Newton001: Targeting the surface proteome of Trypanosoma cruzi

The surface of protozoan parasites represent the interface with the host, with many roles in invasion, immune evasion, signalling, and environmental sensing. We recently identified a conserved ubiquitylation pathway in trypanosomes that controls the surface copy number of important trans-membrane domain proteins, and which provides a means for the first time to manipulate trans-membrane domain protein abundance in these parasites in a rational manor, and to assess their importance to host cell invasion and virulence.
This application seeks to assess the significance of the surface protein ubiquitylation pathway in Trypanosoma cruzi for:
1. Parasite viability, host cell invasion and life cycle progression,
2. Maintenance of the surface coat, specificallycopy number of trans-membrane domain proteins and their impact on the global surface proteome,
3. Possible value as a therapeutic target, by assessment of viability in vitro.
The work will combine state of the art genome editing using CRISPR/Cas9 approaches, together with SILAC proteomics for unbiased analysis of the surface/cellular proteome.

The surface of all cells represents the site for interaction with the environment, but for protozoan parasites the surface also has a fundamental role in interactions with the host. For T. cruzi, specific interactions with host cells to mediate cell invasion, are an essential component of the T. cruzi life cycle. Mediating this process are a number of cell surface molecules that participate in a plethora of interactions; the flexibility of this process likely accounts both for the ability of T. cruzi to invade a wide range of cell lineages/tissues as well as many mammalian species. Significantly, the cell surfaces of trypanosomes are highly distinct, with few proteins conserved and the major known antigens being involved in host cell invasion (mucins/trans-sialidases).
We propose the following experiments, to be performed in T. cruzi, that will combine technology development together with the discreet analysis of the impact of the Usp33/Rbx1 ligase on the surface protein composition of T. cruzi and the evaluation as a potential drug target.
1. We will develop a CRISPR/Cas9 system for the conditional knockout of the Usp33 and Rbx1 genes in T. cruzi. 2. We will analyse the impact of Usp33 and Rbx1 knockout on the surface proteome of T. cruzi using SILAC methodology.
3. Both Usp33 and Rbx1 will be tagged with GFP (endogenous) for purposes of localisation, as well as providing an affinity handle for the isolation of complexes, and in particular to ascertain if Usp33 and the Rbx1/Cullen/Elongin interact directly (where there is evidence they do in mammalian cells) and potentially also to identify substrate proteins.
4. We will study the impact of the genomic modifications of the differentiation of T. cruzi from the insect to infective stage (metacyclic trypomastigotes), the capacity to attach, invade and proliferate in mammalians cells.

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