Elucidating molecular determinants of Trypanosoma cruzi persisters. Chagas disease is endemic in 21 countries with around 70 million people at risk of infection, and an estimated mortality rate of over 10,000 people per year. This neglected tropical disease is caused by the intracellular protozoan parasite Trypanosoma cruzi. Current drugs (benznidazole and nifurtimox) have serious adverse side-effects and long-treatment duration resulting in many patients not completing appropriate treatment. There is thus an urgent need for new, shorter-course, efficacious and safe drugs, but unfortunately the current drug discovery pipeline is very sparse, reflecting the many challenges faced by the Chagas disease drug discovery community. Chronic Chagas disease is associated with a very low parasite burden as the immune system effectively clears most parasites during the acute phase of the disease. It is therefore thought that the most effective new treatments will be the ones that kill every single parasite in the patient. This is made even more challenging by the presence of a small sub-population of parasites that is less susceptible to drugs. We, and others, have recently identified these "persister" parasites and have demonstrated in vitro that they can withstand long drug exposures that otherwise quickly kill the majority of intracellular parasites. The current understanding is that these persister parasites are quiescent (i.e. do not divide), are able to exit from the persister state, and can spread the disease. A key issue for Chagas disease drug discovery is the complete lack of understanding of the biology of these persister parasites. We do not know how they are triggered, what genes they express, what cellular pathways are active and what proteins are suitable drug targets for this population.  To start addressing these questions, the proposed research will study the persister parasites at the molecular level. Specifically, this work will use state-of-the-art single-cell methodologies to determine which genes are expressed in the persisters and compare this with the total parasite population. Understanding this will shed light on the fundamental biology of the persister parasites and will help develop better assays and choose suitable drug targets for new drug discovery efforts. Knowing which genes are expressed and which are not will also allow us to define a set of marker genes to specifically detect the persister population, something that is currently not possible.  Once we have identified markers for the persister population we will carry out experiments to show that the persisters we see in the laboratory are relevant to the ones seen in Chagas disease, through in vivo studies using animal models of the disease.  The final main objective of this proposal is to identify compounds that can either induce the persister state or force persisters into a replicative state. Understanding how compounds that induce persisters act may reveal the mechanisms underlying persister formation. Compounds that release parasites from the persister state can be used to validate a new treatment concept where a compound that induces replication of persisters is combined with existing drugs that kill replicative parasites. By characterising the T. cruzi persisters at the molecular level this proposal will enable better, more targeted, drug discovery approaches for Chagas disease. It will allow development of fit-for-purpose assays for drug discovery, assist with the identification of suitable drug discovery targets and allow tracking of the effect of drugs against persisters in vivo. This work will also enable future detailed studies of persister biology, such as persister dynamics and the study of proteins that regulate the persister state. The Global Challenges Research Fund (GCRF) supports cutting-edge research to address challenges faced by developing countries. The fund addresses the UN sustainable development goals. It aims to maximise the impact of research and innovation to improve lives and opportunity in the developing world.