Structure And Function Analysis Of GC? Protein During The Gametocyte Activation In Plasmodium Yoelii

Malaria,which results in several hundred thousand deaths and several hundred million clinical cases annually,is a vector-borne blood disease caused by protozoan parasites of the genus Plasmodium and transmitted by blood-feeding Anopheline mosquitoes.Malaria parasites have evolved a complicated life cycle alternating between the vertebrate host and the mosquito vector and a major part of the complexity associated with the plasmodium life cycle is due to the parasite's ability to change its cellular and molecular makeup.The malaria sexual phase begins with the differentiation of the gametocytes into male and female gametes which is induced by a small mosquito molecule,xanthurenic acid(XA),followed by their uptake during the blood meal of the mosquito.The transformation of the fertilized zygote into the infective ookinete subsequently marks the end of the malaria sexual phase.Therefore,gametocytes become mature and fertilizable gametes is the first step in sexual reproduction and obligatory stage required for transmission of the parasite to the mosquito vector.Although there is some evidence that the cGMP signaling pathway is involved in the regulation of gametocytogenesis in P.falciparum and P.berghei,little is known about GCa is the guanylate cyclase which produces cGMP in this signaling pathway.We studied the regulatory role of GCa protein in gametocytogenesis through the analysis of structure and function of PyGCa protein.Firstly,we generated an endogenous tagged strain of gca gene to study the expression and subcellular localization of GCa through the life cycle of Plasmodium.We here detected that GCa expression in trophozoites,merozoites,gametocytes,oocysts and sporozoites except ookinetes,and GCa was localized in the cell membrane and cytoplasm,mainly distributing as spot.Then we used the promoter swap methodology to achieve lower expression of GCa in sexual stages of Plasmodium.We show that GCa is essential for sexual mosquito stages and GC?,not GC?,leads to the synthesis of the second messenger cyclic GMP(cGMP)during gametocytogenesis.Finally,we revealed that GCa protein has cleavage,then AD and GCD become two separated proteins during the life cycle of Plasmodium.Using the rodent malaria parasite Plasmodium yoelii,Py17XNL,and CRISPR/Cas9 system,we confirmed that the GCa is a key regulator during the activation of gametocytes and its cleavage structure property.These findings laid a foundation for further study of GCa and provided a new potential target for the development of malaria transmission blocking vaccine.