Discovery of circulating miRNAs as biomarkers of chronic Chagas heart disease via a small RNA-Seq approach.

Chagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.

The Complete Mitochondrial DNA of Trypanosoma cruzi: Maxicircles and Minicircles.

The mitochondrial DNA of Trypanosomatids, known as the kinetoplast DNA or kDNA or mtDNA, consists of a few maxicircles and thousands of minicircles concatenated together into a huge complex network. These structures present species-specific sizes, from 20 to 40 Kb in maxicircles and from 0.5 to 10 Kb in minicircles. Maxicircles are equivalent to other eukaryotic mitochondrial DNAs, while minicircles contain coding guide RNAs involved in U-insertion/deletion editing processes exclusive of Trypanosomatids that produce the maturation of the maxicircle-encoded transcripts. The knowledge about this mitochondrial genome is especially relevant since the expression of nuclear and mitochondrial genes involved in oxidative phosphorylation must be coordinated. In Trypanosoma cruzi (T. cruzi), the mtDNA has a dual relevance; the production of energy, and its use as a phylogenetic marker due to its high conservation among strains. Therefore, this study aimed to assemble, annotate, and analyze the complete repertoire of maxicircle and minicircle sequences of different T. cruzi strains by using DNA sequencing. We assembled and annotated the complete maxicircle sequence of the Y and Bug2148 strains. For Bug2148, our results confirm that the maxicircle sequence is the longest assembled to date, and is composed of 21 genes, most of them conserved among Trypanosomatid species. In agreement with previous results, T. cruzi minicircles show a conserved structure around 1.4 Kb, with four highly conserved regions and other four hypervariable regions interspersed between them. However, our results suggest that the parasite minicircles display several sizes and numbers of conserved and hypervariable regions, contrary to those previous studies. Besides, this heterogeneity is also reflected in the three conserved sequence blocks of the conserved regions that play a key role in the minicircle replication. Our results using sequencing technologies of second and third-generation indicate that the different consensus sequences of the maxicircles and minicircles seem to be more complex than previously described indicating at least four different groups in T. cruzi minicircles.

Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy.

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

Cyclooxygenase 2 Effector Genes as Potential Inflammation-Related Biomarkers for Colorectal Cancer Circulating Tumor Cells Detection by Liquid Biopsy.

Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.

Trypanosoma Cruzi Genome: Organization, Multi-Gene Families, Transcription, and Biological Implications.

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people. Although its first genome dates from 2005, its complexity hindered a complete assembly and annotation. However, the new sequencing methods have improved genome annotation of some strains elucidating the broad genetic diversity and complexity of this parasite. Here, we reviewed the genomic structure and regulation, the genetic diversity, and the analysis of the principal multi-gene families of the recent genomes for several strains. The telomeric and sub-telomeric regions are sites with high recombination events, the genome displays two different compartments, the core and the disruptive, and the genome plasticity seems to play a key role in the survival and the infection process. Trypanosoma cruzi (T. cruzi) genome is composed mainly of multi-gene families as the trans-sialidases, mucins, and mucin-associated surface proteins. Trans-sialidases are the most abundant genes in the genome and show an important role in the effectiveness of the infection and the parasite survival. Mucins and MASPs are also important glycosylated proteins of the surface of the parasite that play a major biological role in both insect and mammal-dwelling stages. Altogether, these studies confirm the complexity of T. cruzi genome revealing relevant concepts to better understand Chagas disease.

Interaction of Signaling Lymphocytic Activation Molecule Family 1 (SLAMF1) receptor with Trypanosoma cruzi is strain-dependent and affects NADPH oxidase expression and activity.

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease.

Analysis of mRNA processing at whole transcriptome level, transcriptomic profile and genome sequence refinement of Trypanosoma cruzi.

The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.

Comparative proteomic analysis of trypomastigotes from Trypanosoma cruzi strains with different pathogenicity.

Chagas disease, caused by the parasite Trypanosoma cruzi, is one of the most neglected diseases in Latin America, being currently a global health problem. Its immunopathogenesis is still quite unknown. Moreover, there are important differences in pathogenicity between some different T. cruzi strains. For example, in mice, Y strain produces a high acute lethality while VFRA remains in the host mostly in a chronic manner. Comparative proteomic studies between T. cruzi strains represent a complement for transcriptomics and may allow the detection of relevant factors or distinctive functions. Here for the first time, we compared the proteome of trypomastigotes from 2 strains, Y and VFRA, analyzed by mass spectrometry. Gene ontology analysis were used to display similarities or differences in cellular components, biological processes and molecular functions. Also, we performed metabolic pathways enrichment analysis to detect the most relevant pathways in each strain. Although in general they have similar profiles in the different ontology groups, there were some particular interesting differences. Moreover, there were around 10% of different proteins between Y and VFRA strains, that were shared by other T. cruzi strains or protozoan species. They displayed many common enriched metabolic pathways but some others were uniquely enriched in one strain. Thus, we detected enriched antioxidant defenses in VFRA that could correlate with its ability to induce a chronic infection in mice controlling ROS production, while the Y strain revealed a great enrichment of pathways related with nucleotides and protein production, that could fit with its high parasite replication and lethality. In summary, Y and VFRA strains displayed comparable proteomes with some particular distinctions that could contribute to understand their different biological behaviors.

Genomic assemblies of newly sequenced Trypanosoma cruzi strains reveal new genomic expansion and greater complexity.

Chagas disease is a complex illness caused by the protozoan Trypanosoma cruzi displaying highly diverse clinical outcomes. In this sense, the genome sequence elucidation and comparison between strains may lead to disease understanding. Here, two new T. cruzi strains, have been sequenced, Y using Illumina and Bug2148 using PacBio, assembled, analyzed and compared with the T. cruzi annotated genomes available to date. The assembly stats from the new sequences show effective improvement of T. cruzi genome over the actual ones. Such as, the largest contig assembled (1.3 Mb in Bug2148) in de novo attempts and the highest mean assembly coverage (71X for Y). Our analysis reveals a new genomic expansion and greater complexity for those multi-copy gene families related to infection process and disease development, such as Trans-sialidases, Mucins and Mucin Associated Surface Proteins, among others. On one side, we demonstrate that multi-copy gene families are located near telomeric regions of the "chromosome-like" 1.3 Mb contig assembled of Bug2148, where they likely suffer high evolutive pressure. On the other hand, we identified several strain-specific single copy genes that might help to understand the differences in infectivity and physiology among strains. In summary, our results indicate that T. cruzi has a complex genomic architecture that may have promoted its evolution.

Genome Sequence of Trypanosoma cruzi Strain Bug2148.

Trypanosoma cruzi belongs to the group of mitochondrion-containing eukaryotes and has a highly plastic genome, unusual gene organization, and complex mechanisms for gene expression (polycistronic transcription). We report here the genome sequence of strain Bug2148, the first genomic sequence belonging to cluster TcV, which has been related to vertical transmission.