Function Identification And Mechanism Of DHHC2/ISP In Regulating Plasmodium Zygote To Ookinete Differenation

Malaria is a insect-borne infection caused by plasmodium,and so far there are still many malaria-infected people worldwide every year.Plasmodium is a single-celled eukaryote with a complex life cycle,including two different hosts,mosquito and vertebrate.In mosquito,malaria parasites develop sexually,and the polarization of zygote is the key to the malaria parasite transmission from vertebrate to mosquito.The cytoskeleton of the malaria parasite comprises an inner membrane complex(IMC)and a subpellicular microtubule(SPM)beneath the IMC.IMC and SPM play an important role in the morphologic change of malaria parasite.However,the key molecules controlling the reconstruction of the IMC and SPM assemble in ookinete differentiation remain unknown.This study used CRISPR/Cas9 gene-editing technology to reveal two inner membrane complexes subcompartment proteins(ISPs)ISP1 and ISP3 in regulating zygote to ookinete transformation coordinately.ISP Proteins have a specific localization and co-localization in the zygote.The double genes knock-out(Δisp1/3)mutant significantly blocked the zygote-ookinete transformation.Mutations of its N-terminal palmitoylation site confirmed the palmitoylation is essential for ISP1/ISP3 IMC localization.We proved that ISP1 and ISP3 achieved its polar membrane distribution in the zygote through protein palmitoylation by compensation,which is also necessary for its function.We used CRISPR/Cas9 gene-editing technology to screen 11 plasmodium-encoded PATs,only DHHC2 co-localized with ISP1/ISP3 in the zygote polarity patch.Accordingly,dhhc2 knock-down(dhhc2kd)phenocopied Aispl/3.Further we found that the palmitoylation level ISP1 and ISP3 significantly reduced in dhhc2kd,and thus lost the polar membrane localization.Furthermore,we determined DHHC2 regulated ISP1 and ISP3 through its palmitoyl-S-acyl-transferase activity by the complementation experiment.Furthermore,this study identified DHHC2 self-palmitoylation of C-terminal cysteines controlled its localization at IMC after zygote formation.Using the transmission electron microscope,we determined that both dhhc2kd and Δisp1/3 caused abnormal development of ookinete cytoskeleton,mainly in the reduction of the number of microtubules and the disorder of microtubule arrangement.In addition,we used co-immunoprecipitation to verify ISP1 likely interacted with β-tubulin.This study used genetics,molecular biology,cell biology and biochemistry methods to reveal the mechanism of two important palmitoylated protein ISP1 and ISP3 modified by palmitoyltransferase DHHC2.DHHC2 targeted to the IMC through its self-palmitoylation.ISP1/ISP3 make subpellicular microtubules stably anchor at the IMC,thus forming the stable cytoskeletal structure to support the zygote to ookinete differentiation.