The Study On Molecular Mechanism Of Ookinete Motility Regulation By Guanylate Cyclase Beta In Plasmodium

Malaria is a mosquito-borne parasite disease caused by the genus plasmodium spp.The ookinete is a special cellular stage of plasmodium developed from sexual reproduction upon entering mosquito after a blood meal.The ookinete directional gliding motility(ODGM)is fundamental for the mosquito vector transmission of malaria parasites.Guanylate cyclase beta(GCp)has been identified to be critical for ookinete gliding motility,however how GCβ regulates ookinete gliding motility and whether GC β itself is regulated remains unknown.Plasmodium GC β is a bi-functional protein consisting an P4-ATPase like domain(ALD)in its N-lobe and a guanylyl cyclase domain(GCD)in its C-lobe.This N-ALD + C-GCD form of guanylate cyclase is common among many protozoans,with the mysterious waiting to be revealed.In this study,to dissect the mechanism of ODGM,the two plasmodium guanylate cyclase GCα and GCβ are endogenously tagged using CRISPR/Cas9 method in P.yoelii for the first time.GCβ is found as the only guanylate cyclase expressed in ookinetes.Astonishingly,GC β forms a distinct apical-dorsal side(ADS)clustered localization in mature ookinetes.The GC β ADS clustering occurs upon ookinete maturation,coincidences with initiation of ODGM.Mutagenesis assay on the GCD indicated the clustering of cGMP-synthesising enzyme active GC β at ADS is required for local cGMP elevation to drive cGMP dependent protein kinase G(PKG)activation and ODGM.Using a recently developed cGMP probe Green cGull,the cGMP synthesizing activity of GCβ is revealed on the single cell level.Importantly,premature activating PKG during ookinete development through PDEδ knockout or zaprinast treatment lead to ookinete deformation and ODGM disruption,which could be reversed by additional treatment with PKG inhibitor Compound 2(C2).These results indicates the cGMP-PKG signaling must be silenced during ookinete development and precocious activation of this pathway interferes with the development process,revealing a fascinating time-spatial regulation pattern.To further dissect the mechanism of GCβ ADS clustering,the ALD and GCD domains were separated by ribose-skip T2A sequence insertion.The ALD was found indispensable for GCO ADS clustering.However,sequence analysis indicates multiple mutations on the core-residues of P4-ATPase of the ALD domain,indicating ALD as a pseudo-flipping ATPase.Through screening of the P4-ATPase co-factor CDC50 genes in plasmodium,a novel GCβ binding CDC50 family protein was identified and named as CDC50A.CDC50A co-localizes with GC β exactly during life cycle progression.CDC50A binding stabilizes GCβ during ookinete development,but not directly determines its ADS clustering.Further analyse by trypsin test and 2-BMP treatment indicates the inner membrane complex(IMC)may medicate GCβ ADS clustering.A screening of IMC proteins identified the inner-membrane-complex subcompartment protein 1(ISP1)as a critical mediator.By knocking out ISP1 unexpectedly,we proved its critical role for GC β ADS clustering through trapping GCβ and correctly presenting it at ADS.Through C7C8 dual palmitoylation,ISP1 is anchored on the IMC membrane and mediates GCβ ADS clustering.This study systematically revealed the upstream mechanism of ODGM.Firstly,CDC50A binds and stabilizes GCβ during ookinete development,preventing its degradation and(may)promoting GCβ sorting.The cGMP-PKG signaling was silenced at these stages by PDE 8,giving way to ookinete development.Upon ookinete maturation,GCβ translocates to the parasite plasma membrane(PPM)and trapped by IMC-docking ISP 1,forming ADS clustering.The clustering breaks the cGMP synthesizing-hydrolyzing balance and leads to local cGMP elevation,which activates PKG and subsequently initiates ODGM.The new mechanisms of ODGM revealed in this study,and the identification of the novel CDC50 family proteins,new function of ISP1,provides new insights on malaria biology and offers new strategy for malaria transmission intervention.