Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Bottoms up! Malaria parasite invasion the right way around.

A critical part of the malaria parasite's life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs 'narrower end' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs 'wider end' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies.

Identification of potent and selective N-myristoyltransferase inhibitors of Plasmodium vivax liver stage hypnozoites and schizonts.

Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage.

High-content imaging as a tool to quantify and characterize malaria parasites.

In 2021, Plasmodium falciparum was responsible for 619,000 reported malaria-related deaths. Resistance has been detected to every clinically used antimalarial, urging the development of novel antimalarials with uncompromised mechanisms of actions. High-content imaging allows researchers to collect and quantify numerous phenotypic properties at the single-cell level, and machine learning-based approaches enable automated classification and clustering of cell populations. By combining these technologies, we developed a method capable of robustly differentiating and quantifying P. falciparum asexual blood stages. These phenotypic properties also allow for the quantification of changes in parasite morphology. Here, we demonstrate that our analysis can be used to quantify schizont nuclei, a phenotype that previously had to be enumerated manually. By monitoring stage progression and quantifying parasite phenotypes, our method can discern stage specificity of new compounds, thus providing insight into the compound's mode of action.

The same genotype of Sarcocystis neurona responsible for mass mortality in marine mammals induced a clinical outbreak in raccoons (Procyon lotor) 10 years later.

Here, we report the first known outbreak of clinical protozoal myeloencephalitis in naturally infected raccoons by the parasite Sarcocystis neurona. The North American opossum (Didelphis virginiana) and the South American opossum (Didelphis albiventris) are its known definitive hosts. Several other animal species are its intermediate or aberrant hosts. The raccoon (Procyon lotor) is considered the most important intermediate host for S. neurona in the USA. More than 50% of raccoons in the USA have sarcocysts in their muscles, however clinical sarcocystosis in raccoons is rare. In 2014, 38 free-living raccoons were found dead or moribund on the grounds of the Saint Louis Zoo, Missouri, USA. Moribund individuals were weak, lethargic, and mildly ataxic; several with oculo-nasal discharge. Seven raccoons were found dead and 31 were humanely euthanized. Postmortem examinations were conducted on nine raccoons. Neural lesions compatible with acute sarcocystosis were detected in eight raccoons. The predominant lesions were meningoencephalitis and perivascular mononuclear cells. Histologic evidence for the Canine Distemper Virus was found in one raccoon. Schizonts and merozoites were present in the encephalitic lesions of four raccoons. Mature sarcocysts were present within myocytes of five raccoons. In six raccoons, S. neurona schizonts and merozoites were confirmed by immunohistochemical staining with S. neurona-specific polyclonal antibodies. Viable S. neurona was isolated from the brains of two raccoons by bioassay in interferon gamma gene knockout mice and in cell cultures seeded directly with raccoon brain homogenate. Molecular characterization was based on raccoon no. 68. Molecular characterization based on multi-locus typing at five surface antigens (SnSAG1-5-6, SnSAG3 and SnSAG4) and the ITS-1 marker within the ssrRNA locus, using DNA isolated from bradyzoites released from sarcocysts in a naturally infected raccoon (no. 68), confirmed the presence of S. neurona antigen type I, the same genotype that caused a mass mortality event in which 40 southern sea otters stranded dead or dying within a 3 week period in April 2004 with S. neurona-associated disease. An expanded set of genotyping markers was next applied. This study reports the following new genotyping markers at 18S rRNA, 28S rRNA, COX1, ITS-1, RON1, RON2, GAPDH1, ROP20, SAG2, SnSRS21 and TUBA1 markers. The identity of Sarcocystis spp. infecting raccoons is discussed.

Histone modification analysis reveals common regulators of gene expression in liver and blood stage merozoites of Plasmodium parasites.

Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages.

Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts.

Eimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate.

Modified Plasmodium falciparum Ring-Stage Survival Assay with ML10 Kinase Inhibitor.

The ring-stage survival assay is the reference assay to measure in vitro Plasmodium falciparum artemisinin partial resistance. The main challenge of the standard protocol is to generate 0-to-3-h postinvasion ring stages (the stage least susceptible to artemisinin) from schizonts obtained by sorbitol treatment and Percoll gradient. We report here a modified protocol facilitating the production of synchronized schizonts when multiple strains are tested simultaneously, by using ML10, a protein kinase inhibitor, that reversibly blocks merozoite egress.

PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture's 'Malaria Box' and 'Pathogen Box'. Recent screens of the 'Malaria Box' and 'Pathogen Box' revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.

Characterization of the dynamics of Plasmodium falciparum deoxynucleotide-triphosphate pool in a stage-specific manner.

Understanding and characterizing the molecular background of the maintenance of genomic integrity might be a major factor in comprehending the exceptional ability of the malaria parasite, Plasmodium falciparum to adapt at a fast pace to antimalarials. A balanced nucleotide pool is an essential factor for high-fidelity replication. The lack of detailed studies on deoxynucleotide-triphosphate (dNTP) pools in various intraerythrocytic stages of Plasmodium falciparum motivated our present study. Here, we focused on the building blocks of DNA and utilized an EvaGreen-based dNTP incorporation assay to successfully measure the temporal dynamics of dNTPs in every intraerythrocytic stage and in drug-treated trophozoites. Our findings show that the ratio of dNTPs in the ring-stage parasites significantly differs from the more mature trophozoite and schizont stages. We were also able to detect dGTP levels that have never been shown before and found it to be the least abundant dNTP in all stages. Treatment with WR99210, a TS-DHFR inhibitor drug, affected not only dTTP, but also dGTP levels, despite its presumed selective action on pyrimidine biosynthesis. Results from our studies might assist in a better understanding of genome integrity mechanisms and may potentially lead to novel drug related aspects involving purine and pyrimidine metabolic targets.

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts.

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.

SICA-mediated cytoadhesion of Plasmodium knowlesi-infected red blood cells to human umbilical vein endothelial cells.

Zoonotic malaria due to Plasmodium knowlesi infection in Southeast Asia is sometimes life-threatening. Post-mortem examination of human knowlesi malaria cases showed sequestration of P. knowlesi-infected red blood cells (iRBCs) in blood vessels, which has been proposed to be linked to disease severity. This sequestration is likely mediated by the cytoadhesion of parasite-iRBCs to vascular endothelial cells; however, the responsible parasite ligands remain undetermined. This study selected P. knowlesi lines with increased iRBC cytoadhesion activity by repeated panning against human umbilical vein endothelial cells (HUVECs). Transcriptome analysis revealed that the transcript level of one gene, encoding a Schizont Infected Cell Agglutination (SICA) protein, herein termed SICA-HUVEC, was more than 100-fold increased after the panning. Transcripts of other P. knowlesi proteins were also significantly increased, such as PIR proteins exported to the iRBC cytosol, suggesting their potential role in increasing cytoadhesion activity. Transgenic P. knowlesi parasites expressing Myc-fused SICA-HUVEC increased cytoadhesion activity following infection of monkey as well as human RBCs, confirming that SICA-HUVEC conveys activity to bind to HUVECs.

Theileria annulata SVSP455 interacts with host HSP60.

BACKGROUND: Theileria annulata, a transforming parasite, invades bovine B cells, dendritic cells and macrophages, promoting the uncontrolled proliferation of these cells. This protozoan evolved intricate strategies to subvert host cell signaling pathways related to antiapoptotic signaling to enable survival and proliferation within the host cells. However, the molecular mechanisms of the cell transformation induced by T. annulata remain largely unclear. Although some studies have predicted that the subtelomere-encoded variable secreted protein (SVSP) family plays roles in host-parasite interactions, the evidence for this is limited. METHODS: In the present study, the SVSP455 (TA05545) gene, a member of the SVSP gene family, was used as the target molecule. The expression pattern of SVSP455 in different life-cycle stages of T. annulata infection was explored using a quantitative real-time PCR assay, and the subcellular distribution of SVSP455 was observed using confocal microscopy. The host cell proteins interacting with SVSP455 were screened using the Y2H system, and their interactions were verified in vivo and in vitro using both bimolecular fluorescence complementation and confocal microscopy, and co-immunoprecipitation assays. The role played by SVSP455 in cell transformation was further explored by using overexpression, RNA interference and drug treatment experiments. RESULTS: The highest level of the SVSP455 transcript was detected in the schizont stage of T. annulata, and the protein was located both on the surface of schizonts and in the host cell cytoplasm. In addition, the interaction between SVSP455 and heat shock protein 60 was shown in vitro, and their link may regulate host cell apoptosis in T. annulata-infected cells. CONCLUSION: Our findings are the first to reveal that T. annulata-secreted SVSP455 molecule directly interacts with both exogenous and endogenous bovine HSP60 protein, and that the interaction of SVSP455-HSP60 may manipulate the host cell apoptosis signaling pathway. These results provide insights into cancer-like phenotypes underlying Theilera transformation and therapeutics for protection against other pathogens.

The Plasmodium falciparum Nuclear Protein Phosphatase NIF4 Is Required for Efficient Merozoite Invasion and Regulates Artemisinin Sensitivity.

Artemisinin resistance in Plasmodium falciparum has been associated with a mutation in the NLI-interacting factor-like phosphatase PfNIF4, in addition to the mutations in the Kelch13 protein as the major determinant. We found that PfNIF4 was predominantly expressed at the schizont stage and localized in the nuclei of the parasite. To elucidate the functions of PfNIF4 in P. falciparum, we performed PfNIF4 knockdown (KD) using the inducible ribozyme system. PfNIF4 KD attenuated merozoite invasion and affected gametocytogenesis. PfNIF4 KD parasites also showed significantly increased in vitro susceptibility to artemisinins. Transcriptomic and proteomic analysis revealed that PfNIF4 KD led to the downregulation of gene categories involved in invasion and artemisinin resistance (e.g., mitochondrial function, membrane, and Kelch13 interactome) at the trophozoite and/or schizont stage. Consistent with PfNIF4 being a protein phosphatase, PfNIF4 KD resulted in an overall upregulation of the phosphoproteome of infected erythrocytes. Quantitative phosphoproteomic profiling identified a set of PfNIF4-regulated phosphoproteins with functional similarity to FCP1 substrates, particularly proteins involved in chromatin organization and transcriptional regulation. Specifically, we observed increased phosphorylation of Ser2/5 of the tandem repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII) upon PfNIF4 KD. Furthermore, using the TurboID-based proteomic approach, we identified that PfNIF4 interacted with the RNAPII components, AP2-domain transcription factors, and chromatin-modifiers and binders. These findings suggest that PfNIF4 may act as the RNAPII CTD phosphatase, regulating the expression of general and parasite-specific cellular pathways during the blood-stage development. IMPORTANCE Protein phosphorylation regulates a multitude of cellular processes. The eukaryotic FCP1 phosphatase acts as a CTD-phosphatase to critically balance the phosphorylation status of the CTD of the RNAPII, controlling the accurate execution of the transcription process. Here, we identified PfNIF4 as the FCP1-like phosphatase in P. falciparum. PfNIF4 KD specifically downregulated genes involved in merozoite invasion, resulting in the attenuation of this process. Consistent with the earlier finding of the association of PfNIF4 mutations with artemisinin resistance in Southeast Asian parasite populations, PfNIF4 KD significantly increased in vitro susceptibility to artemisinins. The regulation of these cellular processes in P. falciparum by PfNIF4 is likely realized through RNAPII-mediated transcription, because PfNIF4 was found to interact with RNAPII subunits and KD of PfNIF4 caused CTD hyperphosphorylation. Our results reveal the functions of the PfNIF4 phosphatase in controlling the transcription of invasion- and resistance-related genes in the malaria parasite.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes.

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 96 clinical isolates from 4 populations with various levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator ap2-g. Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing mspdbl2 did not express ap2-g, consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. IMPORTANCE These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial ex vivo generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and mspdbl2 gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission. Single-cell transcriptome data confirm the absence of a direct correlation with the ap2-g master regulator of sexual development, indicating that the MSPDBL2-positive subpopulation has a separate function in asexual survival and replication under conditions that promote terminal sexual differentiation.

Theileria annulata histone deacetylase 1 (TaHDAC1) initiates schizont to merozoite stage conversion.

A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins.

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.