CSP Resists To The IFN-γ-inducing Killing Of Pre-erythrocytic Stage Through Down-regulation Of Autophagy Related Proteins

Malaria, which is caused by the infection of Plasmodium, remains to be one of the three most serious infectious diseases in the world. The life cycle of Plasmodium includes liver stage(pre-erythrocytic stage), blood stage and mosquito stage. The liver stage is the first step of plasmodium infection in mammalian host, and blockage of liver stage infection could prevent the infection of malaria. Therefore, understanding the underlying mechanism of malaria parasite to escape or suppress the host innate immune responses will shed us with new light on the designation of prophylactic strategy against pre-erythrocytic stage. Due to the limitation of the Anopheles rearing and infection platform, the interaction between Plasmodium and host is still largely unknown. Current studies demonstrated that the CSP(circumsporozoite protein) of sporozoite enclosed in PV would be released and cleavage to ΔCSP, and then exported into the hepatocyte cytoplasm through its pexel domain. Furthermore, the ΔCSP translocated into the hepatocytic cytoplasm could promote the development of the EEFs(Exo-erythrocytic Forms). However, our studies showed that the expression of either recombinant CSP or ΔCSP plasmid could not directly promote the development of P.yoelii 265 BY EEF,but could resist to the IFN-γ-inducing killing of pre-erythrocytic stage. Our further study showed that autophagy related proteins of HepG2-CD81 could be down-regulated by ΔCSP. However, it is still unknown whether the autophagy of hepatocyte is involved in regulating the development of EEF and the killing effect of IFN-γ on EEF.To answer this question, we firstly investigated the effect of hepatocyte autophagy level on the development of EEF and the IFN-γ-induced killing of EEF, and then was confirmed in vivo by using CSP pexel mutant sporozoite and hepatocyte ATG5 conditional knockout mice. Finally, we screened the possible molecules directly interacted with ΔCSP through eXact-His tag purification system and high resolution mass spectrometer. This could not only shed new light on the escape strategy of pre-erythrocytic stage, but also provide novel clues to design prophylactic strategies against the liver-stage.1. The effect of ΔCSP on the development of EEF, the IFN-γ-induced killing of EEF and host hepatocyte autophagy1.1 Transfection and expression of recombinant CSP and ΔCSP plasmid could not directly promote the development of P.yoelii 265 BY EEFThe recombinant plasmids of P.yoelii 265 BYCSP and ΔCSP were constructed and transfected into HepG2-CD81 cell. Then purified sporozoites were added to infect the cells, 42 h after infection, the parasite load was detected by probe quantitative PCR. The results suggest that transfection and expression of recombinant CSP and ΔCSP plasmid could not directly promote the development of P.yoelii 265 BY EEF.1.2 Transfection and expression of recombinant CSP and ΔCSP plasmid could limit the IFN-γ-killing of EEFThe recombinant plasmids of P.yoelii 265 BY CSP and ΔCSP were transfected into HepG2-CD81 cell with or without IFN-γ treatment. Then purified sporozoites were added to infect the cells, 24 h after infection, the diameter, area and the rate of LC3 and LAMP-1 co-location of EEF were analyzed;, the parasite load was detected by probe quantitative PCR 42 h after infection. The results suggest that the diameter, area and the parasite load of EEF in the ΔCSP plasmid transfected cells were not changed after the IFN-γ treatment compared to the control group.1.3 Transfection and expression of recombinant CSP and ΔCSP plasmid could down-regulate the expression level of autophagy related proteinTo investigate the mechanism of ΔCSP resist to the IFN-γ-induced killing of EEF. The recombinant plasmids of P.yoelii 265 BY ΔCSP were transfected into HepG2-CD81 cell. After 0h, 6h, 12 h, 24 h of transfection, the lysate of transfected cells were collected, and the expression level of autophagy related protein LC3, ATG7, Beclin-1 and P62(SQSTM1) were detected by western blot. The results suggested that the transfection of ΔCSP could down-regulate the expression level of autophagy related protein LC3, ATG7.2. The effect of autophagy level of hepatocyte on the development of EEF and the IFN-γ-induced killing of EEF:2.1 The development of EEFs was limited in hepatocyte ATG5 conditional knockout miceTo investigate the effect of hepatocyte autophagy on the development of EEFs, we have cross-bred the genetically modified mice to obtain the hepatocyte ATG5 conditional knockout mice. The control and hepatocyte ATG5 conditional knockout mice were infected with sporozoites i.v. 42 h after infection, the liver parasite burden of each mice were detected by probe quantitative PCR. The results suggested that the development of EEF was limited in hepatocyte ATG5 conditional knockout mice and the hepatocyte autophagy is necessary for EEF to develop in the hepatocyte.2.2 Either induction or suppression of hepatic autophagy has no affect on the development of EEFsThe autophagy of Hep G2-CD81 cells were induced by rapamycin or suppressed by LY294002 prior to the incubation of the purified sporozoites. 24 h after the incubation of HepG2-CD81 cells with sporozoites, the diameter, area and the rate of LC3 co-localization of EEF were analysed; 42 h after infection, the parasite load was detected by probe quantitative PCR. The results indicated that either down-regulating or up-regulation of hepatic autophagy has no affect on the development of EEFs.2.3 Either the induction or suppression the hepatic autophagy has an obvious effect on the IFN-γ-induced killing of EEFsThe different results of 2.1 and 2.2 could be explained that the important innate immune effector IFN-γ might play an essential role in vivo assay. To investigate the whether hepatocyte autophagy could modulate the IFN-γ-induced killing of EEFs, the autophagy level of HepG2-CD81 cells was induced by rapamycin or inhibited by LY294002, and then infected with sporozoites and treated with IFN-γ. 24 h after infection, the diameter, area and the rate of LC3 co-location of EEF were analysed; 42 h after infection, the parasite load was detected by probe quantitative PCR. The results indicated that autophagy induced by rapamycin could enhance 0.2U/ml IFN-γ-induced killing of EEF and the autophagy suppressed by LY294002 could limit 0.5U/ml and 1U/ml IFN-γ-induced killing of EEF. This suggested that either the induction or suppression of hepatocyte autophagy has an obvious affect on the IFN-γ-induced killing of EEF.2.4 IFN-γ-killing of EEFs were greatly suppressed in the hepatocyte ATG5 conditional knockout miceThen we confirmed the results by using hepatocyte ATG5 conditional knockout mice, whose IFN-γ was depleted or not. 42 h after the mice were infected sporozoites i.v., the liver parasite burden of each mouse were detected by probe quantitative PCR. The results suggested that the capacity of IFN-γ-killing of EEFs was greatly suppressed in the hepatocyte ATG5 conditional knockout mice.3. CSP resists to the IFN-γ-inducing killing of EEF through down-regulation of autophagy related proteins3.1 Overexpression of recombinant ATG3/5/7 could rescue the inhibitory effect of on IFN-γ-induced killing of EEFsTo investigate whether the CSP resists to the IFN-γ-inducing killing of EEF through down-regulation of autophagy related proteins or not, HepG2-CD81 cells were co-transfected with the recombination ATG3/5/7 and ΔCSP plasmids, and then infected with sporozoites and treated with IFN-γ. 42 h after infection, the parasite load was detected by probe quantitative PCR. The results indicated that over-expression of ATG3, ATG5 or ATG7 could greatly rescuethe inhibitory effect of on IFN-γ-induced killing of EEFs.3.2 Construction of CSP pexel function domain mutant sporozoiteThe results of studies in vitro indicated that CSP resists to the IFN-γ-inducing killing of pre-erythrocytic stage through down-regulation of autophagy related proteins. As the malaria parasite knockout of CSP could not develop into sporozoites in mosquito, CSP pexel function domain mutant sporozoite was constructed. We would like to confirm the results by comparison of mutant and wild type parasite load in mice when IFN-γ was depleted or not.4. Screening the possible molecules directly interacted with ΔCSP:4.1Construction of P.yoelii 265 BY ΔCSP stable-transfected HepG2-CD81 cell lines: To understand the underlying mechanism of ΔCSP to down-regulate the expression level of autophagy protein, the P.yoelii 265 BY ΔCSP stable-transfected HepG2-CD81 cell lines were established by using lentivirus system.4.2 Screening the possible molecules directly interacted with ΔCSP through high resolution mass spectrometer1x10~7eXact-His ΔCSP stable-transfectedand control HepG2-CD81 cells were ruptured and purified by Profinity eXact Purification Resin(Bio-rad) and Ni-NTA Agarose(Qiagen). Then the eluent were analysed by high resolution mass spectrometer. We found that the expression level of autophagy related protein was closely associated with the down-regulation of HSPA8 by ΔCSP.In conclusion, we first reported that transfection and expression of recombination ΔCSP plasmid could limit the IFN-γ-induced killing through down-regulation of the expression level of autophagy protein. Further study indicated that ΔCSP may down-regulate the expression level of autophagy related protein through HSPA8. The liver stage is the first step of plasmodium infection in mammalian host, and blockage of liver stage infection could prevent the infection of malaria. Therefore, understanding the underlying mechanism of malaria parasite to escape or suppress the host innate immune responses will shed us with new light on the designation of prophylactic strategy against pre-erythrocytic stage.