Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.
Author summary Schistosomiasis is a serious and often fatal disease that is caused by infection with parasitic worms of the Schistosoma species. It affects several hundreds of millions of people world-wide, mostly in the tropics, through contact of skin with infected water, usually lakes and rivers. Only one drug, praziquantel, exists for its treatment, so resistance to it would leave the disease untreatable and new drugs are urgently needed. Here, we report on a strategy to develop new anti-schistosomal agents by inhibiting the regulatory systems of the worm. Cyclic nucleotides cAMP and cGMP are key regulators of cellular activity, and their activity is determined by enzymes called phosphodiesterases or PDEs, that can degrade them. We identified and cloned the genes coding for ten of these PDEs from Schistosoma mansoni, a species causing a very high disease burden. By expressing these genes in specialized cell lines of the yeast Saccharomyces cerevisiae and the protozoan parasite Trypanosoma brucei, under conditions where growth becomes conditional on the expression of the introduced Schistosoma PDE, we show that most of these genes indeed code for functional PDEs. For several PDEs we also determined, using a third expression system, the yeast Schizosaccharomyces pombe, whether they regulate cAMP or cGMP. We also identify a number of inhibitors for one of these PDEs and show that all are expressed more in male than in female worms. This work is essential to map and characterise the regulatory pathways of schistosomes and will facilitate the development of inhibitors of the key enzymes.