A Diversity Covering (DiCo) Plasmodium vivax apical membrane antigen-1 vaccine adjuvanted with RFASE/RSL10 yields high levels of growth-inhibitory antibodies.

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10.

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages.

New therapeutics are a priority for preventing and eliminating Plasmodium vivax (Pv) malaria because of its easy transmissibility and dormant stages in the liver. Relapses due to the dormant liver stages are the major contributor to reoccurring Pv. Therefore, therapies that reduce the establishment of dormant parasites and blood-stage infection are important for controlling this geographically widespread parasite. Here, we isolated 12 human monoclonal antibodies (humAbs) from the plasma of a Pv-exposed individual that recognized Pv apical membrane antigen 1 (PvAMA1). PvAMA1 is important for both sporozoite invasion of hepatocytes and merozoite invasion of reticulocytes. We identified one humAb, 826827, that blocked invasion of human erythrocytes using a transgenic P. falciparum line expressing PvAMA1 (IC 50 = 3 µg/mL) and all Pv clinical isolates in vitro . This humAb also inhibited sporozoite invasion of a human hepatocyte cell line and primary human hepatocytes (IC 50 of 0.3 - 3.7 µg/mL). The crystal structure of recombinant PvAMA1 with the antigen-binding fragment of 826827 at 2.4 Å resolution shows that the humAb partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. HumAb 826827 binds to PvAMA1 with higher affinity than RON2, accounting for its potency. To our knowledge, this is the first reported humAb specific to PvAMA1, and the PvAMA1 residues it binds to are highly conserved across different isolates, explaining its strain-transcendent properties.

Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines.

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a ß-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1-RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1-RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

Characterisation of PfCZIF1 and PfCZIF2 in Plasmodium falciparum asexual stages.

Plasmodium falciparum exerts strong temporal control of gene expression across its lifecycle. Proteins expressed exclusively during late schizogony of blood stages, for example, often have a role in facilitating merozoite invasion of the host red blood cell (RBC), through merozoite development, egress, invasion or early establishment of infection in the RBC. Here, we characterise P. falciparum C3H1 zinc finger 1 (PfCZIF1, Pf3D7_1468400) and P. falciparum C3H1 zinc finger 2 (PfCZIF2, Pf3D7_0818100) which we identified as the only C3H1-type zinc finger proteins with peak expression at schizogony. Previous studies reported that antibodies against PfCZIF1 inhibit merozoite invasion, suggesting this protein may have a potential role during RBC invasion. We show using C-terminal truncations and gene knockouts of each of Pfczif1 and Pfczif2 that neither are essential for blood stage growth. However, they could not both be knocked out simultaneously, suggesting that at least one is needed for parasite growth in vitro. Immunofluorescence localisation of PfCZIF1 and PfCZIF2 indicated that both proteins occur in discrete foci on the periphery of the parasite's cytosol and biochemical assays suggest they are peripherally associated to a membrane. Transcriptomic analyses for the C-terminal truncation mutants reveal no significant expression perturbations with PfCZIF1 truncation. However, modification of PfCZIF2 appears to modify the expression for some exported proteins including PfKAHRP. This study does not support a role for PfCZIF1 or PfCZIF2 in merozoite invasion of the RBC and suggests that these proteins may help regulate the expression of proteins exported into the RBC cytosol after merozoite invasion.

Advances in Plasmodium research, an update: Highlights from the Malaria in Melbourne 2021 conference.

The Malaria in Melbourne 2021 conference was held online in October. This conference aims to provide a platform for students and early career researchers to share their research and develop new collaborative networks. The program covered a broad range of topics including antimalarial drug development, epidemiology, immunology, molecular and cellular biology, and other emerging technologies. This article summarises recent advances in Plasmodium research presented at the Malaria in Melbourne 2021 conference.

Histone modifications associated with gene expression and genome accessibility are dynamically enriched at Plasmodium falciparum regulatory sequences.

BACKGROUND: The malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear. RESULTS: Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity. CONCLUSIONS: The dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.

Developments in drug design strategies for bromodomain protein inhibitors to target Plasmodium falciparum parasites.

Introduction: Bromodomains (BRDs) bind to acetylated lysine residues, often on histones. The BRD proteins can contribute to gene regulation either directly through enzymatic activity or indirectly through recruitment of chromatin-modifying complexes or transcription factors. There is no evidence of direct orthologues of the Plasmodium falciparum BRD proteins (PfBDPs) outside the apicomplexans. PfBDPs are expressed during the parasite's life cycle in both the human host's blood and in the mosquito. PfBDPs could also prove to be promising targets for novel antimalarials, which are urgently required to address increasing drug resistance.Areas covered: This review discusses recent studies of the biology of PfBDPs, current target-based strategies for PfBDP inhibitor discovery, and different approaches to the important step of validating the specificity of hit compounds for PfBDPs.Expert opinion: The novelty of Plasmodium BRDs suggests that they could be targeted by selective compounds. Chemical series that showed promise in screens against human BRDs could be leveraged to create targeted compound libraries, as could hits from P. falciparum phenotypic screens. These targeted libraries and hits could be screened in target-based strategies aimed at discovery and optimization of novel inhibitors of PfBDPs. A key task for the field is to generate parasite assays to validate the hit compounds' specificity for PfBDPs.

Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance.

Increased tolerance of Plasmodium falciparum to front-line artemisinin antimalarials (ARTs) is associated with mutations in Kelch13 (K13), although the precise role of K13 remains unclear. Here, we show that K13 mutations result in decreased expression of this protein, while mislocalization of K13 mimics resistance-conferring mutations, pinpointing partial loss of function of K13 as the relevant molecular event. K13-GFP is associated with ∼170 nm diameter doughnut-shaped structures at the parasite periphery, consistent with the location and dimensions of cytostomes. Moreover, the hemoglobin-peptide profile of ring-stage parasites is reduced when K13 is mislocalized. We developed a pulse-SILAC approach to quantify protein turnover and observe less disruption to protein turnover following ART exposure when K13 is mislocalized. Our findings suggest that K13 regulates digestive vacuole biogenesis and the uptake/degradation of hemoglobin and that ART resistance is mediated by a decrease in heme-dependent drug activation, less proteotoxicity, and increased survival of parasite ring stages.

Alternative splicing is required for stage differentiation in malaria parasites.

BACKGROUND: In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into variable cell types during their life cycles. The role of alternative splicing in transitions between cell types and establishing cellular identity is currently unknown in any unicellular organism. RESULTS: To test whether alternative splicing in unicellular protists plays a role in cellular differentiation, we conduct RNA-seq to compare splicing in female and male sexual stages to asexual intraerythrocytic stages in the rodent malaria parasite Plasmodium berghei. We find extensive changes in alternative splicing between stages and a role for alternative splicing in sexual differentiation. Previously, general gametocyte differentiation was shown to be modulated by specific transcription factors. Here, we show that alternative splicing establishes a subsequent layer of regulation, controlling genes relating to consequent sex-specific differentiation of gametocytes. CONCLUSIONS: We demonstrate that alternative splicing is reprogrammed during cellular differentiation of a unicellular protist. Disruption of an alternative splicing factor, PbSR-MG, perturbs sex-specific alternative splicing and decreases the ability of the parasites to differentiate into male gametes and oocysts, thereby reducing transmission between vertebrate and insect hosts. Our results reveal alternative splicing as an integral, stage-specific phenomenon in these protists and as a regulator of cellular differentiation that arose early in eukaryotic evolution.

Alternative Splicing in Apicomplexan Parasites.

Alternative splicing is a widespread, essential, and complex component of gene regulation. Apicomplexan parasites have long been recognized to produce alternatively spliced transcripts for some genes and can produce multiple protein products that are essential for parasite growth. Recent approaches are now providing more wide-ranging surveys of the extent of alternative splicing; some indicate that alternative splicing is less widespread than in other model eukaryotes, whereas others suggest levels comparable to those of previously studied groups. In many cases, apicomplexan alternative splicing events appear not to generate multiple alternative proteins but instead produce aberrant or noncoding transcripts. Nonetheless, appropriate regulation of alternative splicing is clearly essential in Plasmodium and Toxoplasma parasites, suggesting a biological role for at least some of the alternative splicing observed. Several studies have now disrupted conserved regulators of alternative splicing and demonstrated lethal effects in apicomplexans. This minireview discusses methods to accurately determine the extent of alternative splicing in Apicomplexa and discuss potential biological roles for this conserved process in a phylum of parasites with compact genomes.

Comparative transcriptomics of female and male gametocytes in Plasmodium berghei and the evolution of sex in alveolates.

BACKGROUND: The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation. RESULTS: RNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development. CONCLUSIONS: We characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways.

The Import of Proteins into the Mitochondrion of Toxoplasma gondii.

Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.

A serine-arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii.

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine-rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.

A dual-targeted aminoacyl-tRNA synthetase in Plasmodium falciparum charges cytosolic and apicoplast tRNACys.

Plasmodium parasites possess two endosymbiotic organelles: a mitochondrion and a relict plastid called the apicoplast. To accommodate the translational requirements of these organelles in addition to its cytosolic translation apparatus, the parasite must maintain a supply of charged tRNA molecules in each of these compartments. In the present study we investigate how the parasite manages these translational requirements for charged tRNACys with only a single gene for CysRS (cysteinyl-tRNA synthetase). We demonstrate that the single PfCysRS (Plasmodium falciparum CysRS) transcript is alternatively spliced, and, using a combination of endogenous and heterologous tagging experiments in both P. falciparum and Toxoplasma gondii, we show that CysRS isoforms traffic to the cytosol and apicoplast. PfCysRS can recognize and charge the eukaryotic tRNACys encoded by the Plasmodium nucleus as well as the bacterial-type tRNA encoded by the apicoplast genome, albeit with a preference for the eukaryotic type cytosolic tRNA. The results of the present study indicate that apicomplexan parasites have lost their original plastidic cysteinyl-tRNA synthetase, and have replaced it with a dual-targeted eukaryotic type CysRS that recognizes plastid and nuclear tRNACys. Inhibitors of the Plasmodium dual-targeted CysRS would potentially offer a therapy capable of the desirable immediate effects on parasite growth as well as the irreversibility of inhibitors that disrupt apicoplast inheritance.