Determining function and mechanism of lncRNA Chast in Trypanosoma cruzi infection and dissemination

Chagas disease (CD), caused by parasite Trypanosoma cruzi (Tc), is endemic in Latin America. CD has become a global health problem because of the human migration from endemic to nonendemic areas. The delayed and sup-par immune activation of macrophages (Mf) allows parasite dissemination and persistence that ultimately results in cardiomyopathy and heart failure in infected patients. Understanding of how Tc deregulates Mf function will aid in the development of novel therapeutics with broader relevance to control of intracellular pathogens. Long non-coding RNAs (lncRNAs) have emerged as regulators of multiple biological functions via their effects on epigenetic, transcriptional, and translational programs. Yet, the role of lncRNAs in the regulation of host response to pathogens is not well studied. Our preliminary studies using global and targeted molecular approaches and publicly available databases identified that LncRNA Chast is downregulated in Tc infected Mf. Genomic locus of lncRNA Chast forms a chromatin loop with a nearby protein-coding gene Plekhm1, and RNA binding protein EZH2 has a binding motif near the promoter of the Plekhm1 sequence. Experiments using primary and cultured murine Mf showed that Plekhm1 gene expression was downregulated, EZH2 was enriched at the Plekhm1 locus, and Chast-EZH2 binding was decreased following Tc infection. Further, RNAi mediated Chast deficiency enhanced parasite survival in Mf. Building on these preliminary results, it is hypothesized that T. cruzi deregulates lncRNA Chast to repress EZH2-mediated Plekhm1 gene expression which ensures parasite survival in and escape from Mf. Cutting-edge molecular biology and immunology tools along with in vitro and in vivo models of Tc infection will be utilized to test this hypothesis in two aims. Studies in Aim 1 will examine whether Tc deregulates Chast-Plekhm1 to hijack host defense mechanisms for its intracellular survival and dissemination. Studies in Aim 2 will uncover the molecular mechanisms by which Chast-EZH2 regulate the Plekhm1 gene expression and influence Mf functional activation vs. parasite dissemination. Rigor of the findings will be tested by including cardiomyocytes and heart tissues under similar experimental conditions. The proposed work is highly innovative and will reveal fundamental molecular mechanisms driving T. cruzi to evade immune responses and establish persistent infection in the host. The experiments outlined in the proposal will establish a new role of lncRNA Chast in parasite survival and will lead to development of potential novel therapeutics against T. cruzi infection.