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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 -
Frontiers in Cellular and Infection... 2019In the lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively....
In the lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively. The apical structure of merozoites and sporozoites contains rhoptry and microneme secretory organelles, which are conserved with other infective forms of apicomplexan parasites. During merozoite invasion of erythrocytes, some rhoptry proteins are secreted to form a tight junction between the parasite and target cell, while others are discharged to maintain subsequent infection inside the parasitophorous vacuole. It has been questioned whether the invasion mechanisms mediated by rhoptry proteins are also involved in sporozoite invasion of two distinct target cells, mosquito salivary glands and mammalian hepatocytes. Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex, are commonly expressed in merozoites and sporozoites in spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells. Combined with sporozoite-stage specific gene silencing strategies, the contribution of rhoptry proteins in invasion mechanisms can be described.
Topics: Animals; Anopheles; Blotting, Western; Cells, Cultured; Epithelial Cells; Gene Expression Profiling; Hepatocytes; Mammals; Merozoites; Microscopy, Immunoelectron; Organelles; Plasmodium berghei; Protein Transport; Protozoan Proteins; Real-Time Polymerase Chain Reaction; Sporozoites
PubMed: 31552198
DOI: 10.3389/fcimb.2019.00316 -
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 -
Free Radical Biology & Medicine Jun 2016Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use...
Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions.
Topics: Animals; Antioxidants; Cell Nucleus; DNA Damage; DNA, Mitochondrial; Gene Knockout Techniques; Glutamate-Cysteine Ligase; Glutathione; Glutathione Reductase; Life Cycle Stages; Malaria; Oxidative Stress; Plasmodium berghei; Thioredoxins
PubMed: 26952808
DOI: 10.1016/j.freeradbiomed.2016.02.032 -
Open Biology Aug 2022Protein phosphatase 1 (PP1) is a key enzyme for development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the...
Protein phosphatase 1 (PP1) is a key enzyme for development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of oocyst development through its interaction with PbLRR1.
Topics: Animals; Leucine-Rich Repeat Proteins; Oocysts; Phosphorylation; Plasmodium berghei; Protein Phosphatase 1
PubMed: 35920043
DOI: 10.1098/rsob.220015 -
International Journal of Molecular... May 2022The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited...
The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces hemolymph sporozoite infectivity to mice. Moreover, we found that knockout (-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The - defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.
Topics: Animals; Culicidae; Erythrocytes; Membrane Proteins; Mice; Plasmodium berghei; Protozoan Proteins; Receptors, Cell Surface; Sporozoites
PubMed: 35628522
DOI: 10.3390/ijms23105711 -
Parasites & Vectors Jan 2017Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted...
BACKGROUND
Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle.
METHODS
We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene.
RESULTS
Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts.
CONCLUSIONS
The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates.
Topics: Animals; Antibodies, Protozoan; Antigens, Protozoan; Disease Models, Animal; Disease Transmission, Infectious; Gene Deletion; Immunization, Passive; Malaria; Malaria Vaccines; Mice; Plasmodium berghei; Protozoan Proteins
PubMed: 28057055
DOI: 10.1186/s13071-016-1932-4 -
Nature Communications Mar 2023Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational...
Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCF) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCF plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.
Topics: Humans; Erythrocytes; Plasmodium berghei; Protein Binding; S-Phase Kinase-Associated Proteins; Ubiquitination
PubMed: 36898988
DOI: 10.1038/s41467-023-36999-8