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The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment.

Plasmodium parasites have extensive needs from their host hepatocytes during the obligate liver stage of infection, yet there remains sparse knowledge of specific host regulators. Here we assess 34 host-targeted kinase inhibitors for their capacity to eliminate Plasmodium yoelii -infected hepatocytes. Using pre-existing activity profiles of each inhibitor, we generate a predictive computational model that identifies host kinases, which facilitate Plasmodium yoelii liver stage infection. We predict 47 kinases, including novel and previously described kinases that impact infection. The impact of a subset of kinases is experimentally validated, including Receptor Tyrosine Kinases, members of the MAP Kinase cascade, and WEE1. Our approach also predicts host-targeted kinase inhibitors of infection, including compounds already used in humans. Three of these compounds, VX-680, Roscovitine and Sunitinib, each eliminate >85% of infection. Our approach is well-suited to uncover key host determinants of infection in difficult model systems, including field-isolated parasites and/or emerging pathogens. Host kinases facilitate Plasmodium liver stage (LS) infection, but systematic accounting of important players is lacking. Here, the authors use a computational approach and kinase activity profiles to identify host kinase regulators of LS infection and drugs that could eliminate parasite burden.

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.

New World hantaviruses cause severe infections in humans, with case fatality rates approaching 40%. Previous structural studies have advanced our understanding of hantavirus glycoprotein architecture and function, however, the lack of high-resolution in situ structures of the glycoprotein tetramer and its lattice organization has limited mechanistic insights into viral assembly, entry, and antigenicity. Here, we leveraged a virus-like particle (VLP) system to establish a cryo-electron microscopy workflow for lattice-forming viral glycoproteins. This enabled the determination of a 2.35 Å resolution structure of the membrane-embedded Andes virus (ANDV) glycoprotein tetramer, as well as structures of dimers of tetramers and a complex with antibody ADI-65534. These structures reveal previously uncharacterized features of glycoprotein organization, stability, and pH-sensing. Immunization of mice with self-amplifying replicon RNA (repRNA) encoding ANDV-VLPs elicited high levels of glycoprotein-binding antibodies but equivalent titers of neutralizing antibodies compared to repRNA-encoded native ANDV glycoprotein complex. Collectively, these findings advance our understanding of hantavirus glycoprotein assemblies and their function, laying a foundation for structure-based vaccine design efforts.

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosomal-associated membrane protein (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of the SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates new parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen, Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens.

The facets of host control during Plasmodium liver infection remain largely unknown and conventional innate regulatory pathways are only minimally effective at eliminating parasites. Ferroptosis, a recently described form of iron-dependent cell death that drives accumulation of reactive oxygen species and lipid peroxides, but has not yet been shown to function as an innate immune response. Inducing ferroptosis with pharmacologicals or by genetic perturbation of its negative regulators, GPX4 and SLC7a11, dramatically reduces survival of the Plasmodium Liver Stage. In contrast, knockdown or knockout of NOX1 or knockdown of TFR1, which are required for ferroptosis, increases the number of Liver Stage parasites. Moreover, we demonstrate that blocking ferroptosis renders parasite-infected hepatocytes resistant to P53 mediated hepatocyte death. Our work establishes that ferroptotic signaling serves to control Plasmodium infection in the liver and raises the possibility that ferroptosis operates as an axis of the innate immune system to defend against intracellular pathogens.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources. We show that host microtubules dynamically reorganize around the developing liver stage (LS) parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, ER/Golgi stress and lipid biogenesis that regulate Plasmodium LS development. These novel regulators of infection, including Centromere Protein J (CENPJ), led us to interrogate how microtubule organizing centers (MTOCs) are regulated during infection. Foci of γ-tubulin localized to the parasite periphery; depletion of CENPJ exacerbated this re-localization and increased infection. Further, we show that the Golgi acts as a non-centrosomal MTOC by organizing γ-tubulin and stimulating microtubule nucleation at the parasite periphery. Collectively, we show that the Plasmodium LS recruits the host Golgi to form MT mediated conduits along which host organelles are recruited to the PVM, to support liver stage development. Our findings suggest many host-targeted pharmacological inhibitors may inhibit LS infection.

Summary Prior to initiating symptomatic malaria, Plasmodium parasites infect and develop within hepatocytes. We performed a forward genetic, genome-wide CRISPR-Cas9 screen to identify host regulators of Plasmodium liver infection. Single guide RNAs targeting genes involved in vesicle trafficking, cytoskeleton organization and lipid biogenesis altered Plasmodium liver development. We observed a redistribution of Golgi-derived vesicles and fragmented Golgi stacks with the parasitophorous vacuolar membrane (PVM). The host microtubule network and non-centrosomal microtubule organizing centers (ncMTOC) also re-localized following infection, closely associating with the parasite. Knocking out the centrosomal MTOC protein CENPJ exasperated the re-localization of MTOCs to the parasite and increased infection, suggesting that the parasite relies on ncMTOC assembly. Thus, we have uncovered a mechanism by which parasites sequester host material for survival and development. Our data provide a wealth of yet untested hypotheses about the elusive biology of the liver stage parasite and serves as a foundation for future investigation. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://data.mendeley.com/datasets/75brhkh25r