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Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC 50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

The emergence of resistance against current antimalarial treatments has necessitated the need for the development of novel antimalarial chemotypes. Toward this goal, we recently optimised the antimalarial activity of the dihydroquinazolinone scaffold and showed it targeted PfATP4. Here, we deconstruct the lactam moiety of the tricyclic dihydroquinazolinone scaffold and investigate the structure-activity relationship of the truncated scaffold. It was shown that SAR between scaffolds was largely transferrable and generated analogues with potent asexual stage activity. Evaluation of the truncated analogues against PfATP4 mutant drug-resistant parasite strains and in assays measuring PfATP4-associated ATPase activity demonstrated retention of PfATP4 as the molecular target. Analogues exhibited activity against both male and female gametes and multidrug resistant parasites. Limited efficacy of analogues in a P. berghei asexual stage mouse model was attributed to their moderate metabolic stability and low aqueous stability. Further development is required to address these attributes toward the potential use of the dihydroquinazolinone class in a curative and transmission blocking combination antimalarial therapy., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S -WJM992, were shown to inhibit PfATP4-associated Na + -ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S -WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action., (© 2024. The Author(s).)

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum , is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817 -resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817 , and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial., Competing Interests: The authors declare no conflict of interest.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Brad Sleebs reports financial support was provided by National Health and Medical Research Council. Susan Charman reports financial support was provided by National Health and Medical Research Council. Paul Gilson reports financial support was provided by National Health and Medical Research Council. Alan Cowman reports financial support was provided by National Health and Medical Research Council. William Nguyen reports financial support was provided by National Health and Medical Research Council. Tania de Koning Ward reports financial support was provided by National Health and Medical Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pitman, Counihan, Modak, Chowdury, Gilson, Webb and de Koning-Ward.)

Cerebral malaria (CM) is a severe neurological complication caused by Plasmodium falciparum parasites; it is characterized by the sequestration of infected red blood cells within the cerebral microvasculature. New findings, combined with a better understanding of the central nervous system (CNS) barriers, have provided greater insight into the players and events involved in CM, including site-specific T cell responses in the human brain. Here, we review the updated roles of innate and adaptive immune responses in CM, with a focus on the role of the perivascular macrophage-endothelium unit in antigen presentation, in the vascular and perivascular compartments. We suggest that these events may be pivotal in the development of CM., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P 1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P 2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

Importance: The malaria parasite Plasmodium falciparum relies on variant expression of members of multi-gene families as a strategy for environmental adaptation to promote parasite survival and pathogenesis. These genes are located in transcriptionally silenced DNA regions. A limited number of these genes escape gene silencing, and switching between them confers variant fitness on parasite progeny. Here, we show that PfSir2 histone deacetylases antagonize DNA-interacting acetylated alternative histones at the boundaries between active and silent DNA. This finding implicates acetylated alternative histones in the mechanism regulating P. falciparum variant gene silencing and thus malaria pathogenesis. This work also revealed that acetylation of alternative histones at promoters is dynamically associated with promoter activity across the genome, implicating acetylation of alternative histones in gene regulation genome wide. Understanding mechanisms of gene regulation in P. falciparum may aid in the development of new therapeutic strategies for malaria, which killed 619,000 people in 2021., Competing Interests: The authors declare no conflict of interest.

The Cytokine-inducible Src homology 2 domain-containing (CISH) protein is a negative feedback regulator induced by cytokines that play key roles in immunity and erythropoiesis. Single nucleotide polymorphisms (SNPs) in the human CISH gene have been associated with increased susceptibility to severe malaria disease. To directly assess how CISH might influence outcomes in the BALB/c model of malaria anemia, CISH knockout ( Cish -/- ) mice on this background were infected with Plasmodium berghei and their hematopoietic responses, cytokine production and ability to succumb to severe malaria disease evaluated. Despite basal erythrocytic disruption, upon P. berghei infection, the Cish -/- mice were better able to maintain peripheral blood cell counts, hemoglobin levels and a steady-state pattern of erythroid differentiation compared to wild-type ( Cish +/+ ) mice. Ablation of CISH, however, did not influence the outcome of acute malaria infections in either the BALB/c model or the alternative C57BL/6 model of experimental cerebral malaria, with the kinetics of infection, parasite load, weight loss and cytokine responses being similar between Cish +/+ and Cish -/- mice, and both genotypes succumbed to experimental cerebral malaria within a comparable timeframe., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Lakkavaram, Maymand, Naser, Ward and de Koning-Ward.)

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dans et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cerebral malaria (CM) is the most severe form of malaria with the highest mortality rate and can result in life-long neurological deficits and ongoing comorbidities. Factors contributing to severity of infection and development of CM are not fully elucidated. Recent studies have indicated a key role of the gut microbiome in a range of health conditions that affect the brain, but limited microbiome research has been conducted in the context of malaria. To address this knowledge gap, the impact of CM on the gut microbiome was investigated in mice. C57BL/6J mice were infected with Plasmodium berghei ANKA (PbA) parasites and compared to non-infected controls. Microbial DNA from faecal pellets collected daily for 6-days post-infection were extracted, and microbiome comparisons conducted using 16S rRNA profiling. We identified significant differences in the composition of bacterial communities between the infected and the non-infected groups, including a higher abundance of the genera Akkermansia, Alistipes and Alloprevotella in PbA-infected mice. Furthermore, intestinal samples were collected post-cull for morphological analysis. We determined that the caecal weight was significantly lower, and the small intestine was significantly longer in PbA-infected mice than in the non-infected controls. We concluded that changes in microbial community composition were primarily driven by the infection protocol and, to a lesser extent, by the time of infection. Our findings pave the way for a new area of research and novel intervention strategies to modulate the severity of cerebral malaria disease., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Most eukaryotic proteins undergo post-translational modifications (PTMs) that significantly alter protein properties, regulate diverse cellular processes and increase proteome complexity. Among these PTMs, lipidation plays a unique and key role in subcellular trafficking, signalling and membrane association of proteins through altering substrate function, and hydrophobicity via the addition and removal of lipid groups. Three prevalent classes of lipid modifications in Plasmodium parasites include prenylation, myristoylation, and palmitoylation that are important for regulating parasite-specific molecular processes. The enzymes that catalyse these lipid attachments have also been explored as potential drug targets for antimalarial development. In this review, we discuss these lipidation processes in Plasmodium spp. and the methodologies that have been used to identify these modifications in the deadliest species of malaria parasite, Plasmodium falciparum. We also discuss the development status of inhibitors that block these pathways., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase Pf A-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of Pf A-M17 to show that Pf A-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of Pf A-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate Pf A-M17 as a potential novel drug target., Competing Interests: RE, GS, KH, TM, NV, CW, CH, CM, HC, WS, NC, DC, PS, SM, Td No competing interests declared, (© 2022, Edgar et al.)

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell's physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite's ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite's ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite's ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101's specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte., Competing Interests: The authors have declared that no competing interests exist.

Plasmodium falciparum malaria remains a global health problem as parasites continue to develop resistance to all antimalarials in use. Infection causes clinical symptoms during the intra-erythrocytic stage of the lifecycle where the parasite infects and replicates within red blood cells (RBC). During this stage, P. falciparum digests the main constituent of the RBC, hemoglobin, in a specialized acidic compartment termed the digestive vacuole (DV), a process essential for survival. Many therapeutics in use target one or multiple aspects of the DV, with chloroquine and its derivatives, as well as artemisinin, having mechanisms of action within this organelle. In order to better understand how current therapeutics and those under development target DV processes, techniques used to investigate the DV are paramount. This review outlines the involvement of the DV in therapeutics currently in use and focuses on the range of techniques that are currently utilized to study this organelle including microscopy, biochemical analysis, genetic approaches and metabolomic studies. Importantly, continued development and application of these techniques will aid in our understanding of the DV and in the development of new therapeutics or therapeutic partners for the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Edgar, Counihan, McGowan and de Koning-Ward.)

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane., (© 2021 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Plasmodium parasites responsible for the disease malaria reside within erythrocytes. Inside this niche host cell, parasites internalize and digest host hemoglobin to source amino acids required for protein production. However, hemoglobin does not contain isoleucine, an amino acid essential for Plasmodium growth, and the parasite cannot synthesize it de novo . The parasite is also more metabolically active than its host cell, and the rate at which some nutrients are consumed exceeds the rate at which they can be taken up by erythrocyte transporters. To overcome these constraints, Plasmodium parasites increase the permeability of the erythrocyte membrane to isoleucine and other low-molecular-weight solutes it requires for growth by forming new permeation pathways (NPPs). In addition to the erythrocyte membrane, host nutrients also need to cross the encasing parasitophorous vacuole membrane (PVM) and the parasite plasma membrane to access the parasite. This review outlines recent advances that have been made in identifying the molecular constituents of the NPPs, the PVM nutrient channel, and the endocytic apparatus that transports host hemoglobin and identifies key knowledge gaps that remain. Importantly, blocking the ability of Plasmodium to source essential nutrients is lethal to the parasite, and thus, components of these key pathways represent potential antimalaria drug targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Counihan, Modak and de Koning-Ward.)

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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 Protein 2 (EXP2) is critical for invasion of hepatocytes. EXP2 is a pore-forming protein that is secreted from the sporozoite upon contact with the host cell milieu. EXP2-deficient sporozoites are impaired in invasion, which can be rescued by the exogenous administration of recombinant EXP2 and alpha-hemolysin (an S. aureus pore-forming protein), as well as by acid sphingomyelinase. The latter, together with the negative impact of chemical and genetic inhibition of acid sphingomyelinase on invasion, reveals that EXP2 pore-forming activity induces hepatocyte membrane repair, which plays a key role in parasite invasion. Overall, our findings establish a novel and critical function for EXP2 that leads to an active participation of the host cell in Plasmodium sporozoite invasion, challenging the current view of the establishment of liver stage infection.

Severe malaria anemia is one of the most common causes of morbidity and mortality arising from infection with Plasmodium falciparum . The pathogenesis of malarial anemia is complex, involving both parasite and host factors. As mouse models of malaria also develop anemia, they can provide a useful resource to study the impact of Plasmodium infections and the resulting host innate immune response on erythropoiesis. In this study, we have characterized the bone marrow and splenic responses of the erythroid as well as other hematopoietic lineages after an acute infection of Balb/c mice with Plasmodium berghei. Such characterization of the hematopoietic changes is critical to underpin future studies, using knockout mice and transgenic parasites, to tease out the interplay between host genes and parasite modulators implicated in susceptibility to malaria anemia. P. berghei infection led to a clear perturbation of steady-state erythropoiesis, with the most profound defects in polychromatic and orthochromatic erythroblasts as well as erythroid colony- and burst-forming units (CFU-E and BFU-E), resulting in an inability to compensate for anemia. The perturbation in erythropoiesis was not attributable to parasites infecting erythroblasts and affecting differentiation, nor to insufficient erythropoietin (EPO) production or impaired activation of the Signal transducer and activator of transcription 5 (STAT5) downstream of the EPO receptor, indicating EPO-signaling remained functional in anemia. Instead, the results point to acute anemia in P. berghei -infected mice arising from increased myeloid cell production in order to clear the infection, and the concomitant release of pro-inflammatory cytokines and chemokines from myeloid cells that inhibit erythroid development, in a manner that resembles the pathophysiology of anemia of chronic disease., (Copyright © 2020 Lakkavaram, Lundie, Do, Ward and de Koning-Ward.)

With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na + ) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na + dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na + inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na + into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na + within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

Plasmodium parasites must export proteins into their erythrocytic host to survive. Exported proteins must cross the parasite plasma membrane (PPM) and the parasitophorous vacuolar membrane (PVM) encasing the parasite to access the host cell. Crossing the PVM requires protein unfolding and passage through a translocon, the Plasmodium translocon of exported proteins (PTEX). In this study, we provide the first direct evidence that heat shock protein 101 (HSP101), a core component of PTEX, unfolds proteins for translocation across the PVM by creating transgenic Plasmodium parasites in which the unfoldase and translocation functions of HSP101 have become uncoupled. Strikingly, while these parasites could export native proteins, they were unable to translocate soluble, tightly folded reporter proteins bearing the Plasmodium export element (PEXEL) across the PVM into host erythrocytes under the same conditions. In contrast, an identical PEXEL reporter protein but harboring a transmembrane domain could be exported, suggesting that a prior unfolding step occurs at the PPM. Together, these results demonstrate that the export of parasite proteins is dependent on how these proteins are presented to the secretory pathway before they reach PTEX as well as their folded status. Accordingly, only tightly folded soluble proteins secreted into the vacuolar space and not proteins containing transmembrane domains or the majority of erythrocyte-stage exported proteins have an absolute requirement for the full unfoldase activity of HSP101 to be exported. IMPORTANCE The Plasmodium parasites that cause malaria export hundreds of proteins into their host red blood cell (RBC). These exported proteins drastically alter the structural and functional properties of the RBC and play critical roles in parasite virulence and survival. To access the RBC cytoplasm, parasite proteins must pass through the Plasmodium translocon of exported proteins (PTEX) located at the membrane interfacing the parasite and host cell. Our data provide evidence that HSP101, a component of PTEX, serves to unfold protein cargo requiring translocation. We also reveal that addition of a transmembrane domain to soluble cargo influences its ability to be translocated by parasites in which the HSP101 motor and unfolding activities have become uncoupled. Therefore, we propose that proteins with transmembrane domains use an alternative unfolding pathway prior to PTEX to facilitate export., (Copyright © 2019 Matthews et al.)

Plasmodium spp. parasites that cause malaria disease remain a significant global-health burden. With the spread of parasites resistant to artemisinin combination therapies in Southeast Asia, there is a growing need to develop new antimalarials with novel targets. Invasion of the red blood cell by Plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. Red blood cell invasion requires a co-ordinated series of protein/protein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. As these steps occur in the bloodstream, they are directly susceptible and exposed to drugs. A number of invasion inhibitors against a diverse range of parasite proteins involved in these different processes of invasion have been identified, with several showing potential to be optimised for improved drug-like properties. In this review, we discuss red blood cell invasion as a drug target and highlight a number of approaches for developing antimalarials with invasion inhibitory activity to use in future combination therapies., (© FEMS 2019.)

Plasmodium parasites that cause the disease malaria have developed an elaborate trafficking pathway to facilitate the export of hundreds of effector proteins into their host cell, the erythrocyte. In this review, we outline how certain effector proteins contribute to parasite survival, virulence, and immune evasion. We also highlight how parasite proteins destined for export are recognised at the endoplasmic reticulum to facilitate entry into the export pathway and how the effector proteins are able to transverse the bounding parasitophorous vaculoar membrane via the Plasmodium translocon of exported proteins to gain access to the host cell. Some of the gaps in our understanding of the export pathway are also presented. Finally, we examine the degree of conservation of some of the key components of the Plasmodium export pathway in closely related apicomplexan parasites, which may provide insight into how the diverse apicomplexan parasites have adapted to survival pressures encountered within their respective host cells., (© 2019 John Wiley & Sons Ltd.)

The inner membrane complex and the apical secretory organelles are defining features of apicomplexan parasites. Despite their critical roles, the mechanisms behind the biogenesis of these structures in the malaria parasite Plasmodium falciparum are still poorly defined. We here show that decreasing expression of the P. falciparum homologue of the conserved endolysomal escorter Sortilin-VPS10 prevents the formation of the inner membrane complex and abrogates the generation of new merozoites. Moreover, protein trafficking to the rhoptries, the micronemes, and the dense granules is disrupted, which leads to the accumulation of apical complex proteins in the endoplasmic reticulum and the parasitophorous vacuole. We further show that protein export to the erythrocyte and transport through the constitutive secretory pathway are functional. Taken together, our results suggest that the malaria parasite P. falciparum Sortilin has potentially broader functions than most of its other eukaryotic counterparts., (© 2018 John Wiley & Sons Ltd.)

The pathogenic nature of malaria infections is due in part to the export of hundreds of effector proteins that actively remodel the host erythrocyte. The Plasmodium translocon of exported proteins (PTEX) has been shown to facilitate the trafficking of proteins into the host cell, a process that is essential for the survival of the parasite. The role of the auxiliary PTEX component PTEX88 remains unclear, as previous attempts to elucidate its function through reverse genetic approaches showed that in contrast to the core components PTEX150 and HSP101, knockdown of PTEX88 did not give rise to an export phenotype. Here, we have used biochemical approaches to understand how PTEX88 assembles within the translocation machinery. Proteomic analysis of the PTEX88 interactome showed that PTEX88 interacts closely with HSP101 but has a weaker affinity with the other core constituents of PTEX. PTEX88 was also found to associate with other PV-resident proteins, including chaperones and members of the exported protein-interacting complex that interacts with the major virulence factor PfEMP1, the latter contributing to cytoadherence and parasite virulence. Despite being expressed for the duration of the blood-stage life cycle, PTEX88 was only discretely observed at the parasitophorous vacuole membrane during ring stages and could not always be detected in the major high molecular weight complex that contains the other core components of PTEX, suggesting that its interaction with the PTEX complex may be dynamic. Together, these data have enabled the generation of an updated model of PTEX that now includes how PTEX88 assembles within the complex., (© 2018 Federation of European Biochemical Societies.)

The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects.

We describe an MHC class II (I-A b )-restricted TCR transgenic mouse line that produces CD4 + T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4 + T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human ( Plasmodium falciparum ) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8 + T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4 + T cells and the previously described PbT-I CD8 + T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8 + DC (a subset of XCR1 + DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4 + T cell responses. Depletion of CD8 + DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4 + T cell immunity during malaria and provides evidence that CD4 + T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8 + DC., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill-defined due to the lack of functional studies. In this study, we show that the rhoptry-associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post-invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra-erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite-host cell interface are not simply by-products of the invasion process but contribute to the growth of Plasmodium in vivo., (© 2017 John Wiley & Sons Ltd.)

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer's clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.

Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium -infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival.

When in their human hosts, malaria parasites spend most of their time housed within vacuoles inside erythrocytes and hepatocytes. The parasites extensively modify their host cells to obtain nutrients, prevent host cell breakdown and avoid the immune system. To perform these modifications, malaria parasites export hundreds of effector proteins into their host cells and this process is best understood in the most lethal species to infect humans, Plasmodium falciparum. The effector proteins are synthesized within the parasite and following a proteolytic cleavage event in the endoplasmic reticulum and sorting of mature proteins into the correct vesicular trafficking pathway, they are transported to the parasite surface and released into the vacuole. The effector proteins are then unfolded before extrusion across the vacuole membrane by a unique translocon complex called Plasmodium translocon of exported proteins. After gaining access to the erythrocyte cytoplasm many effector proteins continue their journey to the erythrocyte surface by utilising various membranous structures established by the parasite. This complex trafficking pathway and a large number of the effector proteins are unique to Plasmodium parasites. This pathway could, therefore, be developed as new drug targets given that protein export and the functional role of these proteins are essential for parasite survival. This review explores known and potential drug targetable steps in the protein export pathway and strategies for discovering novel drug targets., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex., (© 2016 John Wiley & Sons Ltd.)

Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host.

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection., (© 2015 John Wiley & Sons Ltd.)

Pathogenesis of malaria infections is linked to remodeling of erythrocytes, a process dependent on the trafficking of hundreds of parasite-derived proteins into the host erythrocyte. Recent studies have demonstrated that the Plasmodium translocon of exported proteins (PTEX) serves as the central gateway for trafficking of these proteins, as inducible knockdown of the core PTEX constituents blocked the trafficking of all classes of cargo into the erythrocyte. However, the role of the auxiliary component PTEX88 in protein export remains less clear. Here we have used inducible knockdown technologies in P. falciparum and P. berghei to assess the role of PTEX88 in parasite development and protein export, which reveal that the in vivo growth of PTEX88-deficient parasites is hindered. Interestingly, we were unable to link this observation to a general defect in export of a variety of known parasite proteins, suggesting that PTEX88 functions in a different fashion to the core PTEX components. Strikingly, PTEX88-deficient P. berghei were incapable of causing cerebral malaria despite a robust pro-inflammatory response from the host. These parasites also exhibited a reduced ability to sequester in peripheral tissues and were removed more readily from the circulation by the spleen. In keeping with these findings, PTEX88-deficient P. falciparum-infected erythrocytes displayed reduced binding to the endothelial cell receptor, CD36. This suggests that PTEX88 likely plays a specific direct or indirect role in mediating parasite sequestration rather than making a universal contribution to the trafficking of all exported proteins.

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function., (© 2013 John Wiley & Sons Ltd.)