Abstract Babesia are tick-borne intracellular apicomplexan parasites that infect a wide range of wild and domestic animals (e.g., cattle), resulting in significant economic losses to the livestock industry. Humans are considered accidental hosts for a few Babesia species. Babesia microti and B. divergens are the most prevalent causes of human babesiosis that are showing a broadening geographic distribution. Due to the complex life cycle of Babesia species, their survival depends on the precise control of gene expression, which is primarily regulated by epigenetic, transcriptional, and post-transcriptional mechanisms. High-resolution structural information on key components of the translation machinery, such as ribosomes, could aid in the development of antiparasitic drugs. Here, we report cryogenic electron microscopy ribosome structures from B. divergens , showing associated tRNAs, an mRNA fragment, and RACK1, a signaling scaffold crucial to translation regulation. Density map analysis displays ribosome regions at high resolution, which, when combined with nanopore sequencing, enabled the comprehensive identification of rRNA modifications. The rRNA modifications localize not only to the reduced B. divergens rRNA expansion segments but also to functionally essential ribosomal sites.

The clinical and parasitological factors underlying severe malaria pathogenesis remain incompletely understood, particularly in Plasmodium vivax (P. vivax). To address this issue, clinical and parasitological metrics, including circulating peripheral parasitemia and the parasite biomass (the total number of parasites in the body consisting of both circulating peripheral and noncirculating parasites), were measured in patients with severe and uncomplicated monoinfections of P. vivax and Plasmodium falciparum (P. falciparum) malaria who presented at a hospital in southwest India. Peripheral parasitemia was positively correlated with disease severity in P. falciparum but negatively correlated with disease severity in P. vivax. Contrarily, the parasite biomass, estimated by the levels of circulating antigens P. vivax lactate dehydrogenase and P. falciparum histidine-rich protein 2 for P. vivax and P. falciparum, respectively, was positively correlated with both the degree of severity and the length of hospital stay in both P. vivax and P. falciparum cases. Together, these observations indicate a significant hidden parasite biomass in P. vivax infections and suggest that parasite biomass is a more robust parasitological marker of severity than parasitemia. Notably, thrombocytopenia was also positively correlated with parasite biomass and longer hospital stay in both P. vivax and P. falciparum infections. These results highlight the importance of parasite biomass as a leading factor in the disease severity of malaria infections in India.

Egress from host cells is fundamental for the spread of infection by apicomplexan parasites, including Babesia species. These tick-borne pathogens represent emerging zoonoses, but treatment options are limited. Here, using microscopy, transcriptomics and chemical genetics, we identified signalling, proteases and gliding motility as key drivers of egress by Babesia divergens. We developed reverse genetic tools in B. divergens to perform a knockdown screen of putative mediators of egress, identifying kinases and proteases involved in distinct steps of egress (aspartyl protease (ASP) 3 and kinases cGMP-dependent protein kinase (PKG) and calcium-dependent protein kinase (CDPK4)) and invasion (ASP2, ASP3 and PKG) of red blood cells. Inhibition of egress stimulates additional rounds of intracellular replication, indicating that exit from the replication cycle is uncoupled from egress. Chemical genetics validated PKG, CDPK4, ASP2 and ASP3 as druggable targets in Babesia spp. and identified promising compounds for babesiosis treatment. Taken together, egress in B. divergens more closely resembles egress in Toxoplasma gondii than in the more evolutionarily related Plasmodium spp.

Invasion of erythrocytes by members of the Plasmodium genus is an essential step of the parasite lifecycle, orchestrated by numerous host-parasite interactions. In P. falciparum Rh5, with PfCyRPA, PfRipr, PfCSS, and PfPTRAMP, forms the essential PCRCR complex which binds basigin on the erythrocyte surface. Rh5 is restricted to P. falciparum and its close relatives; however, PTRAMP, CSS and Ripr orthologs are present across the Plasmodium genus. We investigated PTRAMP, CSS and Ripr orthologs from three species to elucidate common features of the complex. Like P. falciparum, PTRAMP and CSS form a disulfide-linked heterodimer in both P. vivax and P. knowlesi with all three species forming a complex with Ripr by binding its C-terminal region, termed the PTRAMP-CSS-Ripr (PCR) complex. Cross-reactive antibodies targeting the PCR complex differentially inhibit merozoite invasion. The crystal structure of a cross-reactive antibody reveals an inhibitory epitope on the C-terminal tail of PvRipr. Cryo-EM visualization of the P. knowlesi PCR complex confirms predicted models and demonstrates a core invasion scaffold in Plasmodium spp. with implications for vaccines targeting multiple species of malaria-causing parasites.

Cryptosporin, a fungal metabolite, exhibited potent antimalarial activity against both asexual blood stage Plasmodium falciparum and liver-stage Plasmodium berghei with minimal human HepG2 toxicity. Unlike atovaquone, cryptosporin's mechanism is independent of mitochondrial electron transport. Minimum inoculum of resistance showed a low risk of resistance development. RNA-Seq analysis revealed the upregulation of genes associated with sexual development including many canonical markers such as Pfs25, and PfCCp3, suggesting a stress response that is also seen when parasites are treated with artemisinin. In vitro evolution and whole genome sequencing analysis identified a mutation (F138Y) in PfAQP (PF3D7_1132800) and duplications of the two superoxide dismutase genes, PfSOD-1 (PF3D7_0814900) and PfSOD-2 (PF3D7_0623500). CRISPR/Cas9 editing confirmed that the F138Y mutation in PfAQP was sufficient to confer resistance to cryptosporin. Alignment of the P. falciparum structure with that of HsAQP3 suggests the mutation may impact transport of hydrogen peroxide and the transition between open and closed conformations. Indeed, studies with BY4742 Δfps1 yeast expressing PfAQP showed that the permeability of PfAQP was not affected by cryptosporin and that it is likely not a direct target. Taken together, this study highlights the role of PfAQP in the resistance development of cryptosporin. In addition, cryptosporin likely induces high levels of oxidative stress which results in the duplications of oxidative dismutase genes as part of the parasite's defense response. These findings highlight the role of PfAQP in mediating drug resistance, the mechanism of which warrants further research.

Digital holographic microscopy (DHM) has emerged as a powerful, label-free technique for visualizing and analyzing biological samples. By extracting the intrinsic optical properties of red blood cells (RBCs), DHM enables the detection of infection-induced morphological and biophysical changes. Traditional classification methods often rely on feature-specific analysis, which can lead to misclassification when a single parameter fails to differentiate between uninfected and infected cells. In this study, we present a novel features-based approach that integrates multiple features to classify Plasmodium falciparum-infected RBCs obtained using lensless inline DHM. Our analysis shows that phase-based features classification provides a more reliable indicator of infected RBCs compared to morphological features. Additionally, our features-based approach outperforms feature-specific methods that rely on individual attributes. The parasitemia detection rate improved from 48% (feature-specific method) to 61% (phase-based features method) on the same sample set, demonstrating enhanced detection accuracy. Furthermore, the proposed method achieved high specificity (98-100%), ensuring reliable identification of uninfected cells. Although our method slightly underestimates the parasitemia detection rate compared to Giemsa staining (90%), it offers a significant advantage as a real-time, label-free imaging tool, presenting a promising avenue for rapid and automated malaria diagnosis.

Malaria continues to pose significant health challenges globally despite advances in control measures. Plasmodium falciparum, the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for P. falciparum invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null MkMk blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent P. falciparum strains, but not the primate malaria parasite Plasmodium knowlesi. However, different P. falciparum parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.

Plasmodium vivax vaccine discovery lags behind Plasmodium falciparum due to the absence of a reliable in vitro culture system. We developed a miniature 384-well assay for the evolutionarily related parasite species Plasmodium cynomolgi and Plasmodium knowlesi, deploying it for screening invasion inhibition efficacy of antibodies elicited against 8 P. vivax proteins. All 8 antibodies showed consistent inhibition across both P. knowlesi and P. cynomolgi species, correlating with inhibition in ex vivo P. vivax isolates. The use of simian malaria parasites and this assay represent a robust, high-throughput method for prioritizing P. vivax blood-stage vaccine candidates.

Babesia is a tick-borne intracellular apicomplexan parasite responsible for diseases ranging from mild to fatal, with a broadening geographic distribution. Due to the complex life cycle of Babesia species, their survival depends on the precise control of gene expression, which is primarily regulated by epigenetic, transcriptional, and post-transcriptional mechanisms. High-resolution structural information on key components of the translation machinery, such as ribosomes, could aid in the development of antiparasitic drugs. Here, we report cryo-EM ribosome structures (2.6 Å) from the tick-borne apicomplexan pathogen Babesia divergens , showing associated tRNAs, an mRNA fragment, and RACK1, a signaling scaffold crucial to translation regulation. Density map analysis displays ribosome regions at atomic resolution (1.7 Å), which, when combined with nanopore sequencing, enabled the comprehensive identification of rRNA modifications, including modifications unreported in other organisms. The new rRNA modifications localize not only to the reduced Babesia rRNA expansion segments but also to functionally essential ribosomal sites, uncovering new avenues for therapeutic intervention against babesiosis.

Measures to combat the parasites that cause malaria have become compromised because of reliance on a small arsenal of drugs and emerging drug resistance. We conducted a transposon mutagenesis screen in the primate malaria parasite Plasmodium knowlesi , producing the most complete classification of gene essentiality in any Plasmodium spp. to date, with the resolution to define truncatable genes. We found conservation in the druggable genome between Plasmodium spp. and divergences in mitochondrial metabolism. Perturbation analyses with the frontline antimalarial artemisinin revealed modulators that both increase and decrease drug susceptibility. Our findings aid prioritization of drug and vaccine targets for the Plasmodium vivax clade and reveal mechanisms of resistance that can inform therapeutic development.

Human-derived induced pluripotent stem cells (iPSCs) are an invaluable resource in both two-dimensional (2D) and three-dimensional (3D) tissue engineering due to their multilineage potential in culture systems. To date, modeling red blood cell (RBC) disorders such as sickle cell disease (SCD) from iPSCs has been challenging due to the tendency for differentiation protocols to produce immature erythrocytes that lack robust β-globin expression and enucleate poorly. Here, we demonstrated an optimized three-stage erythroid differentiation protocol that generates enucleated, β-globin-expressing RBCs from somatically sourced iPSCs, derived from both healthy donors and patients with homozygous SCD. Induced RBCs (iRBCs) present phenotypically as GlyA+Band3+CD71lo and express adult hemoglobin tetramers. SCD iRBCs displayed sickling phenotypes in vitro when exposed to hypoxia. RNA-sequencing analysis of iPSC-derived SCD reticulocytes revealed dysregulated disease-relevant molecular pathways, suggesting future therapeutic avenues of investigation can be identified in this model. We further refined the protocol into a xeno-free formulation by replacing albumin sources with polyvinyl alcohol (PVA), significantly enhancing iRBC production without loss of terminal maturation. The ability to generate patient-specific iRBCs from somatic cell sources provides a valuable in vitro tool for the study of SCD and the development of novel treatments.

Malaria continues to pose significant health challenges globally despite advances in control measures. , the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null M M blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent strains, but not the primate malaria parasite . However, different parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

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.

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite's unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

BackgroundChemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases.MethodsOur overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs.ResultsWe identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite.ConclusionsWe report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.