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Model for the action of Plasmodium berghei EXP2 Plasmodium berghei sporozoites, after reaching the liver, traverse hepatocytes before invading the final one inside which they will replicate. Once they encounter the definitive hepatocyte, and stimulated for ~1 hour by the host cell milieu conditions (increase in temperature and presence of mammalian serum components), sporozoites secrete the pore-forming protein EXP2. a EXP2 might create pores at the membrane of hepatocytes, allowing the influx of calcium, leading to the activity of host aSMase that facilitates the invasion of the hepatocyte by the sporozoite. b Alternatively, EXP2 might bind to the sporozoite membrane, inducing a calcium influx in the sporozoite, which triggers the release of rhoptry and other proteins, allowing for the sporozoite to invade the hepatocyte. c Another possibility is that EXP2 is discharged by the sporozoite during vacuole formation, similarly to what happens during the blood stage, being critical for the survival of the sporozoite during the initial stages of hepatocyte infection.
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Plasmodium parasites possess a translocon that exports parasite proteins into the infected erythrocyte. Although the translocon components are also expressed during the mosquito and liver stage of infection, their function remains unexplored. Here, using a combination of genetic and chemical assays, we show that the translocon component Exported Pr...
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Citations
... Furthermore, pore-forming proteins are crucial in disease infections. For example, during the plasmodium parasite infection of the liver, exported protein 2 secreted by sporozoites is a critical factor in the invasion of hepatocytes [100]. ...
The pathogenesis of various diseases often involves an intricate interplay between membrane proteins and membrane curvature. Understanding the underlying mechanisms of this interaction could offer novel perspectives on disease treatment. In this review, we provide an introduction to membrane curvature and its association with membrane proteins. Furthermore, we delve into the impact and potential implications of this interaction in the context of disease treatment. Lastly, we discuss the prospects and challenges associated with harnessing these interactions for effective disease management, aiming to provide fresh insights into therapeutic strategies.
... EXP2 has recently been reported to facilitate hepatocyte invasion by P. berghei sporozoites 40 . To further investigate the function ofPTEX150 and EXP2 in sporozoites, we measured the rates of host cell traversal and invasion using our mutants. ...
... Disruption of ptex150 and exp2 also did not affect sporozoite infectivity for hepatocytes under our conditions, in agreement with these proteins having no role in erythrocyte invasion by merozoites. This result contrasts with a previous study showing that P. berghei EXP2-knockdown sporozoites generated via FlpL/FRT-mediated excision of the 3'UTR of exp2 in mosquitoes were inhibited for invasion of hepatocytes 40 . In merozoites, PTEX members including EXP2 are localized in DGs 15 . ...
Plasmodium falciparum assembles a protein translocon (PTEX) at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported. The preceding Plasmodium liver stage develops in hepatocytes within a PVM; however, the importance of PTEX and identification of exported proteins in P. falciparum liver stages remains unexplored. Here, we apply the FlpL/ FRT system to P. falciparum NF54 to conditionally excise genes in sporozoites, enabling studies at the liver stage. Conditional disruption of PTEX components PTEX150 and EXP2 in sporozoites does not affect their development or infectivity but attenuates liver stage growth. While PTEX150-deficiency significantly reduces liver load in humanized mice, EXP2-deficiency conferred a severe fitness cost, demonstrating that PTEX is essential for P. falciparum liver stage development. We show that liver specific protein 2 (LISP2) and circumsporozoite protein (CSP) contain putative PEXEL sequences cleaved by plasmepsin V, yet they localize to the PVM of infected hepatocytes. The abundance of LISP2 is reduced in PTEX-deficient liver stages, suggesting this protein is degraded in the absence of a functioning PTEX complex. This study employs the FlpL/ FRT system for functional analysis of P. falciparum pre-erythrocytic biology, revealing that the protein export translocon required for growth in erythrocytes is essential for P. falciparum development in hepatocytes and normal LISP2 expression. It also describes two P. falciparum proteins that contain putative PEXEL motifs that are targeted to the PVM.
... Furthermore, blood-stage exported proteins and reporters are not translocated beyond the liver-stage PVM, even when HSP101 is ectopically expressed at this stage, suggesting a distinct export mechanism (15,(18)(19)(20)(21). Inactivation of exp2 expression in sporozoites using the FLP/FRT stage-specific conditional knockdown system in Plasmodium berghei demonstrated a critical role for EXP2 in the transition from the mosquito host to the vertebrate blood stage (15). However, subsequent work surprisingly reported this to be the result of an invasion defect in EXP2deficient sporozoites which could be rescued by exogenous supplementation with recombinant EXP2 or bacterial pore-forming toxins, implicating extracellular secretion of EXP2 in hepatocyte invasion through host membrane wounding (22). ...
... While nutrient transport and protein export across the intraerythrocytic PVM both depend on the PTEX membrane pore EXP2 (10,14), the intrahepatic function of EXP2/ PTEX has remained unclear. A recent study unexpectedly reported that EXP2 is secreted by extracellular sporozoites and is important for hepatocyte invasion to initiate the liver stage (22). As EXP2 knockdown in the blood stage was not observed to impact erythrocyte invasion (10,14), this additional EXP2 function is apparently unique to hepatocyte entry. ...
During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.
... A comprehensive understanding of the key processes required for successful Pf invasion and maturation within the hepatocyte is lacking, including the molecular mechanisms that define host specificity (Mota et al., 2001;Mello-Vieira et al., 2020). Although key host receptors such as cluster of differentiation 81 (CD81) (Silvie et al., 2003), class B scavenger receptor type 1 (SR-B1) (Rodrigues et al., 2008), Ephrin A2 (Kaushansky et al., 2015), and highly sulfated heparin sulfate proteoglycans (HSPGs) have been identified as playing a role, their reciprocal interacting partners in the parasite remain largely unknown. ...
... Mello-Vieira et al. (2020) show that EXP2 is required for liver invasion and maturation and for establishing blood stage infection in a mouse model, which corroborates with our findings of its importance in maturation (Mello-Vieira et al., 2020). Furthermore, the EXP1 C-terminal region exposed to the host cytoplasm interacts with host Apolipoprotein A (ApoH) which is pivotal for successful liver-stage development in Pb, although it is unclear whether it performs the same role in Pf (Saé t al., 2017;Meseń-Ramıŕez et al., 2019;Wolanin et al., 2019). ...
During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development.
... This work showed that the poreforming activity of the parasite's exported protein 2 (EXP2) triggers a response from the hepatic host cell, which leads to the production of acid sphingomyelinase. This enzyme then plays a critical role in the repair of the host cell membrane, which is key for sporozoite invasion and establishment in hepatocytes (Mello-Vieira et al., 2020). This finding is a perfect illustration of Maria's ability to push the boundaries of knowledge beyond the status quo, by unveiling an active role for the hepatic host cell in a process long thought to depend solely on the parasite. ...
... 5 Furthermore, while the existing therapies exclusively target the blood stage of infection, limited efforts have been made to target the liver and transmission stages of the parasite. 6 Conventional antimalarial therapeutics were primarily developed and implemented based on their in vitro activity without extensive knowledge of their molecular mechanism. 2 Drug-resistant mutations arise in sexual stages of the parasite's life cycle where they are diploid and enter the asexual stages. ...
... Functional analysis of EXP2 in the liver-stage by a stage-specific conditional knockdown revealed EXP2 is important for transition from the sporozoite to the blood-stage, but it is unclear if this involves a function in protein export and/or small molecule transport within the hepatocyte [152]. Surprisingly, this stage transition defect was recently tied to an unexpected role for EXP2 in hepatocyte invasion [155]. In this study, extracellular sporozoites were shown to secrete EXP2 into the supernantant upon serum stimulation, and reexamination of the same conditional EXP2 knockdown parasites revealed a specific impact on invasion but not intracellular development. ...
... The apparent nonessential role of EXP2 during intracellular liver-stage development [155] is suprising given that even if liver-stage protein export relies on a distinct translocon, EXP2 might still be expected to serve an important role in small molecule transport across the PVM. This would parallel the situation in Toxoplasma, where EXP2 orthologs GRA17 and GRA23 (the only PTEX-related components present; several additional HSP100s are encoded but are not found in the PV [156,157]) are only important for small molecule transport [59], while a distinct set of proteins is required for effector translocation into the host cell. ...
Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.
Productive invasion of hepatocytes by Plasmodium sporozoites is a key step of infection. The parasites traverse hepatocytes before targeting one of them to form a parasitophorous vacuole for parasite expansion. Schepis et al. show the induction of membrane ruffling via host Rho GTPases by Plasmodium sporozoites facilitating productive invasion.
Plasmodium sporozoites are the motile forms of the malaria parasites that infect hepatocytes. The initial invasion of hepatocytes is thought to be actively driven by sporozoites, but host cell processes might also play a role. Sporozoite invasion triggers a host plasma membrane invagination that forms a vacuole around the intracellular parasite, which is critical for subsequent intracellular parasite replication. Using fast live confocal microscopy, we observed that the initial interactions between sporozoites and hepatocytes induce plasma membrane ruffles and filopodia extensions. Importantly, we find that these host cell processes facilitate invasion and that Rho GTPase signaling, which regulates membrane ruffling and filopodia extension, is critical for productive infection. Interestingly, sporozoite cell traversal stimulates these processes, suggesting that it increases hepatocyte susceptibility to productive infection. Our study identifies host cell signaling events involved in plasma membrane dynamics as a critical host component of successful malaria parasite infection of hepatocytes.
During the vertebrate stage of the Plasmodium life cycle, obligate intracellular malaria parasites establish a vacuolar niche for replication, first within host hepatocytes at the pre-patent liver-stage and subsequently in erythrocytes during the pathogenic blood-stage. Survival in this protective microenvironment requires diverse transport mechanisms that enable the parasite to transcend the vacuolar barrier. Effector proteins exported out of the vacuole modify the erythrocyte membrane, increasing access to serum nutrients which then cross the vacuole membrane through a nutrient-permeable channel, supporting rapid parasite growth. This review highlights the most recent insights into the organization of the parasite vacuole to facilitate the solute, lipid and effector protein trafficking that establishes a nutrition pipeline in the terminally differentiated, organelle-free red blood cell.
