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Uncovering the components and interactors of a novel cAMP signalling pathway and characterisation of its role in cytokinesis in Trypanosoma brucei

Final Report Summary - TRYPCARPINTERACTORS (Uncovering the components and interactors of a novel cAMP signalling pathway and characterisation of its role in cytokinesis in Trypanosoma brucei.)

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), colloquially known as ‘Sleeping Sickness’ and is lethal to humans without treatment. Current drugs are very unsatisfactory, having often deadly side effects, or have become increasingly less effective due to drug resistance.
Recently, cAMP metabolism was validated genetically and pharmacologically as an excellent drug target against HAT: cAMP-synthesizing adenylyl cyclases of the parasite were shown to be important for cell division of mother and daughter cells. Similarly, knockdown of phosphodiesterase enzymes (that degrade cAMP) kill the parasite, causing a severe cell division defect. Finally, a newly identified trypanosomal-specific PDE inhibitor is lethal to the parasite and also results in the same severe cytokinesis phenotype. However, how changes to cAMP concentrations translate to a cell division 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 objectives of this research project are:
• To identify more components of the cyclic-AMP signalling cascade in this medically important, pathogenic protozoan
• To characterise these newly identified proteins with respect to known phenotypes associated with cAMP signalling
• To uncover how these proteins interact with each other and their respective order in the cAMP pathway(s)
• To assess the potential for any newly identified cAMP signalling proteins to be good drug targets.

Work performed and main results achieved
To identify more downstream effectors of the cAMP signalling pathway, a re-analysis of a genome-wide RNAi screen for resistance to Cpd A was carried out using Illumina next generation sequencing. Reassuringly, this analysis identified the 4 previously documented CARPs, but also uncovered many other potential candidate genes as cAMP effectors. A further 6 of these genes have now been validated as genuine CARPs through RNAi and drug sensitivity assays, bringing the total number of CARPs identified to 10. Phylogenetic analysis of all 10 CARPs clearly shows that the cAMP signalling cascade in trypanosomes is remarkably divergent from higher eukaryotes and is unique to the kinetoplastida.
A second approach was used to simultaneously identify more CARPs as well as uncover how they interact. The BioID protocol utilises a mutated bacterial ligase (BirA*) that biotinylates neighbouring proteins in a proximity dependent manner. Once the BirA*gene is fused to one of the CARPs, on addition of biotin and expression of the fusion protein, those proteins that directly interact with the target CARP are biotinylated and can be purified and identified by Mass Spectrometry.
To aid the BioID project, it was necessary to develop a molecular toolkit of plasmid constructs. These plasmids allow the rapid and simple generation of the BirA*-target gene fusions and protein expression and have become a valuable resource for the wider trypanosome research community, with Material Transfer requests from research groups in France, the United Kingdom and Brazil.
Multiple CARP-BirA* fusion cell lines have now been generated and samples extracted from both the mammalian infective bloodstream stage and the insect infective procyclic stage of the parasite’s life cycle. Mass spectrometry and bioinformatics analysis will allow us to identify and map interacting proteins and larger complexes as well as how the membership of the interacting cohort changes between the life cycle stages.
Epistasis experiments, where the effect of knocking out and repressing pairs of CARPs simultaneously, have helped us to determine that the cAMP cascade, at least in the bloodstream form of the parasite, is complex. It is not a simple linear pathway with one effector protein following the other, but is actually more like a network with more than one pathway working in parallel.
So far two phenotypes associated with cAMP signalling in trypanosomes have been described: In bloodstream forms, extreme increases or decreases to cAMP concentrations result in reduced cell growth and death following a block in cell division. In procyclic forms, elevating or decreasing cAMP levels disrupts a newly described phenomenon called Social Motility, where thousands of procyclic cells move in a coordinated and responsive fashion on agar plates.
Repressing the expression of one of the CARPs in bloodstream forms results in a slower rate of population growth and the same block in cell division seen when manipulating the intracellular cAMP concentration. This particular CARP is highly conserved across phyla, including in humans, but remains entirely uncharacterised in any model organism, with this research representing its first functional assignment.
Repression of a different CARP in procyclic form trypanosomes resulted in a strong inhibition of Social Motility. While no effect on growth rate was observed, this finding validates the cAMP pathway as important for this phenotype. Further research is on-going with a collaborating group in Belgium to assess whether this gene, and consequently the Social Motility phenotype, is essential in further life cycle stages within the insect host.
As described in the project context, the need for new and effective drugs against Human African Trypanosomiasis is urgent. Current drug discovery projects targeting the cAMP modulating machinery (phosphodiesterases) are continuing and have confirmed that the signalling cascade plays an essential role in cell division; however, the specific effector gene responsible for the lethal effect has never been identified. During the project the researcher has validated a single CARP as being essential and giving the same cell division phenotype on repression as observed when varying the cAMP concentration. The disruption of the function of this CARP potentially represents the ultimate mode of action of PDE inhibitors, although further research will be required to confirm this. It also validates this CARP itself as a potentially more precise drug target.