The Effect Of Hepatocyte Autophagy On The Development Of Liver Stage And The Underlying Mechanism

Malaria is still one of the most devastating diseases worldwide. Approximately40%of the world’s population is at risk for malaria, and225million new cases with650,000deaths occur each year. Malaria begins with the bite of plasmodium-infected mosquitoes. After being injected into the host’s skin, sporozoites rapidly invade the liver and transform into the EEFs (exo-erythrocytic forms) in hepatocytes, then the matured schizonts are released from hepatocytes and invade the red blood cells, leading to the initiation of the blood-stage infection. Although the clinical symptoms of patients are resulted from the blood-stage infection, patients are always clinical silent at the pre-erythrocytic stage. Thus, understanding the mechanism interaction of host immune responses with the pre-erythrocytic stage could provide us with clues to design prophylactic strategies.Autophagy is a bulk degradation system that delivers cytoplasmic constituents and organelles into lysosomes for hydrolysis, and it is essential for cell survival, development and homeostasis. Interestingly, host cell autophagy also restricts a variety of viral infections[4]and the replication of intracellular bacteria and protozoa. However, emerging evidence also showed some viruses could utlize the components of autophagic pathway to facilitate their own replication. Although trigger of macrophagy autophagy was reported to restrict the development of amplexan parasiteToxplasma gondii, the autophagy of nonprofessional phagotocytes could foster the growth of Trypanosomatids brucei, Trypanosomatides cruzi and Toxplasma gondii possibly through providing nutrients. However, it is still largely unknown whether sporozoite infection could induce hepatocyte autophagy, if so, what’s its role in the development of pre-erythrocytic stage.In this study, we firstly investigated whether sporozoite infection could induce the host cell autophagy, and the effect of autophagy on the development of liver stage and the underlying mechanisms.1. Rapamycin promoted the hepatocyte autophagy induced by sporozoites and the development of liver stage.1.1Sporozoite infection induces the hepatocyte autophagy of EEFsTo investigate whether sporozoite infection could induce the autophagy of the parasites by hepatocyte, the autophagy-specfic marker LC3(microtubule-associated protein1light chain3) surrounding the parasite was detected after P.y BY265-RFPsporozoites were incubated with Hepal-6vitro for6,14and24h, respectively. As a result, we found autophagy of EEFs could be detected as early as6h post incubation of sporozoites with Hepal-6, and20-30%of autophgy EEFs were found at24h post incubation of sporozoite with Hepal-6. This data suggested that sporozoite infection could induce the host autophagy of liver stage.1.2Rapamycin promotes the host cell autophagy induced by sporozoite infectionRapamycin, the pharmacological inhibtor of mTOR (mammalian target of rapamycin), is always used to induce the autophagy. To investigate whether it could enchance the autophagy of EEFs by hepatocyte, rapamycin was added at3h post incubation of sporozoites with Hepal-6. As a result, the rate of autophagic EEFs was increased from20-30%to60-70%at24h after incubation with rapamycin alone or along with Cq. The autophagic EEFs induced by rapamycin with Cq were significantly reduced when the autophagy inhibitor3-MA was added. Interestingly,3-MA could also reduce the host autophagy induced by rapamycin, as well as sporozoite infection. Thus, our data strongly suggested that the autophagy of EEFs could be greatly enhanced by the induction of rapamycin.1.3Host autophagy induces the fusion of autophagic EEFs with lysosomeTo test whether induction of host autophagy could also induce the fusion of EEFs with lysosome, lysosome marker LAMP1around the parasite were observed when cells were treated with rapamycin alone or along with Cq. Although relative low level (30%) of the fusion of EEFs with lysosome at24h post the incubation of sporozoites with hepatocytes, treatment of rapamycin remarkably increase the number of EEFs fused with lysosome. Without rapamycin treatment, about20%autophagic EEFs were fused with lysosome, as indicated as the merge of LC3and LAMP1arround the EEFs. In contrast, autophagic EEFs fusing with lysosome were increased to70%when treated either with rapamycin alone or along with Cq, but3-MA could reduce the rate of the fusion of autophagic EEFs with lysosome to baseline. Therefore, our data strongly supported that induction of autophagy rapamycin could significantly promote the fusion of parasitophorous vaculoes with lysosome.1.4Host cell autophagy promotes the development of EEF in vitroTo investigate whether autophagy induced by rapamycin could also suppress the development of pre-erythrocytic stage, the amount and growth of EEF in hepatocyte treated with or without rapamycin were dected by immunofluorescence microscopy of P.y BY265-RFP and real-time PCR of P.y BY265-specific18srRNA gene at42h post incubation, respectively. As a result, both the number of EEF and the amount of P.y BY265-specific18srRNA gene were much higher than those of control, and the co-adminstration of3-MA could reduce the number of EEF and the amount of18srRNA gene to baseline. This data suggested the induction of hepatocyte autophagy could promote the development of liver stage.2.The mechanism of autophagy promote EEFs development in liver stage.2.1EEFs normally survive and replicate in autophagosomeTo answer why induction of autophagy could promote the development of exo-erythrocytic stage, we fistly investigated whether EEFs could survive in the autophagsome. After sporozoites were incubated with Hepal-6for3h and then treated with or without rapamycin for24h, malaria parasite was stained with LC3, along with either DAPI or Edu that is incorpated into replicating DNA. DAPI staining showed the nucleis of EEFs, either in encapsulated by LC3or not, were divided, indicating the survival and replication of EEFs in autolysosome. This was further confirmed by Edu staining, which showed Edu was incorpated into hepatocyt nuclei, as well as EEFs nuclei in LC3-positive autophagosome. Thus, the data suggested that malaria parasite could survive and replicate in the autophagosome. 2.2. EEFs inhibited the acidification of autolysomeIt is strange to find that EEFs could still survive in the autolysosome, as most microorganisms were reported to be degraded in the acidic lysosome. Therefore, we next investigated the acidification of autolysosome containing EEFs by staining both early lysosome marker LAMP1and lysosome acidification marker Cathe D, at24h after incubation of sporozoite with Hep1-6treated with or without rapamycin. As a result, most autolysosome containing parasite was LC3positive, but no one was found to be stained with Cath D, indicating that malaria parasite could inhibit the acidificaition of autolysosome. This data also strongly suggested that survival and repliaction of EEF might be closely associated with its ability to inhibit the acidificaition of autolysosome.Therefore, we firstly provide evidence that sporozoite infection could induce host cell autophagy of malaria parasite, and the induction of hepatocyte autophagy promoted the development of pre-erythrocytic stage, possibly through suppressing acidification of autolysosome. This data indicated the induction of autophagy as a novel escape strategy of exo-erythrocytic stage, and shed new light on the prophylatic therapy against liver stage.