Recent years have witnessed a great gain in knowledge regarding parasite–host cell interactions during Plasmodium liver stage development. It is now an accepted fact that a large percentage of sporozoites invading hepatocytes fail to form infectious merozoites. There appears to be a delicate balance between parasite survival and elimination and we now start to understand why this is so. Plasmodium liver stage parasites replicate within the parasitophorous vacuole (PV), formed during invasion by invagination of the host cell plasma membrane. The main interface between the parasite and hepatocyte is the parasitophorous vacuole membrane (PVM) that surrounds the PV. Recently, it was shown that autophagy marker proteins decorate the PVM of Plasmodium liver stage parasites and eliminate a proportion of them by an autophagy-like mechanism. Successfully developing Plasmodium berghei parasites are initially also labeled but in the course of development, they are able to control this host defense mechanism by shedding PVM material into the tubovesicular network (TVN), an extension of the PVM that releases vesicles into the host cell cytoplasm. Better understanding of the molecular events at the PVM/TVN during parasite elimination could be the basis of new antimalarial measures.

SummaryEukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei- infected ATG5−/− host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway. | INTRODUCTIONMalaria, with more than 200 million estimated cases and more than 400 thousand deaths per year, remains one of the most devastating diseases worldwide (World Health Organization, 2015). Once Plasmodium sporozoites are injected by an infected female Anopheles mosquito during blood feeding, they migrate to the liver and infect hepatocytes by the formation of a parasitophorous vacuole (PV).Although the PV membrane (PVM) originates from invagination of the host cell plasma membrane, it is remodelled by the parasite upon infection (Spielmann, Montagna, Hecht, & Matuschewski, 2012).Inside the vacuole, the parasite starts its exoerythrocytic development by transforming into a multinuclear schizont that undergoes massive replication. Finally, several thousand erythrocyte-infective merozoites are formed and safely released into the blood within merosomes (Sturm et al., 2006). Within the hepatocyte, the Plasmodium parasite shows one of the fastest replication rates among eukaryotes, necessitating that massive amounts of nutrients are obtained from the host cell (Bano, Romano, Jayabalasingham, & Coppens, 2007).At the same time, the parasite needs to escape defence mechanisms of the host cell. homeostasis, cells can use selective autophagy to remove damaged or deleterious elements from the cytoplasm. Selective autophagy can also serve as a cellular antimicrobial defence against intracellular pathogens, a process called xenophagy (Deretic & Levine, 2009;Knodler & Celli, 2011;Levine, Mizushima, & Virgin, 2011;Mostowy, 2013). Starvation-induced and selective autophagy are both characterised by the formation of double membrane autophagosomes that sequester the autophagic cargo (Levine et al., 2011;Mizushima & Komatsu, 2011). In mammalian cells, starvation initiates autophag...

The hepatic stage of the malaria parasite Plasmodium is accompanied by an autophagy-mediated host response directly targeting the parasitophorous vacuolar membrane (PVM) harbouring the parasite. Removal of the PVM-associated autophagic proteins such as ubiquitin, p62, and LC3 correlates with parasite survival. Yet, it is unclear how Plasmodium avoids the deleterious effects of selective autophagy. Here we show that parasites trap host autophagic factors in the tubovesicular network (TVN), an expansion of the PVM into the host cytoplasm. In proliferating parasites, PVM-associated LC3 becomes immediately redirected into the TVN, where it accumulates distally from the parasite’s replicative centre. Finally, the host factors are shed as vesicles into the host cytoplasm. This strategy may enable the parasite to balance the benefits of the enhanced host catabolic activity with the risk of being eliminated by the cell’s cytosolic immune defence.

Liver stage Plasmodium parasites reside in a parasitophorous vacuole (PV) that associates with lysosomes. It has previously been shown that these organelles can have beneficial as well as harmful effects on the parasite. Yet it is not clear how the association of lysosomes with the parasite is controlled and how interactions with these organelles lead to the antagonistic outcomes. In this study we used advanced imaging techniques to characterize lysosomal interactions with the PV. In host cells harboring successfully developing parasites we observed that these interaction events reach an equilibrium at the PV membrane (PVM). In a population of arrested parasites, this equilibrium appeared to shift towards a strongly increased lysosomal fusion with the PVM witnessed by strong PVM labeling with the lysosomal marker protein LAMP1. This was followed by acidification of the PV and elimination of the parasite. To systematically investigate elimination of arrested parasites, we generated transgenic parasites that express the photosensitizer KillerRed, which leads to parasite killing after activation. Our work provides insights in cellular details of intracellular killing and lysosomal elimination of Plasmodium parasites independent of cells of the immune system.

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.