Haem Biosynthesis and Uptake in Schistosoma mansoni

Abstract

Schistosomiasis is a major public health problem in many developing countries. The disease is endemic in 74 countries and infects nearly 250 million people worldwide. Among the five main species affecting humans, Schistosoma mansoni and S. japonicum are responsible for the majority of hepato-intestinal schistosomiasis. Praziquantel is the only available drug against this chronic debilitating disease and many vaccines are being trialled in the search for an effective control of the disease. Adult schistosomes are dependent on the blood of their definitive host for nutrition. In the process of ingesting and breaking down host erythrocytes, large quantities of haem are released. Despite being a potentially toxic molecule, haem is an essential factor for numerous biological reactions and may also serve as an important source of iron. The molecules involved in the catabolism of red blood cells (RBC) and release of its end-product haem, are potential targets for development of anthelmintic drugs or vaccines. By contrast, knowledge on haem metabolism and the associated uptake in schistosomes is limited and further research may identify potential targets among the molecules involved in haemoglobin breakdown and haem utilization or sequestration. In the first part of this thesis, the de novo haem biosynthetic capability of schistosomes was determined by investigating two enzymes of the eight-enzyme haem biosynthetic pathway, d- aminolevulinic acid dehydratase (ALAD) and ferrochelatase (FC). The function of the two enzymes was investigated using a combination of three techniques: (1) in silico analysis of protein sequences; (2) complementation assays using mutant E. coli strains defective in haem biosynthesis; (3) enzymatic assays using the recombinant schistosome proteins. While the in silico analysis suggested that these proteins are likely to be active enzymes, complementation and enzymatic assays were unable to verify their function. In the second part of the thesis, it was hypothesized that an adaptive haem uptake mechanism is present in the schistosomes to support their haem needs. This hypothesis was confirmed with a pulsechase experiment using a fluorescent haem analogue, palladium mesoporphyrin IX (Pd-mP). The results of this experiment indicated that the transmembrane uptake of haem in schistosomes is likely to be an active process that is specific to haem. Further, it was demonstrated that Pd-mP is taken up and accumulated in the vitellaria and ovary of females, suggesting that haem uptake and metabolism is an essential biological process that supports reproduction in schistosomes. Further investigation of the underlying mediating this transmembrane transport of haem was presented by targeting this pathway pharmacologically. It was demonstrated that cyclosporin A (CsA) inhibits the uptake of Pd-mP, a fluorescent haem analogue, by the ovary of S. mansoni in concentration-dependent manner. Furthermore, it was shown that worms treated with CsA had significant reduction in egg production and the eggs produced by the treated worms showed delays in development and maturation. This suggested that the antifecundity effect of CsA on schistosomes is related to the inhibitory effect of CsA on haem uptake in the parasites. A putative haem transporter protein, S. mansoni haem responsive gene-1 (SmHRG-1), was identified based on loose sequence homology with the C. elegans haem transporter protein, HRG-4. In the knock-down study, it was found that worms treated with Smhrg-1 dsRNA had significant decrease in egg production and maturation of the laid eggs. This result implied that SmHRG-1 could be involved in embryogenesis and vitellogenesis. The functionality of SmHRG-1 as a transmmembrane haem transporter was confirmed by complementation assays using a mutant yeast strain in both liquid and solid media. Overall, my thesis suggest that SmHRG-1 is a potential haem transporter protein in S. mansoni and this uptake of haem is required in the egg production and may affect subsequent maturation of the eggs. It was concluded that SmHRG-1 may represent a promising target for drug and vaccine development in the control of schistosomiasis, given its involvement in egg production and subsequent maturation of egg.