Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global becoming a new worldwide challenge. For more than a century since its discovery, it has remained neglected with no effective drugs or vaccines. The mechanisms by which Trypanosoma cruzi regulates and uses the extracellular matrix (ECM) to invade cells and cause disease are just beginning to be understood. Here we critically review and discuss the regulation of the ECM interactome by T. cruzi, the use of the ECM by T. cruzi and analyze the molecular ECM/T. cruzi interphase during the early process of infection. It has been shown that invasive trypomastigote forms of T. cruzi use and modulate components of the ECM during the initial process of infection. Infective trypomastigotes up-regulate the expression of laminin γ-1 (LAMC1) and thrombospondin (THBS1) to facilitate the recruitment of trypomastigotes to enhance cellular infection. Silencing the expression of LAMC1 and THBS1 by stable RNAi dramatically reduces trypanosome infection. T. cruzi gp83, a ligand that mediates the attachment of trypanosomes to cells to initiate infection, up-regulates LAMC1 expression to enhance cellular infection. Infective trypomastigotes use Tc85 to interact with laminin, p45 mucin to interact with LAMC1 through galectin-3 (LGALS3), a human lectin, and calreticulin (TcCRT) to interact with TSB1 to enhance cellular infection. Silencing the expression of LGALS3 also reduces cellular infection. Despite the role of the ECM in T. cruzi infection, almost nothing is known about the ECM interactome networks operating in the process of T. cruzi infection and its ligands. Here, we present the first elucidation of the human ECM interactome network regulated by T. cruzi and its gp83 ligand that facilitates cellular infection. The elucidation of the human ECM interactome regulated by T. cruzi and the dissection of the molecular ECM/T. cruzi interphase using systems biology approaches are not only critically important for the understanding of the molecular pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.

Advances in understanding disease pathogenesis correlates to modifications in gene expression within different tissues and organ systems. In depth knowledge about the dysregulation of gene expression profiles is fundamental to fully uncover mechanisms in disease development and changes in host homeostasis. The body of knowledge surrounding mammalian regulatory elements, specifically regulators of chromatin structure, transcriptional and translational activation, has considerably surged within the past decade. A set of key regulators whose function still needs to be fully elucidated are small non-coding RNAs (sncRNAs). Due to their broad range of unfolding functions in the regulation of gene expression during transcription and translation, sncRNAs are becoming vital to many cellular processes. Within the past decade, a novel class of sncRNAs called PIWI-interacting RNAs (piRNAs) have been implicated in various diseases, and understanding their complete function is of vital importance. Historically, piRNAs have been shown to be indispensable in germline integrity and stem cell development. Accumulating research evidence continue to reveal the many arms of piRNA function. Although piRNA function and biogenesis has been extensively studied in Drosophila, it is thought that they play similar roles in vertebrate species, including humans. Compounding evidence suggests that piRNAs encompass a wider functional range than small interfering RNAs (siRNAs) and microRNAs (miRNAs), which have been studied more in terms of cellular homeostasis and disease. This review aims to summarize contemporary knowledge regarding biogenesis, and homeostatic function of piRNAs and their emerging roles in the development of pathologies related to cardiomyopathies, cancer, and infectious diseases.

Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1, which is up-regulated by the parasite.

The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy. Examining the very early molecular events of T. cruzi cellular infection may provide disease biomarkers which will aid clinicians in patient assessment and identification of patient subpopulation at risk of developing Chagasic cardiomyopathy. About 30% of Trypanosoma cruzi infected individuals will develop Chagas heart disease cardiomyopathy which is similar to other cardiomyopathies. T. cruzi induced heart disease can lead to cardiac arrhythmias, heart failure and death. These cardiac disorders can be caused by several host-parasite interaction factors that lead to myocardial inflammation which remains poorly understood from the initial phase of infection. In this study, we challenged primary human cardiomyocyte (PHCM) with T. cruzi trypomastigotes and evaluated changes in gene and protein expression profile occurring early during the process of cellular infection. Paying attention to genes that have the potential of tilting the PHCM towards a fibrogenic phenotype, we observed that the parasite upregulates the expression of several transcription factors and cytokines/chemokines that have been suggested to be implicated in the development of fibrogenic responses. The validated microarray data were confirmed at the protein level where we demonstrated an increase in the protein expression of EGR1, SNAI1, JunB and IL6. Pathway analyses of our results show that early during T. cruzi infection, the parasite induces multiple factors that are profibrotic in PHCM. The operating fibrotic interactome presented in our work advances our understanding of the initiation of cardiovascular pathology in Chagas disease patients.

The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, causes severe morbidity and mortality in afflicted individuals. About 30% of T. cruzi-infected individuals present with cardiac, gastrointestinal tract, and/or neurological disorders. Megacolon, one of the major pathologies of Chagas disease, is accompanied by gastrointestinal motility disorders. The molecular mechanism of T. cruzi-mediated megacolon in Chagas disease is currently unknown. To decipher the molecular mechanism of T. cruzi-induced alteration in the colon during the early infection phase, we exposed primary human colonic epithelial cells (HCoEpiC) to invasive T. cruzi trypomastigotes at multiple time points to determine changes in the phosphoprotein networks in the cells following infection using proteome profiler Human phospho-kinase arrays. We found significant changes in the phosphorylation pattern that can mediate cellular deregulations in colonic epithelial cells after infection. We detected a significant increase in the levels of phosphorylated heat shock protein (p-HSP) 27 and transcription factors that regulate various cellular functions, including c-Jun and CREB. Our study confirmed significant upregulation of phospho (p-) Akt S473, p-JNK, which may directly or indirectly modulate CREB and c-Jun phosphorylation, respectively. We also observed increased levels of phosphorylated CREB and c-Jun in the nucleus. Furthermore, we found that p-c-Jun and p-CREB co-localized in the nucleus at 180 minutes post infection, with a maximum Pearson correlation coefficient of 0.76±0.02. Increased p-c-Jun and p-CREB have been linked to inflammatory and profibrotic responses. T. cruzi infection of HCoEpiC induces an increased expression of thrombospondin-1 (TSP-1), which is fibrogenic at elevated levels. We also found that T. cruzi infection modulates the expression of NF-kB and JAK2-STAT1 signaling molecules which can increase pro-inflammatory flux. Bioinformatics analysis of the phosphoprotein networks derived using the phospho-protein data serves as a blueprint for T. cruzi-mediated cellular transformation of primary human colonic cells during the early phase of T. cruzi infection. Trypanosoma cruzi is a hemoflagellate that is now considered a global health threat in all industrialized regions of the world. Some chagasic patients present with digestive, neurological, and/or cardiac disorders. The mechanisms of T. cruzi-induced pathology remain to be elucidated. In this study, we challenged primary human colonic epithelial cells with T. cruzi and evaluated changes in the phosphorylated kinases and phosphoprotein levels that may induce cellular and molecular alterations leading to cellular transformations during the early phase of infection. The parasite induced significant increases in levels of phosphorylated kinases and phosphoproteins that govern multiple cellular pathways associated with immunological, stress, neuronal, and intercellular interactions as well as fibrogenic responses. The parasite also enhanced the levels of p-AKT, p-HSP27, p-JNK, and downstream transcription factors like p-c-Jun and p-CREB during the early infection phase. Additionally, we observed that the phosphorylated transcription factors are translocated to and colocalized in the nucleus in a time-dependent manner. These transcription factors regulate the expression of genes, including genes encoding extracellular matrix proteins, which play a role in the onset of colon pathology observed in some chagasic patients. Our study provides novel insights into the interactome that occurs during acute phase of T. cruzi infection of primary human colon cells.

Trypanosoma cruzi dysregulates the gene expression profile of primary human cardiomyocytes (PHCM) during the early phase of infection through a mechanism which remains to be elucidated. The role that small non-coding RNAs (sncRNA) including PIWI-interacting RNA (piRNA) play in regulating gene expression during the early phase of infection is unknown. To understand how T. cruzi dysregulate gene expression in the heart, we challenged PHCM with T. cruzi trypomastigotes and analyzed sncRNA, especially piRNA, by RNA-sequencing. The parasite induced significant differential expression of host piRNAs, which can target and regulate the genes which are important during the early infection phase. An average of 21,595,866 (88.40%) of clean reads mapped to the human reference genome. The parasite induced 217 unique piRNAs that were significantly differentially expressed (q ≥ 0.8). Of these differentially expressed piRNAs, 6 were known and 211 were novel piRNAs. In silico analysis showed that some of the dysregulated known and novel piRNAs could target and potentially regulate the expression of genes including NFATC2, FOS and TGF-β1, reported to play important roles during T. cruzi infection. Further evaluation of the specific functions of the piRNAs in the regulation of gene expression during the early phase of infection will enhance our understanding of the molecular mechanism of T. cruzi pathogenesis. Our novel findings constitute the first report that T. cruzi can induce differential expression of piRNAs in PHCM, advancing our knowledge about the involvement of piRNAs in an infectious disease model, which can be exploited for biomarker and therapeutic development.