Research Projects

Toxoplasma gondii is one of the leading parasites in terms of absolute number of infections, with a prevalence of 20-30 % worldwide and as high as 80-90% in a few South and Central American countries. In India, estimates of prevalence vary between 20-40%, with an estimated 56,737–176,882 children per year born at risk of congenital Toxoplasmosis, with symptoms including mental deficiencies and fatality, according to a 2014 study. The parasite can infect a wide range of hosts, including humans, and within the host, a wide range of tissues and cell types. Though clinical manifestations are mostly restricted to immunocompromised individuals, ocular toxoplasmosis is one of the major causes of posterior uveitis in immunocompetent individuals, leading to vision loss. Despite its importance, the molecular mechanisms underlying the pathogenesis of ocular toxoplasmosis are not well understood. Recent studies from animal models and other cellular models have shown dysregulation of host microRNAs (miRNAs) upon Toxoplasma infection, highlighting a potential role in regulating host-pathogen interactions, but the functional relevance of these miRNAs in toxoplasmosis in general remains unknown. Recent research challenges the notion that the presence or absence of specific microRNAs (miRNAs) directly implies functional significance. Instead, they can be considered functional only when loaded onto the RISC complex and are bound by Argonaute (AGO). The proposed project aims to investigate the role of host miRNAs in ocular toxoplasmosis, with a focus on identifying AGO-bound dysregulated miRNAs (functional miRNAs) upon T. gondii infection. We propose to use RPE-1 (Retinal Pigment epithelial) cells as a good model for ocular toxoplasmosis. We will elucidate the mechanism of regulation of host-parasite interaction by dysregulated miRNAs and, in the process, will identify host miRNAs as potential targets for host-directed therapy. Our study differs from previous studies, which have been mostly descriptive, and we propose to elucidate the functional implications of dysregulated miRNAs. Using AGO IP-sRNA-seq and dual luciferase reporter assays, we will identify and validate AGO-bound dysregulated miRNAs and the impact of infection on their activity in RPE-1 cells. We will experimentally identify mRNA targets directly misregulated by host miRNAs upon parasite infection. We will further design miRNA mimics and inhibitors to modulate host miRNA levels and analyze their impact on parasite invasion, proliferation, and egress. Our study will also elucidate mechanisms of host miRNA dysregulation and the impact of candidate miRNAs’ modulation on host transcriptome and translatome, and thus identify the pathways impacted by the dysregulation of candidate miRNAs. Identification of AGO-bound miRNAs dysregulated upon T. gondii infection will provide novel insights into the role of host miRNAs in ocular toxoplasmosis, identify potential therapeutic targets, and contribute to the development of novel treatment strategies. The proposed project is innovative, timely, and has the potential to make a significant impact on our understanding of ocular toxoplasmosis, with implications for public health and the development of new therapeutic approaches. The project will be carried out by the PI at the Indian Institute of Science, Bangalore, and the co-PI from BITS-Pilani, Hyderabad, leveraging the highly complementary strengths of both institutions in molecular biology and genomics. Our teams have expertise in Toxoplasma biology, miRNA regulation, and sequencing-based molecular approaches, along with high-resolution imaging and CRISPR-based gene editing approaches. We anticipate that our collaborative research will identify functionally dysregulated miRNAs that play a crucial role in determining the outcome of T. gondii infection in ocular tissues, and thus, we will be able to identify potential miRNA modulators to subvert infection.