Metabolism and drug resistance probed with new genetic tools in the neglected animal pathogen Trypanosoma vivax.

Animal African Trypanosomiasis (AAT) is a debilitating disease of cattle and other livestock, caused mainly by two species of single-celled parasite: Trypanosoma congolense and Trypanosoma vivax. There are ~70 million cases of AAT each year, causing ~3 million deaths and creating a great economic burden on rural communities. As well as causing disease in Africa, T. vivax has also spread to South America, where it is an increasing problem. Despite being a globally important pathogen, T. vivax is severely neglected in terms of knowledge and research effort - due largely to the lack of good systems in place for maintaining the parasite outside of animals or for investigating the function of its individual genes. T. vivax is genetically distinct from better-studied trypanosomes, with around one third of all T. vivax genes not being found in related species - this is mirrored in important differences in the biology of the parasite, including its transmission through the insect vector, evasion of the host immune system, and its resistance to drugs. Advancement of both fundamental knowledge and laboratory capabilities are needed to transform the ability to work meaningfully with T. vivax , and to design tools to consequently reduce the impact of AAT. This project will do both; by generating new knowledge on key areas of T. vivax biology and developing a toolkit for culturing and genetic transformation of the parasite.

We will characterise the core metabolism of T. vivax by analysing the pattern of gene expression and what chemicals the parasites use/produce. This will enable us to define the chemical processes going on inside T. vivax cells, and we will test these predictions with targeted chemical inhibitors and interfering with expression of specific genes. We will also characterise the proteins on the surface of T. vivax, which are the parts of the cell in direct contact with the host, including those responsible for key metabolite and drug uptake. We will generate parasites resistant to three drugs (the two drugs currently widely used in the field, and one in clinical development) and identify genetic changes responsible for resistance. By comparing these to information on T. vivax metabolism, surface composition, and data from functional studies in other trypanosomes, we will decode mechanisms of drug resistance in T. vivax for the first time. Finally, we aim to develop critical laboratory resources for T. vivax. Information on parasite metabolism and surface complement will be used to design chemical formulations that will support growth of T. vivax outside of animals, as has been done for other trypanosomes. We will also develop a genetic toolkit for T. vivax to enable functional analysis of its genes, which will feed directly into our aims of understanding of metabolism and drug resistance.

The project will build upon relevant expertise of the team, who have experience in successfully tackling similar questions for the related parasites T. brucei and T. congolense, and complements the drug development efforts of our industrial partner, the Global Alliance for Livestock Veterinary Medicines (GALVmed). The outputs will be particularly relevant to drug development and drug sustainability - understanding drug resistance to existing and new drugs enables informed design of drug usage that can maximise the lifetime of treatments. Additionally, advancing our knowledge of core metabolism has the potential to identify novel drug targets, and the ability to culture T. vivax would enable the scaling up of drug discovery efforts. Therefore, achieving these aims will transform our ability to work with T. vivax, generating foundational knowledge and datasets, and advancing fundamental and applied research capabilities for this significantly neglected pathogen.

Trypanosoma vivax is one of the main causes of Animal African Trypanosomiasis, but is sorely neglected despite its substantial impact. This project will bridge the critical knowledge and capability gaps in order to shift paradigms for basic and applied research on this important pathogen. The project will build on the experience of the applicants, and the industrial partner, the Global Alliance for Livestock Veterinary Medicines (GALVmed), to generate new knowledge on key areas of T. vivax biology and develop a laboratory toolkit for in vitro culture and genetic transformation, through a series of integrated aims. We will comprehensively characterise the metabolism of bloodstream-form T. vivax using RNAseq and LC-MS/GC-MS, enabling the production of a genome-scale model of metabolism. Predictions will be validated in genetic mutants or chemical inhibition of key metabolic steps. In parallel, the T. vivax surface proteome will be defined to identify proteins expected to be involved in metabolite and drug transport. Resistance induced both in vivo and in vitro to three drugs - the widely used diminazene aceturate and isometamidium chloride, and a benzoxaborole in clinical development - will be used in combination with data on parasite metabolism and surface proteome to identify mechanisms of drug uptake and resistance. These outputs will provide novel and foundational information on how T. vivax interacts with its environment, and particularly how it responds to exposure to drugs. Finally, we will generate laboratory capabilities to transform the ability to work meaningfully with T. vivax. Building upon the applicants' expertise with related trypanosomes, we will use the data generated to design culture medium that will support the growth of bloodstream-form T. vivax, and will generate a toolkit for functional genetics in this important parasite.