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Military Medicine Jan 2021The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved...
BACKGROUND
The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved understanding of P. berghei life cycle stage growth and development would provide new opportunities to evaluate antimalarial vaccines and drugs.
METHODS
Blood stage samples from C57BL/6 albino mice infected with P. berghei sporozoites were singly stained with a high binding affinity deoxyribonucleic acid dye, YOYO-1, and measured by flow cytometry (FCM). Duplicate slides were made from samples and stained with diluted Giemsa's and YOYO-1, respectively. Correlated results were compared by FCM, light microscopy, and fluorescent microscopy.
RESULTS
Complete life cycle stage determination and analysis by FCM is reported to include merozoites, ring forms, trophozoites, immature, and mature schizonts. FCM demonstrated a clear separation between each stage using their unique fluorescence distribution. When compared to light microscopy, a strong correlation (r 2 = 0.925 to 0.974) was observed in determining the ring forms, trophozoites, and schizonts phases, but only a moderate correlation (r 2 = 0.684 to 0.778) was observed for merozoites. The identification and measurement of merozoites suggest that FCM is a useful technique to monitor the entire life stage of the parasite. Initial stage-specific data demonstrated that mefloquine has a mode of action on mature parasite forms, and artesunic acid was rapidly effective against merozoites and other immature and mature parasite forms with higher killing.
CONCLUSION
Blood stage parasites in each individual life stage, including merozoites, are reliably identified and quantified quickly by FCM, making this technique an ideal alternative to microscopy. This integrated whole life stage model, particularly with confirmed determination of merozoite population, could widely be used for drug and vaccine research in malaria therapy and prophylaxis.
Topics: Animals; Cell Cycle; Flow Cytometry; Malaria; Merozoites; Mice; Plasmodium berghei
PubMed: 33499463
DOI: 10.1093/milmed/usaa272 -
Scientific Reports Aug 2023Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host,...
Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.
Topics: Animals; Oocysts; Plasmodium berghei; Mosquito Vectors; Malaria, Falciparum; Anopheles; Fatty Acids; Protozoan Proteins
PubMed: 37543672
DOI: 10.1038/s41598-023-39708-z -
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 -
Angewandte Chemie (International Ed. in... Jul 2020Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a...
Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell- and tissue-specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one-pot intein-mediated ligation (OPL) to obtain GPI-anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP1 were prepared. Glypiation did not alter the structure of eGFP and MSP1 proteins in solution, but it induced a strong pro-inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.
Topics: Bacterial Proteins; Bacterial Vaccines; Carbohydrate Sequence; Glycolipids; Glycosylphosphatidylinositols; Green Fluorescent Proteins; Humans; Membrane Proteins; Models, Molecular; Plasmodium berghei; Protein Processing, Post-Translational
PubMed: 32307806
DOI: 10.1002/anie.202002479 -
Nature Nov 2022Malaria infection involves an obligatory, yet clinically silent liver stage. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene...
Malaria infection involves an obligatory, yet clinically silent liver stage. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing and single-molecule transcript imaging to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.
Topics: Animals; Hepatocytes; Liver; Malaria; Parasites; Plasmodium berghei; Single-Cell Analysis; Single Molecule Imaging; Sequence Analysis, RNA; Gene Expression Regulation; Iron; Fatty Acids; Transcription, Genetic; Genes, Protozoan; Host-Parasite Interactions
PubMed: 36352220
DOI: 10.1038/s41586-022-05406-5 -
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 -
Chinese Journal of Integrative Medicine Apr 2020To study the antimalarial effects and mechanisms of artemisinin (Qinghaosu in Chinese, QHS) on mitochondria in mice infected with Plasmodium berghei.
OBJECTIVE
To study the antimalarial effects and mechanisms of artemisinin (Qinghaosu in Chinese, QHS) on mitochondria in mice infected with Plasmodium berghei.
METHODS
A total of 108 C57 mice infected with Plasmodium berghei were randomly divided into 3 groups by weight: the control group, 200 and 400 mg/kg QHS groups. The two QHS treatment groups were further divided into 4 sub-groups with 12 animals each time according to the treatment time, 0.5, 1, 2, and 4 h. Normal saline was intragastrically (i.g.) administered to the control group. The other two groups received different doses of QHS by i.g. administration. Animals were treated once with QHS for different detection time as follows: 0.5, 1, 2, and 4 h. The mitochondrial energy metabolism, oxidative damage, membrane potential, and membrane permeability and other indexes were detected.
RESULTS
After administration of 200 and 400 mg/kg QHS, adenosine triphosphate (ATP) levels in Plasmodium and its mitochondria were reduced (P<0.05), the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were increased (P<0.05), and the activity of superoxide dismutase (SOD) was also increased (P<0.05). At the same time, the membrane potential of the mitochondria was reduced and the degree to which the membrane permeability transition pore was opened was irreversibly increased (P<0.05).
CONCLUSIONS
Mitochondria in Plasmodium were the targets of QHS, which can adversely affect mitochondrial energy metabolism, oxidative damage, membrane potential, and membrane opening, and ultimately exert an antimalarial effect.
Topics: Animals; Antimalarials; Artemisinins; Energy Metabolism; Malaria, Falciparum; Membrane Potentials; Mice; Mitochondria; Oxidative Stress; Plasmodium berghei; Reactive Oxygen Species; Superoxide Dismutase
PubMed: 31227963
DOI: 10.1007/s11655-019-3164-x -
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