Identification of domains in Plasmodium falciparum proteins of unknown function using DALI search on AlphaFold predictions.

Plasmodium falciparum, the causative agent of malaria, poses a significant global health challenge, yet much of its biology remains elusive. A third of the genes in the P. falciparum genome lack annotations regarding their function, impeding our understanding of the parasite's biology. In this study, we employ structure predictions and the DALI search algorithm to analyse proteins encoded by uncharacterized genes in the reference strain 3D7 of P. falciparum. By comparing AlphaFold predictions to experimentally determined protein structures in the Protein Data Bank, we found similarities to known domains in 353 proteins of unknown function, shedding light on their potential functions. The lowest-scoring 5% of similarities were additionally validated using the size-independent TM-align algorithm, confirming the detected similarities in 88% of the cases. Notably, in over 70 P. falciparum proteins the presence of domains resembling heptatricopeptide repeats, which are typically involvement in RNA binding and processing, was detected. This suggests this family, which is important in transcription in mitochondria and apicoplasts, is much larger in Plasmodium parasites than previously thought. The results of this domain search provide a resource to the malaria research community that is expected to inform and enable experimental studies.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum.

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/ß-fold with a mixed five-stranded ß-sheet that is sandwiched between 4 helices in a ß1 α1 ß2 α2 ß3 α3 ß4 ß5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1.

mSphere of Influence: Deciphering purine auxotrophy in protozoan parasites.

Bruno Martorelli Di Genova works in parasitology, focusing on Toxoplasma gondii metabolism. In this mSphere of Influence article, he reflects on how the articles "Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani" and "Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites" impacted him, informing his research strategies and understanding of metabolic flexibility in Toxoplasma gondii.

A targetable receptor-binding site on PfCyRPA to aid in the fight against malaria.

Recently, Day et al. identified a receptor-binding site on the malaria parasite protein PfCyRPA that binds the host sugar Neu5Ac, and they found that disrupting this interaction impedes parasite growth. A map of the receptor-binding site identifies an attractive target for antimalarial vaccines and therapeutics.

Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion.

Remodeling the erythrocyte membrane and skeleton by the malarial parasite Plasmodium falciparum is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not ß-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of P. falciparum, from the ring to the schizont stage. We further observed an upregulated expression of P. falciparum phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during P. falciparum development. We further revealed that inhibition of the activity of PfPI3K impaired P. falciparum development in vitro and Plasmodium berghei infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of Plasmodium species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria. IMPORTANCE: Plasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while ß-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Insights from structure-activity relationships and the binding mode of peptidic α-ketoamide inhibitors of the malaria drug target subtilisin-like SUB1.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 µM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC50 value of 23 µM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds.

Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.

Plasmodium falciparum heat shock proteins as antimalarial drug targets: An update.

Global efforts to eradicate malaria are threatened by multiple factors, particularly the emergence of antimalarial drug resistant strains of Plasmodium falciparum. Heat shock proteins (HSPs), particularly P. falciparum HSPs (PfHSPs), represent promising drug targets due to their essential roles in parasite survival and virulence across the various life cycle stages. Despite structural similarities between human and malarial HSPs posing challenges, there is substantial evidence for subtle differences that could be exploited for selective drug targeting. This review provides an update on the potential of targeting various PfHSP families (particularly PfHSP40, PfHSP70, and PfHSP90) and their interactions within PfHSP complexes as a strategy to develop new antimalarial drugs. In addition, the need for a deeper understanding of the role of HSP complexes at the host-parasite interface is highlighted, especially heterologous partnerships between human and malarial HSPs, as this opens novel opportunities for targeting protein-protein interactions crucial for malaria parasite survival and pathogenesis.

Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania.

BACKGROUND: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. METHODS: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. RESULTS: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. CONCLUSION: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Role for gene conversion in the evolution of cell-surface antigens of the malaria parasite Plasmodium falciparum.

While the malaria parasite Plasmodium falciparum has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell-infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical P. falciparum transmission bottleneck.