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Cell Host & Microbe Feb 2023Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the...
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.
Topics: Animals; Female; Male; Parasites; Malaria; Plasmodium berghei; Sexual Development; Culicidae; Protozoan Proteins
PubMed: 36634679
DOI: 10.1016/j.chom.2022.12.011 -
Science (New York, N.Y.) Aug 2019Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We...
Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.
Topics: Animals; Anopheles; Atlases as Topic; Genes, Protozoan; HeLa Cells; Humans; Life Cycle Stages; Malaria; Plasmodium berghei; Single-Cell Analysis; Transcriptome
PubMed: 31439762
DOI: 10.1126/science.aaw2619 -
Cell Reports Nov 2023Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized...
Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.
Topics: Humans; Proteomics; Malaria; Proteome; Plasmodium berghei; Erythrocytes
PubMed: 37952150
DOI: 10.1016/j.celrep.2023.113419 -
Nature Communications Sep 2023The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities...
The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.
Topics: Animals; Chromosome Segregation; Cell Nucleus Division; Plasmodium berghei; Cell Proliferation; Meiosis; Aurora Kinases; Eukaryota
PubMed: 37704606
DOI: 10.1038/s41467-023-41395-3 -
Proceedings of the National Academy of... Aug 2022Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical...
Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.
Topics: Aminoacyltransferases; Animals; Culicidae; Glutamic Acid; Glutamine; Humans; Malaria; Plasmodium berghei; Protein Processing, Post-Translational; Protozoan Proteins; Sporozoites
PubMed: 35994647
DOI: 10.1073/pnas.2209729119 -
Antimicrobial Agents and Chemotherapy Aug 2023Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H from the cell. This...
Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials.
Topics: Animals; Mice; Monocarboxylic Acid Transporters; Plasmodium falciparum; Malaria, Falciparum; Antimalarials; Parasites; Lactates; Plasmodium berghei; Protozoan Proteins
PubMed: 37428074
DOI: 10.1128/aac.00356-23 -
Cell Host & Microbe Apr 2022In 2015, we reported the development of a rapid protein degradation tool in the malaria parasite Plasmodium berghei. This commentary discusses the questions and events...
In 2015, we reported the development of a rapid protein degradation tool in the malaria parasite Plasmodium berghei. This commentary discusses the questions and events that led to developing this technology, as well as future outlooks.
Topics: Plasmodium berghei
PubMed: 35421346
DOI: 10.1016/j.chom.2022.03.021 -
Parasites & Vectors Jul 2022Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes,...
BACKGROUND
Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium.
METHODS
A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony.
RESULTS
The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites.
CONCLUSIONS
Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.
Topics: Animals; Culicidae; Female; Homologous Recombination; Life Cycle Stages; Malaria; Mammals; Mice; Oocysts; Parasites; Plasmodium berghei
PubMed: 35804459
DOI: 10.1186/s13071-022-05357-w -
ELife Oct 2022Malaria is among the deadliest infectious diseases, and , the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to...
Malaria is among the deadliest infectious diseases, and , the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express antimalarial effector molecules, and paratransgenesis, consisting of introducing into the mosquito commensal bacteria engineered to express antimalarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors - scorpine and the MP2 peptide - into the mosquito gut and salivary glands. We also engineered , a commensal bacterium capable of spreading through mosquito populations to secrete effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of from infected to naïve mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promises to become a powerful approach to combat malaria.
Topics: Animals; Mice; Antimalarials; Anopheles; Mosquito Vectors; Malaria; Plasmodium falciparum; Plasmodium berghei; Gene Transfer Techniques
PubMed: 36281969
DOI: 10.7554/eLife.77584 -
Parasites & Vectors Aug 2023Malaria caused by Plasmodium species is a prominent public health concern worldwide, and the infection of a malarial parasite is transmitted to humans through the saliva...
BACKGROUND
Malaria caused by Plasmodium species is a prominent public health concern worldwide, and the infection of a malarial parasite is transmitted to humans through the saliva of female Anopheles mosquitoes. Plasmodium invasion is a rapid and complex process. A critical step in the blood-stage infection of malarial parasites is the adhesion of merozoites to red blood cells (RBCs), which involves interactions between parasite ligands and receptors. The present study aimed to investigate a previously uncharacterized protein, PbMAP1 (encoded by PBANKA_1425900), which facilitates Plasmodium berghei ANKA (PbANKA) merozoite attachment and invasion via the heparan sulfate receptor.
METHODS
PbMAP1 protein expression was investigated at the asexual blood stage, and its specific binding activity to both heparan sulfate and RBCs was analyzed using western blotting, immunofluorescence, and flow cytometry. Furthermore, a PbMAP1-knockout parasitic strain was established using the double-crossover method to investigate its pathogenicity in mice.
RESULTS
The PbMAP1 protein, primarily localized to the P. berghei membrane at the merozoite stage, is involved in binding to heparan sulfate-like receptor on RBC surface of during merozoite invasion. Furthermore, mice immunized with the PbMAP1 protein or passively immunized with sera from PbMAP1-immunized mice exhibited increased immunity against lethal challenge. The PbMAP1-knockout parasite exhibited reduced pathogenicity.
CONCLUSIONS
PbMAP1 is involved in the binding of P. berghei to heparan sulfate-like receptors on RBC surface during merozoite invasion.
Topics: Humans; Female; Animals; Mice; Plasmodium berghei; Merozoites; Protozoan Proteins; Erythrocytes; Carrier Proteins; Plasmodium falciparum
PubMed: 37563696
DOI: 10.1186/s13071-023-05896-w