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Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites.Scientific Reports Feb 2021In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian...
In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite's development in the mosquito.
Topics: Computational Biology; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Genetic Heterogeneity; High-Throughput Nucleotide Sequencing; Malaria; Organ Specificity; Plasmodium berghei; Single-Cell Analysis; Sporozoites; Transcriptome
PubMed: 33619283
DOI: 10.1038/s41598-021-82914-w -
PloS One 2015Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the...
Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ) and artemisinin (ART) are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ) and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT) and the multidrug resistant gene (pfmdr), CQ resistance has also been associated with increased parasite glutathione (GSH) levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs) encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko) or higher (pbggcs-oe) levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ). Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.
Topics: Animals; Artemisinins; Chloroquine; Drug Resistance; Glutamate-Cysteine Ligase; Glutathione; Malaria; Mice; Plasmodium berghei; Protozoan Proteins
PubMed: 26010448
DOI: 10.1371/journal.pone.0128212 -
Life Science Alliance Dec 2020Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of...
Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model DEH is evolutionarily conserved across eukaryotes, with a single DEH in spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δ mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δ oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.
Topics: Animals; Anopheles; Cell Division; Coenzyme A Ligases; Endoplasmic Reticulum; Enoyl-CoA Hydratase; Fatty Acids; Female; Life Cycle Stages; Malaria; Mice; Mitosis; Oocysts; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 33106323
DOI: 10.26508/lsa.202000879 -
Malaria Journal Jun 2017Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo...
BACKGROUND
Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.
RESULTS
First, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.
CONCLUSIONS
These findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.
Topics: Animals; Blood-Brain Barrier; Erythrocytes; Female; Fumarate Hydratase; Fumarates; Malaria, Cerebral; Malates; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Oxaloacetic Acid; Oxidoreductases; Plasmodium berghei; Specific Pathogen-Free Organisms
PubMed: 28606087
DOI: 10.1186/s12936-017-1898-5 -
MBio Jun 2016Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei...
UNLABELLED
Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.
IMPORTANCE
Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite's successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite's autophagic pathway may represent a novel approach to control malarial infections.
Topics: Acyl Carrier Protein; Animals; Apicoplasts; Autophagy; Autophagy-Related Protein 8 Family; Hepatocytes; Humans; Liver; Malaria; Membrane Proteins; Merozoites; Mice, Transgenic; Mutation; Organelles; Plasmodium berghei; Protozoan Proteins
PubMed: 27353755
DOI: 10.1128/mBio.00682-16 -
MSphere Aug 2023During invasion, parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A...
During invasion, parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver . Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, , we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver . Combined with investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in merozoites and tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of parasites.
Topics: Animals; Mice; Plasmodium berghei; Liver; Malaria; Sporozoites; Protozoan Proteins; Hepatocytes
PubMed: 37272704
DOI: 10.1128/msphere.00587-22 -
Molecular Microbiology Jun 2006Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and...
Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3- parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3- ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3- sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3.
Topics: Animals; Anopheles; Cell Movement; Epithelium; Gene Deletion; Insect Vectors; Intestines; Mice; Plasmodium berghei; Protein Kinases; Protozoan Proteins
PubMed: 16796674
DOI: 10.1111/j.1365-2958.2006.05189.x -
Experimental Parasitology Dec 2014We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate...
We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate of the human parasite, Plasmodium falciparum. We analyzed the whole coding sequence of Plasmodium berghei chloroquine resistance transporter (Pbcrt) and Plasmodium berghei multidrug resistance gene 1(Pbmdr-1) for polymorphisms. These genes are associated with quinoline resistance in Plasmodium falciparum. No polymorphic changes were detected in the coding sequences of Pbcrt and Pbmdr1 or in the mRNA transcript levels of Pbmdr1. However, our data demonstrated that PQ and LM resistance is achieved by multiple mechanisms that include elevated mRNA transcript levels of V-type H(+) pumping pyrophosphatase (vp2), Ca(2+)/H(+) antiporter (vcx1), gamma glutamylcysteine synthetase (ggcs) and glutathione-S-transferase (gst) genes, mechanisms also known to contribute to chloroquine resistance in P. falciparum and rodent malaria parasites. The increase in ggcs and gst transcript levels was accompanied by high glutathione (GSH) levels and elevated activity of glutathione-S-transferase (GST) enzyme. Taken together, these results demonstrate that Pbcrt and Pbmdr1 are not associated with PQ and LM resistance in P. berghei ANKA, while vp2, vcx1, ggcs and gst may mediate resistance directly or modulate functional mutations in other unknown genes.
Topics: Animals; Antimalarials; Antiporters; Cation Transport Proteins; Cloning, Molecular; DNA, Protozoan; Drug Resistance, Multiple; Ethanolamines; Fluorenes; Gene Expression Regulation, Enzymologic; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Lumefantrine; Male; Mice; Parasitic Sensitivity Tests; Plasmodium berghei; Quinolines; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 25448357
DOI: 10.1016/j.exppara.2014.10.008 -
Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission.EMBO Reports Jul 2022Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual...
Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.
Topics: Animals; Culicidae; Malaria, Falciparum; Nonmuscle Myosin Type IIA; Phosphorylation; Plasmodium berghei; Protozoan Proteins; Serine; Sporozoites
PubMed: 35506479
DOI: 10.15252/embr.202254857 -
Cellular Microbiology Jan 2021The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole...
The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterised by a long-lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3-phosphate (PI3P)-labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P-labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3-labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.
Topics: Animals; Autophagy; Cell Line; Female; HeLa Cells; Hepatocytes; Host-Parasite Interactions; Humans; Malaria; Mice; Microtubule-Associated Proteins; Organisms, Genetically Modified; Phosphatidylinositol Phosphates; Plasmodium berghei; Sporozoites; Vacuoles
PubMed: 32979009
DOI: 10.1111/cmi.13271