotting representative images using sera pools (n = 10) after 2, 10, and 12 passages in mice of CL-Bnener <i>T. cruzi</i>; predicted peptides were covalently synthesized in pre-activated cellulose membranes according to the SPOT synthesis technique (Frank <i>et al.</i>, 1992); circled spots: peptides with the highest Rd values.</p

Dendrogram of hierarchical analysis of MASP expression libraries.

<p>The uncertainty of the hierarchical cluster analysis of all expression libraries was calculated using Pvclust package <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001779#pntd.0001779-Timenetsky1" target="_blank">[10]</a>. AU values (in red): approximately unbiased p-value.</p

Sequencing results of the MASP libraries of culture and bloodstream trypomastigotes.

*<p>The majority of non-MASP hits are derived from short hypothetical proteins that seem unreal since were predicted within MASP 3′UTR.</p>**<p>Redundant matches taken into account.</p>***<p>Excluding redundant matches among the different libraries.</p><p>Valid sequences: quality tested sequences used in the analysis; MASP hits: valid sequences identified as MASP genes; 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 trypomastigotes 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

Expression analysis by qRT-PCR of selected MASP genes.

<p>Relative quantity (Rq) calculations were based on specific standard curves for each MASP gene. Rq values of each cDNA sample (MASP Rq) were normalized with the average of two constitutive genes MSH2 (MSH2 Rq) and RAD51 (RAD51 Rq). <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001779#pntd-0001779-g003" target="_blank"><b>Figure 3A</b>:</a> Expression analysis by qRT-PCR of six MASP genes in 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-g003" target="_blank"><b>Figure 3B</b>:</a> Expression analysis by qRT-PCR of six MASP genes in culture trypomastigotes derived from distinct cell types. 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

MASP peptide ELISA.

<p>Seven MASP peptides and SAPA were submitted to soluble synthesis (Peptide 2.0) and ELISA experiments. Flexible ELISA plates (Falcon) were sensitized with 2 µg of soluble peptides, incubated with uninfected and infected mouse serum pools from three passages, IgG or IgM secondary antibodies, and revealed with OPD solution. Absorbance values were normalized with values resulting from ELISA of each passage against total trypomastigote extract.</p

Numbers of ORFs identified in <i>A. deanei</i> and <i>S. culicis</i> and their symbionts, according to the mechanisms of DNA replication and repair, signal transduction, transcription and translation.

<p>Numbers of ORFs identified in <i>A. deanei</i> and <i>S. culicis</i> and their symbionts, according to the mechanisms of DNA replication and repair, signal transduction, transcription and translation.</p