Novel Genetic Approaches to Elucidate Critical Signaling Pathways in Leishmania donovani

About This Grant

Project Summary Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as one million people per year. At least 20,000 are killed each year by the deadly visceral form of the disease, which is caused by Leishmania donovani. The signal transduction pathways used by these parasites to sense and respond to their host environments are poorly understood. Identification of signaling pathways essential for parasite survival may lead to new targets for antileishmanial drug development. Genetic screens targeting signaling pathways offer a powerful means of discovering genes encoding important signaling proteins. A recent CRISPR/Cas9-mediated knockout screen targeting 204 protein kinases found 44 essential kinases in Leishmania mexicana. Using barcode sequencing (Bar-seq) technology, this study also identified more than 50 kinases that were involved in progression through at least one lifecycle stage, both in cell culture, and in sand fly and mouse models of infection. However, these Loss-of-Function (LoF) knockout screens are tedious and expensive to implement, making it challenging to apply these methods to the hundreds of additional signaling proteins with unknown roles in Leishmania survival. We propose to take a Gain-of-Function (GoF) genetic screening approach to understand Leishmania signal transduction. Like LoF gene knockouts, GoF approaches that express proteins at higher levels than normal (i.e., overexpression) can cause deleterious effects on cell fitness that reveal important pathways and protein functions. We have generated a library of 440 known or predicted L. donovani signaling proteins, cloned into novel barcoded inducible expression vectors developed in our lab. This signaling library will be transfected into L. donovani and then used to identify signaling proteins that cause fitness defects upon induction of overexpression via Bar-seq. We will conduct GoF overexpression screens to discover signaling proteins and pathways critical for progression through in vitro (Aim 1) and in vivo (Aim 2) lifecycle stages. Many of the signaling proteins encoded in our library have not been studied before. Therefore, we anticipate we will uncover previously unknown roles for many signaling proteins in Leishmania lifecycle progression. Our GoF signaling library overexpression platform will be simpler to implement and easier to share with other researchers than LoF knockout libraries. The signaling library can be applied to identify signaling proteins critical for surviving any growth condition, and can be readily moved into other Leishmania species. We predict that the signaling library and the information gleaned from the proposed overexpression screens will serve as valuable resources for the Leishmania community.