Authors

Discussion

Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, belongs to the group of neglected diseases of the world and is a life-threatening disease. There are three different subspecies of T. brucei: T. b. rhodesiense causing East African trypanosomiasis which isthe acute form, T. b. gambiense, which causes West African trypanosomiasis, the chronic form, and T. b. brucei which is only infectious for animals but is used as an experimental model of human trypanosomiasis. The tsetse fly is responsible for the transmission, this means that the disease is only prevalent in the natural biotope of the tsetse fly, rural sub-Saharan Africa. Due to vector control and surveillance measurements, the incidence of HAT was gradually reduced in the last decade. HAT has two disease stages. The first one is the haemolymphatic stage which is accompanied by non-specific symptoms such as fever, lymphadenopathy and headache. The Winterbottom’s sign is a typical sign of the gambiense form. The second stage or neurological stage has sleep disturbance and coma as major symptoms. HAT is a life-threatening disease when left untreated. The most used diagnostic tool for the gambiense form is card agglutination test for trypanosomiasis/T. b. gambiense (CATT) and microscopical conformation of trypanosomes for the rhodesiense form. The current available treatments are old and have toxic side effects. Drug resistance is also seen in the field. There is an urgent need for new, more efficient and safer drugs. Those drugs need to be easy to administer. Cyclic nucleotide phosphodiesterases (PDEs) may present a new group of drug targets. PDEs catalyse the hydrolysis of cyclic adenosine monophosphate (AMP) to AMP. cAMP plays a role in the cell proliferation of the parasites. Adenylyl cyclases are responsible for the production of cAMP from ATP and PDEs are responsible for the degradation of cAMP. The steady state concentration of cellular cAMP is different in the stumpy forms compared to the level in the long slender ones. The catalytic domains of the PDEs are highly conserved between humans and Trypanosoma brucei. This means that the extensive research on human PDEs can be used in the research for PDEs inhibitors against trypanosomiasis. TbrPDEB1/B2 is already validated as drug target. Currently there are no successful experimental models of a double knockout of TbrPDED, this means that TbrPDED is potentially an essential gene for T. brucei and is potentially a new drug target. In this project the aim is to validate TbrPDED as potential drug target by the use of a conditional knockout model. The expression of the enzyme can be turned off by withdrawal of tetracycline. The principal aim is to characterize the conditional construct by the use of drug sensitivity assays, localisation studies and cAMP assays. In addition, a TcrPDED expression construct will be made and tested whether it can complement for TbrPDEB1/2. If it can complement, this will also be characterized in the same way. Acknowledgement: this work was done as part of the EU Famework 7 consiortium PDE4NPD.