This research project will focus on identifying the components of a remarkably divergent cyclic-AMP signalling cascade in the medically important, pathogenic protozoan Trypanosoma brucei. It will map an interaction network for this unique cAMP pathway and biochemically characterise the lethal effect on cytokinesis that disruption of the intracellular cAMP concentration has on the human-infective form of the parasite.
Recently, cAMP metabolism was validated genetically and pharmacologically as an excellent drug target against Trypanosoma brucei: The cAMP-synthesizing adenylyl cyclases of the parasite were shown to be essential for cytokinesis of mother and daughter cells. Similarly, knockdown of phosphodiesterase enzymes (that degrade cAMP) kill the parasite, causing a severe cytokinesis defect. Finally, a newly identified trypanosomal-specific PDE inhibitor, Cpd A, is lethal to the parasite and also results in the same severe cytokinesis phenotype. How changes to cAMP concentrations translate to a cytokinesis defect and the identity of the downstream cAMP effector proteins in trypanosomes remain almost entirely unknown. To rectify this, a newly-developed forward genetics approach was used by the applicant to identify four cAMP response proteins (CARPs) which, when knocked down, give resistance to elevated cAMP and appear to represent part of a Kinetoplastid-specific cAMP pathway. The proposed research project will utilise advanced proteomic and interactomic techniques, combined with optogenetics, high-resolution live-cell confocal fluorescence microscopy and microscale thermophoresis to identify, map and characterize the proteins interacting with CARPs.
These investigations will provide essential information on the evolution of the cAMP pathway in eukaryotic organisms and is likely to identify proteins unique to trypanosomes that will represent excellent future drug targets.
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