FAPESP: Deciphering Leishmania non coding RNA function at the host-parasite interface

Over 1 billion people are at risk of infection with the vector-borne parasitic protozoan, Leishmania. In South and Central America, Leishmania species in the sub-genus Viannia, such as L. braziliensis, cause cutaneous and mucocutaneous leishmaniasis. Unlike many other Neglected Tropical Diseases that have seen great recent success in reduction measures, including malaria and Sleeping Sickness, leishmaniasis is on the rise globally. A major goal of Leishmania discovery research is to understand how the parasite transitions through its complex lifecycle, adapts to its environment, and disables our immune defences. Understanding these mechanisms on a molecular level will enable us to develop novel therapies and vaccines. To date, most Leishmania genetic studies have focussed on the role of protein-coding genes in lifecycle progression. Our research has shown that a number of non-protein-coding RNAs (ncRNAs), known to play central roles in cancer metastatic processes and other physiological and pathological conditions in humans, also play crucial roles in the survival and infectivity of L. braziliensis. We hypothesise that the Leishmania genome encodes many more uncharacterised ncRNAs that act as key regulators of parasite virulence, dissemination and disease potential. To test this hypothesis in aim 1, we will attempt the deletion of 650 candidate ncRNAs identified in L. braziliensis, which are preferentially, or highly, expressed in amastigotes, the proliferative disease-causing stage of the parasite found within the human host. Each null mutant will carry a unique genetic barcode, and pools of these mutants will undergo lifecycle phenotyping in in vitro culture, in macrophages and in mice. By sequencing the barcodes in the population (bar-seq), we will determine which ncRNAs are required at each stage of the life cycle. Importantly, we will do all the in vivo experiments with our recently developed L. braziliensis mouse infection model, which gives rump lesions similar to human cutaneous lesions. In aim 2 we will then select ncRNAs where knockout mutants display loss of fitness compared to wildtype parasites in macrophage or mice infections. We will use 10 - 20 of these key ncRNAs for detailed mechanistic investigation. We will confirm that loss of fitness is specifically due to deletion of the ncRNA and not bystander effects by reintroducing the ncRNA expression using add-back and over-expressing lines. These will be tested, quantified and verified by RNA sequencing and RT-qPCR methods. We will identify the protein partners of these ncRNAs, which will facilitate our understanding of the pathways and networks of proteins and RNAs involved in key aspects of parasite development and mammalian infection. For this analysis, we will employ two different in vitro systems and will develop and compare in vivo pulldown assays. In aim 3 we will examine whether the levels of regulatory ncRNAs and associating proteins impact parasite:host interaction and metastasis specific to L. braziliensis infection. Among the ncRNAs whose knockouts result in impaired infection, 5-10 will be analysed in vivo in mice using bioluminescent L. braziliensis and longitudinal IVIS in vivo imaging. The overall objective is to leverage state-of-the-art approaches to accelerate ncRNAs as molecular targets for novel therapies against leishmaniasis, as demonstrated for other diseases.