Functional Analysis Of PbIMC1g In Plasmodium Berghei Blood Stage Development And Gametogenesis

Objective:Malaria is one of the B infectious diseases in China,also a serious public health-threatening transmitted by Anopheles mosquitoes.In Plasmodium spp.life cycle,gametogenesis is the first bottleneck during sexual stage development.Transmission blocking drugs or vaccines that targeted this stage can efficiently block malaria transmission.The inner membrane complex（IMC）is a double-membrane organelle,located beneath parasite plasma membrane,and together with tubulin,supports parasite’s architecture.IMC located proteins also interact with the glideosome to provide power for parasite’s invasion to host cells.IMC1g protein is a member of the IMC1protein family.Previous results indicates that Pb IMC1g is an essential protein,but its function remains unclear.This study intends to detect the expression pattern and subcellular localization of Pb IMC1g protein in Plasmodium berghei life cycle;Investigate the function of Pb IMC1g protein in asexual stage development and gametogenesis,and also identify the immune-modulate roles of this protein,to provide a theoretical basis for new anti-malarial drug targets development.Methods:The structure and function of Pb IMC1g protein were predicted through bioinformatics analysis in this study.The C-terminal HA-tagged insect strain of Pb IMC1g protein was constructed using CRISPR-Cas9 technology.The localization and expression of Pb IMC1g protein in the asexual and sexual stages were detected using indirect immunofluorescence and immunoblotting techniques.Meanwhile,to study the function of Pb IMC1g protein,we construct a Pb IMC1g^{c KD}strain using E.coli DHFR destabilizing domain（DDD）system to regulate Pb IMC1g expression by trimethoprim（TMP）.The subclass and levels of antibodies in sera of Pb IMC1g^{c KD}-infected mice from+/-TMP group were evaluated by ELISA analysis.The percentage of B220⁺CD138⁺、B220^-CD138⁺and B220⁺GL-7⁺B cells in splenocyte of infected mice was evaluated by flow cytometry analysis.Results:1.It was found through bioinformatics analysis that Pb IMC1g protein exhibited high homology in various genera of Plasmodium and contained conserved IMCp protein domains in amphiphyla such as Plasmodium and Toxoplasma gondii.2.The successful construction and screening of transgenic insect strains of Pb IMC1g ^HAand Pb IMC1g^{c KD}were confirmed through the results of genotype PCR and Western blot.3.Western blot analysis revealed that Pb IMC1g protein was expressed in all life cycles of Plasmodium and was highly expressed in gametocyte and zygote in sexual stage.4.Immunofluorescence assays indicated that Pb IMC1g protein was highly expressed in the merozoite during the late erythrocytic stage and localized in the cytoplasm during the gametophyte and zygote stages.Moreover,Pb IMC1g protein gradually transferred to Plasmodium plasma membrane during the development of zygote to ookinete.According to Image J we know that among the merozoites,Pb IMC1g protein was co-located with IMC membrane localization protein CDPK1（Pearson’s correlation coefficient,R²=0.90）,GAP50（R²=0.88）,and MTIP protein（R²=0.87）.5.The knockdown of Pb IMC1g significantly decreased the number of merozoites by 33.3%（P<0.001）in both the erythrocytic and sexual stages.Furthermore,the knockdown group exhibited a slower increase in infection rate compared to the normal expression group of Pb IMC1g protein,leading to significantly prolonged survival times of mice,and even self-healing of some mice（P<0.01）.6.The gametophyte rate and male/female gametophyte ratio were not affected by the knockdown of Pb IMC1g protein.7.The mitotic centers of male gametes in the-TMP group decreased significantly by 45.2%（P<0.05）compared to the+TMP group.8.The humoral immune response of the host immune system to Pb IMC1g^{c KD}C1strain was evaluated.After 10 days of infection,the serum of mice treated with-TMP exhibited significantly increased levels of total Ig G and Ig G1 antibodies compared to the+TMP control group（P<0.01）.Additionally,the content of B cells in plasma cells increased significantly（P<0.05）.Conclusion:1.The presence of the conserved IMCp domain in Plasmodium and other Apicomplexan parasites is a characteristic feature of Pb IMC1g protein.2.Pb IMC1g protein exhibits expression in all developmental stages of Plasmodium,localized in the parasite plasma membrane during the schizont,female/male gamete,and mature ookinete stages.3.Pb IMC1g protein plays a pivotal role in the proliferation of merozoites during the late erythrocytic stage and in male gametogenesis.4.Knockdown of Pb IMC1g protein elicits a significant humoral immune response in the host,which has a high protective effect against Plasmodium infection,leading to its elimination.