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.

Quiescent, less-drug susceptible T. cruzi parasites pose a key challenge for Chagas disease drug discovery. There currently is no understanding of these persister forms at the molecular level. Here, I propose to use single-cell RNA-sequencing (scRNA-seq) to, for the first time, reveal population heterogeneity in the intracellular amastigote population and to characterise the persister parasites through their gene expression profile. For this I will employ drop-seq technology on methanol-fixed parasites, including the complete intracellular parasite population isolated from mammalian host cells and populations enriched for persisters through fluorescence-activated cell sorting. Expression level differences will be validated both at the mRNA and protein level. The transcriptome data will dramatically increase our understanding of persisters and allow identification of persister markers. To demonstrate physiological relevance of the in vitro persisters, I will use the identified marker genes to probe for persisters in tissues from infected mice. Finally, I propose a large high-content screen with 130,000 compounds to identify compounds that can promote either entry or exit from the persister state. The high-throughput screen will be based on previously demonstrated dye-retention as a persister marker, whereas validation will be carried out with the persister markers found through scRNA-seq. Compounds that stimulate persister replication will be tested in combination with compounds known to kill replicative parasites in intracellular viability assays to test this type of combination as an new treatment approach. Overall this work will provide key information regarding T. cruzi population heterogeneity and the nature of T. cruzi persisters and will enable further research into the fundamental biology of persisters as well as support drug discovery efforts allowing creation of better assays, specific detection of persisters and rational identification of drug targets.

This project will elucidate the molecular determinants of T. cruzi persister parasites and will benefit:

a) Chagas disease patients: The ultimate aim of this work is to enable the discovery and development of new drugs for Chagas disease. Current drug discovery efforts are hampered by the lack of understanding of the persister forms. Successful completion of this project will provide key information to improve drug discovery for Chagas disease and bring new medicines quicker to patients.

b) Clinicians: Clinicians are desperate for new treatments for Chagas disease. Benznidazole and nifurtimox have been used for many decades and have major drawbacks. The only other treatments tested in the clinic (posaconazole and fos-ravuconazole) failed badly. As explained above, the outcomes of this work will help accelerate the development of new drugs.

c) Drug discovery community: Drug discovery for Chagas disease is hampered by the lack of basic understanding of the T. cruzi parasite and its resilience in the face of drug treatment. Based on our evidence, persisters are a rare but key sub-population for drug discovery efforts as they exhibit significantly lower drug susceptibility compared to the bulk amastigote population. The understanding of the persister state that will result from the proposed work will be crucial for the drug discovery community. It will allow development of better assays that monitor effects on persisters, tracking of persister parasites in vitro and in vivo, and identification of better drug targets. The work exploring a new combination treatment paradigm will directly benefit drug discovery efforts as it may provide an alternative route to developing better drugs for Chagas disease.

d) Scientists studying T. cruzi: There is no understanding of the molecular nature of persister parasites and intracellular amastigote population heterogeneity in T. cruzi. This work will for the first time describe this heterogeneity and reveal specific differences between replicative parasites and persisters, thus providing valuable information and tools for scientists studying T. cruzi. The proposed work will also provide valuable methods to the T. cruzi and more widely the kinetoplastid scientific community for state-of-the-art technology such as drop-seq and whole-organ smFISH.

e) Scientists studying persisters in different organisms, including bacteria, other pathogenic protozoa such as Leishmania donovani, Plasmodium falciparum, and Toxoplasma gondii, and even in humans where dormant tumour cells bear resemblance to persisters. Understanding the mechanisms underlying T. cruzi persisters may give insights into general strategies used by persister cells.

f) Academic researchers directly involved: This project will use multiple advanced technologies such as scRNA-seq, smFISH, MRM-MS and whole-organ clearing and staining. Direct involvement of the PDRA with these approaches will broaden their expertise and allow them to develop key skills for their future career. Direct exposure to ongoing drug discovery activities will also help familiarise the PDRA with the drug discovery process and allow them to understand how fundamental science can be critical to supporting successful drug discovery.