The Role Of ER Tubule-forming Protein YOP1 In The Virulence Of Plasmodium Berghei Parasites

Malaria,a serious life-threatening infectious disease,is caused by protozoan pathogens of the genus Plasmodium and transmitted by a bite of female mosquitoes of the Anopheles genus.Malaria still has a profound effect on global human health and economic development,especially in the tropical and subtropical countries.As the occurrence of drug resistance,artemisinin-based combination therapies are insufficient for malaria treatment,novel antimalarial drugs and more efficacious vaccines are the key point for the elimination malaria.Endoplasmic reticulum(ER)is an important organelle in eukaryotic cells,involving in protein synthesis and folding,lipid metabolism,and calcium storage.ER is composed of cistem-like sheet structure,reticular network of tubules and nuclear envelop.Reticulon and REEP family of proteins generate and stabilize ER tubules by inducing the high curvature of the membrane.Plasmodium berghei YOP1(PbYOP1)is a homolog of REEP5,associating with the morphology and function of ER tubules in Plasmodium.The investigation of ER morphology and function is always performed in yeast and mammalian cells and has rarely been studied in protozoan parasite.To investigate the importance of ER morphogenesis in Plasmodium,we generate PbYOP1-deficient parasite(Pbyop1Δ)and analyze the growth rate and virulence of parasites.Our study include three sections:Section one Pbyop1Δparasite construction was performed via standard genetic modification technology by double-crossover homologous recombination.Disruption of Pbyop1 leaded to attenuated growth rate of parasites in blood-stage,the ER tubules and digestive vacuole were significantly enlarged,but the hemozoin production was similar with WT parasites.To analyze the virulence of Pbyop1Δparasites,the morbidity and mortality of experimental cerebral malaria(ECM)was examined.More than 90%WT parasite infected mice developed neurological symptoms of ECM and died within 6-8 days post-infection(dpi),whereas Pbyop1Δparasite infected mice were not succumbed to ECM and died of severe anemia 3 weeks post-infection.To further investigate the role of YOP1 in plasmodium parasites,Pbyop1Δ-rescue parasite was constructed,and the delay of growth and the morbidity and mortality of ECM were restored through complementation of Pbyop1.Section two Disruption of Pbyop1 inhibits the pathogenesis of ECM.Loss of PbYOP1 had no effect on the sequestration of parasites.The frequency and cell number of total CD4~+and CD8~+T cells,and the expression of CXCR3 in each T cell,were increased in peripheral blood and brain,no significant difference was detected between WT and Pbyop1Δ-infected mice.CD4~+T cell subsets were also analyzed,Th1 cells sequestration was significantly decreased in brains of Pbyop1Δparasite infected mice compared with WT,no remarkable difference of Tregs sequestration was detected.In comparison with WT parasites infected mice,the expression of IFN-γand TNF-α,the two important pro-inflammatory cytokines associated with ECM,was down-regulated in brain and serum of Pbyop1Δ-infected mice.Expression of adhesion molecules ICAM-1,VCAM-1 and CD36,the hallmark of endothelial cell activation,was significantly decreased in brainstem of Pbyop1Δparasite infected mice.Expression of cytotoxic molecules such as granzyme B and perforin and activation of caspase-3 were reduced,resulting in alleviated cell apoptosis in brain of Pbyop1Δ-infected mice.In addition,the expression of IFN-γ,TNF-α,granzyme B,and perforin,and the activation of caspase-3,were down-regulated in the brains of Pbyop1Δparasite-infected mice 11 dpi compared to 7 dpi,particularly in 1×10~6Pbyop1Δ-infected mice.Although PbYOP1 deficiency has no effect on the sequestration of parasite,the alleviated inflammation and reduced brain injury,particularly in brainstem,protecting Pbyop1Δ-infected mice from ECM.Section three To investigate whether the Pbyop1Δparasites infection could elicit protective immunity in mice.Since the virulence was attenuated,BALB/c mice were able to clear the Pbyop1Δparasites infection spontaneously and protected from subsequent WT parasites challenge given at high dose.In conclusion,the maintenance of ER morphology has profound effect on the Plasmodium parasite proliferation in blood stage and the pathogenesis of ECM.PbYOP1 deficiency leads to aberrant ER morphology and digestive vacuole,and attenuated growth rate and virulence of parasite.Expression of pro-inflammatory cytokines and cytotoxic molecules and activation of apoptosis molecules are down-regulated in Pbyop1Δparasites infected mice compared with WT parasite,resulting in alleviated brain injury and protecting mice from ECM.Pbyop1Δparasites could be cleared spontaneously in BALB/c mice and elicit protective immunity in convalescence mice.YOP1 and other proteins that play a role in ER morphology and function could be the novel targets for anti-malaria drug and effective vaccine invention.