IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.autophagy | LC3-associated phagocytosis | IFN-γ | malaria S everal hundred million cases of human malaria are reported annually, and nearly 600,000 people die from the disease each year (1). Of the five species that infect humans, Plasmodium vivax is not only the most geographically widespread, but also the most prevalent malarial parasite in areas outside Africa. As such, it has caused massive morbidity in these regions of the world. Although malaria caused by P. vivax was previously regarded as benign compared with that caused by Plasmodium falciparum, the recent alarming increase in both the severity and the drug resistance of P. vivax infection has raised concern (2).The widespread distribution of P. vivax has been attributed to the parasite's ability to remain dormant in the liver for years before reactivation (3). The molecular mechanism responsible for P. vivax dormancy is unknown, and knowledge of Plasmodiumhepatocyte interactions remains very limited. Nonetheless, because the number of liver-stage parasites is in the range of 100, whereas in the blood stage as many as 10 13 organisms may be found (4), intervention at the liver stage would seem to offer a better strategy for parasite elimination. A prerequisite to this route of malaria control and the development of novel therapies is a better understanding of liver-stage Plasmodium and its interactions with host hepatocytes. IFN-γ was previously shown to exhibit antimalarial activity against the liver stag...

BackgroundEradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.MethodsA novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.ResultsimHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.ConclusionsimHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2198-4) contains supplementary material, which is available to authorized users.

objective Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection.methods The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined.results The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. conclusions These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes. keywords dsRNA, malaria, oocyst development, yellow-g Sustainable Development Goals: Good Health and Wellbeing *Contributed equally.

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The modulation of gene expression levels of Anopheles dirus on Plasmodium vivax infection at the ookinete and oocyst stages was previously reported. In the present study, several upregulated An. dirus genes were selected based on their high expression levels and subcellular locations to examine their roles in P. vivax infection. Five An. dirus genes—carboxylesterase, cuticular protein RR‐2 family, far upstream element‐binding protein, kraken, and peptidase212—were knocked down by dsRNA feeding using dsRNA‐lacZ as a control. The dsRNA‐fed mosquitoes were later challenged by P. vivax–infected blood, and the oocyst numbers were determined. The expression of these five genes was examined in many organs of both male and female mosquitoes. The results showed that the decreased expression level of the far upstream element‐binding protein gene could lower the oocyst numbers, whereas the others showed no effect on P. vivax infection. The expression levels of these genes in ovaries were found, and in many organs, they were similar between male and female mosquitoes. The reduction of these five gene expressions did not affect the lifespan of the mosquitoes. In addition, the malaria box compound, MMV000634, demonstrated the lowest binding energy to the far upstream element‐binding protein using virtual screening. This protein might be a target to block malaria transmission.