On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity.

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.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

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.

Unravelling mysteries at the perivascular space: a new rationale for cerebral malaria pathogenesis.

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.

Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

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 P1 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 P2 ' 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.

Cish knockout mice exhibit similar outcomes to malaria infection despite altered hematopoietic responses.

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.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

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.

The P. falciparum alternative histones Pf H2A.Z and Pf H2B.Z are dynamically acetylated and antagonized by PfSir2 histone deacetylases at heterochromatin boundaries.

The Plasmodium falciparum alternative histones Pf H2A.Z and Pf H2B.Z are enriched in the same nucleosomes in intergenic euchromatin but depleted from heterochromatin. They occupy most promoters but are only dynamically associated with expression at var genes. In other organisms, acetylation of H2A.Z is important for its functions in gene expression and chromatin structure. Here, we show that acetylated Pf H2A.Z and Pf H2B.Z are dynamically associated with gene expression at promoters. In addition, acetylated Pf H2A.Z and Pf H2B.Z are antagonized by the sirtuin class III histone deacetylases (HDAC) PfSir2A and B at heterochromatin boundaries and encroach upon heterochromatin in parasites lacking PfSir2A or B. However, the majority of acetylated Pf H2A.Z and Pf H2B.Z are deacetylated by class I or II HDACs. Acetylated Pf H2A.Z and Pf H2B.Z are also dynamically associated with promoter activity of both canonical upstream var gene promoters and var gene introns. These findings suggest that both acetylated Pf H2A.Z and Pf H2B.Z play critical roles in gene expression and contribute to maintenance of chromatin structure at the boundaries of subtelomeric, facultative heterochromatin, critical for the variegated expression of genes that enable rapid adaptation to altered host environments.IMPORTANCEThe 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.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

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.

Altered gastrointestinal tract structure and microbiome following cerebral malaria infection.

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.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

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.

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway.

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 PfA-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 PfA-M17 to show that PfA-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 PfA-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 PfA-M17 as a potential novel drug target.

Malaria is a disease spread by mosquitoes. When infected insects bite the skin, they inject parasites called Plasmodium into the host. The symptoms of the disease then develop when Plasmodium infect host red blood cells. These parasites cannot make the raw materials to build their own proteins, so instead, they digest haemoglobin ­ the protein used by red blood cells to carry oxygen ­ and use its building blocks to produce proteins. Blocking the digestion of haemoglobin can stop malaria infections in their tracks, but it is unclear how exactly Plasmodium parasites break down the protein. Researchers think that a group of four enzymes called aminopeptidases are responsible for the final stage in this digestion, releasing the amino acids that make up haemoglobin. However, the individual roles of each of these aminopeptidases are not yet known. To start filling this gap, Edgar et al. set out to study one of these aminopeptidases, called PfA-M17. First, they genetically modified Plasmodium falciparum parasites so that the levels of this aminopeptidase were reduced during infection. Without the enzyme, the parasites were unable to grow. The next step was to confirm that this was because PfA-M17 breaks down haemoglobin, and not for another reason. To test this, Edgar et al. designed a new molecule that could stop PfA-M17 from releasing amino acids. This molecule, which they called 'compound 3', had the same effect as reducing the levels of PfA-M17. Further analysis showed that the amino acids that PfA- M17 releases match the amino acids found in haemoglobin. Malaria causes hundreds of thousands of deaths per year. Although there are treatments available, the Plasmodium parasites are starting to develop resistance. Confirming the role of PfA-M17 provides a starting point for new studies by parasitologists, biologists, and drug developers. This could lead to the development of chemicals that block this enzyme, forming the basis for new treatments.

Post-translational lipid modifications in Plasmodium parasites.

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.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

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.

A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell.

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.

How Malaria Parasites Acquire Nutrients From Their Host.

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.

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

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.

Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion.

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.

Methods Used to Investigate the Plasmodium falciparum Digestive Vacuole.

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.

Molecular approaches to Malaria 2020.

Twenty years ago the Molecular Approaches to Malaria conference was conceived as a forum to present the very latest advances in malaria research and to consolidate and forge new collaborative links between international researchers. The 6th MAM conference, held in February 2020 in Australia, provided 5 days of stimulating scientific exchange and highlighted the incredible malaria research conducted globally that is providing the critical knowledge and cutting-edge technological tools needed to control and ultimately eliminate malaria.