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Proceedings of the National Academy of... Oct 2023Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that...
Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of development in the mosquito vector. Disruption of AQP2 in the human parasite and the rodent parasite blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in , combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.
Topics: Animals; Aquaporin 2; Malaria; Mosquito Vectors; Phylogeny; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 37883438
DOI: 10.1073/pnas.2304339120 -
Frontiers in Immunology 2023Recent studies have suggested that CD8 liver-resident memory T (T) cells are crucial in the protection against liver-stage malaria. We used liver-directed...
Recent studies have suggested that CD8 liver-resident memory T (T) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver T cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. T cells (CD8 CD44 CD62L CD69 KLRG1) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8 T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease.
Topics: Animals; Mice; CD8-Positive T-Lymphocytes; Memory T Cells; Liver; Malaria; RNA, Messenger; Sporozoites
PubMed: 37680630
DOI: 10.3389/fimmu.2023.1116299 -
Genes Aug 2023Malaria in pregnancy (MiP) is a public health problem in malaria-endemic areas, contributing to detrimental outcomes for both mother and fetus. Primigravida and...
Malaria in pregnancy (MiP) is a public health problem in malaria-endemic areas, contributing to detrimental outcomes for both mother and fetus. Primigravida and second-time mothers are most affected by severe anemia complications and babies with low birth weight compared to multigravida women. Infected erythrocytes (IE) reach the placenta, activating the immune response by placental monocyte infiltration and inflammation. However, specific markers of MiP result in poor outcomes, such as low birth weight, and intrauterine growth restriction for babies and maternal anemia in women infected with are limited. In this study, we identified the plasma proteome signature of a mouse model infected with ANKA and pregnant women infected with infection using quantitative mass spectrometry-based proteomics. A total of 279 and 249 proteins were quantified in murine and human plasma samples, of which 28% and 30% were regulated proteins, respectively. Most of the regulated proteins in both organisms are involved in complement system activation during malaria in pregnancy. CBA anaphylatoxin assay confirmed the complement system activation by the increase in C3a and C4a anaphylatoxins in the infected plasma compared to non-infected plasma. Moreover, correlation analysis showed the association between complement system activation and reduced head circumference in newborns from -infected mothers. The data obtained in this study highlight the correlation between the complement system and immune and newborn outcomes resulting from malaria in pregnancy.
Topics: Infant, Newborn; Pregnancy; Infant; Female; Humans; Animals; Mice; Mice, Inbred CBA; Placenta; Complement Activation; Malaria; Biomarkers
PubMed: 37628675
DOI: 10.3390/genes14081624 -
Malaria Journal Nov 2023Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for...
BACKGROUND
Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays.
METHODS
IC values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 10 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH.
RESULTS
The IC values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification.
CONCLUSIONS
Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system.
Topics: Mice; Animals; Antibodies, Monoclonal; Plasmodium berghei; Plasmodium falciparum; RNA, Ribosomal, 18S; Protozoan Proteins; Animals, Genetically Modified; Antibodies, Protozoan; Malaria Vaccines
PubMed: 37936181
DOI: 10.1186/s12936-023-04765-2 -
Current Research in Pharmacology and... 2024infection is a health challenge. Although, antiplasmodial drugs kill the parasites, information on the effects of infection and drugs on the expression of some genes is...
infection is a health challenge. Although, antiplasmodial drugs kill the parasites, information on the effects of infection and drugs on the expression of some genes is limited. Malaria was induced in two different studies using NK65 (chloroquine-susceptible, study 1), and ANKA (chloroquine-resistant, study 2) strains of in 30 male Swiss mice (n = 5) in each study. Mice orally received 10 mL/kg distilled water, (infected control), Mefloquine (MF) (10 mg/kg), MF and Curcumin (CM) (25 mg/kg), MF and CM (50 mg/kg), CM (25 mg/kg) and CM (50 mg/kg). Five mice (un-infected) were used as the control. After treatment, total Ribonucleic acid (RNA) was isolated from liver and erythrocytes while Deoxyribonucleic acid (DNA)-free RNA were converted to cDNA. Polymerase Chain Reaction (PCR) amplification was performed and relative expressions of oxidoreductase, and cytochrome oxidase expressions were determined. Markers of glycolysis, toxicity and antioxidants were determined using ELISA assays. While the expression of was blunted by MF in the susceptible study, co-treatment with curcumin (25 mg/kg) yielded the same results in the chloroquine-resistant study. Similar results were obtained on in both studies. Curcumin decreased in both studies. infection decreased oxidoreductase and cytochrome oxidase but mefloquine-curcumin restored the expression of these genes. While glycolysis and toxicity were inhibited, antioxidant systems improved in the treated groups. Curcumin is needed for effective therapeutic efficacy and prevention of toxicity. infection and treatment modulate the expressions of some genes in the host. Curcumin combination with mefloquine modulates the expression of some genes in the host.
PubMed: 38725654
DOI: 10.1016/j.crphar.2024.100180 -
Ethiopian Journal of Health Sciences Sep 2023The increasing resistance to most antimalarial drugs suggests a need for better alternatives. This study evaluated in vivo antimalarial and liver antioxidant profile of...
BACKGROUND
The increasing resistance to most antimalarial drugs suggests a need for better alternatives. This study evaluated in vivo antimalarial and liver antioxidant profile of dry plantain leaf extract (Musa paradisiaca) on mice infected with Plasmodium berghei.
METHODS
Six groups of ten mice each grouped as control, P. berghei, artesunate, and P. berghei infected mice were orally administered 250,500 and 1000mg/kg Musa paradisiaca leaf extract for 5 days. Blood smears were evaluated for parasitaemia on the 10 day and the mice sacrificed. Catalase, Malondialdehyde, protein, Glutathione peroxidase and reduced glutathione was estimated using Colorimetric, Biuret and spectrophotometric methods respectively with data analyzed using SPSS version 21.
RESULTS
Catalase activity (umol/ml/mins) was 24.62 ± 0.99, 10.04 ± 0.50, 19.35 ± 0.38, 22.13 ± 0.00, 22.79 ± 0.00 and 23.66 ± 0.20 while Glutathione Peroxidase(u/l) was 332.34± 0.64, 205.22± 4.61, 218.26± 0.63, 310.59± 0.00, 305.20± 0.00. and 295.97± 0.02 at Control, P.berghei, artesunate, 250mg, 500mg and1000mg extracts. Glutathione (mM) was 1.60 ± 0.12, 0.64 ± 0.09, 1.06 ± 0.16, 0.72 ± 0.00, 0.92 ± 0.00 and 1.26 ± 0.08 while Malondialdehye (uM) was 16.93 ± 3.59, 61.65 ± 1.72, 27.80 ± 0.26, 36.90 ± 0.00, 34.30 ± 0.00 and 32.68 ± 0.27 and Protein(g/dl) was 22.37 ± 1.87, 7.91 ± 0.13, 11.78 ± 1.19, 11.79 ± 0.00, 13.20 ± 0.00 and 17.04 ±0.03 at control, P.berghei, artesunate, 250mg, 500mg and1000mg respectively.
CONCLUSION
The study suggested that ethanolic extract of Musa paradisiaca reduced liver oxidative stress caused by P.berghei.
Topics: Animals; Plant Extracts; Mice; Plasmodium berghei; Malaria; Liver; Plant Leaves; Antioxidants; Antimalarials; Musa; Malondialdehyde; Glutathione; Glutathione Peroxidase; Male; Catalase; Ethanol
PubMed: 38784501
DOI: 10.4314/ejhs.v33i5.6 -
Molecular Microbiology Mar 2024Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade...
Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.
Topics: Animals; Culicidae; Parasites; Protozoan Proteins; Oocysts; Plasmodium berghei; Sporozoites
PubMed: 37314965
DOI: 10.1111/mmi.15078 -
BMC Microbiology Sep 2023Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported...
BACKGROUND
Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported to date. Identification of new disease-related genes as many as possible will provide a landscape for better understanding the pathogenesis of P. berghei.
METHODS
Network module analysis method was developed and applied to identify disease-related genes in P. berghei genome. Sequence feature identification, gene ontology annotation, and T-cell epitope analysis were performed on these genes to illustrate their functions in the pathogenesis of P. berghei.
RESULTS
33,314 genes were classified into 4,693 clusters. 4,127 genes shared by six malaria parasites were identified and are involved in many aspects of biological processes. Most of the known essential genes belong to shared genes. A total of 63 clusters consisting of 405 P. berghei genes were enriched in rodent malaria parasites. These genes participate in various stages of parasites such as liver stage development and immune evasion. Combination of these genes might be responsible for P. berghei infecting mice. Comparing with P. chabaudi, none of the clusters were specific to P. berghei. P. berghei lacks some proteins belonging to P. chabaudi and possesses some specific T-cell epitopes binding by class-I MHC, which might together contribute to the occurrence of experimental cerebral malaria (ECM).
CONCLUSIONS
We successfully identified disease-associated P. berghei genes by network module analysis. These results will deepen understanding of the pathogenesis of P. berghei and provide candidate parasite genes for further ECM investigation.
Topics: Humans; Animals; Mice; Plasmodium berghei; Gene Ontology; Genes, Essential; Immune Evasion; Molecular Sequence Annotation
PubMed: 37735351
DOI: 10.1186/s12866-023-03019-0 -
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 -
Nature Communications Dec 2023Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian...
Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.
Topics: Male; Animals; Plasmodium berghei; Haploidy; Germ Cells; Anopheles; Flagella; Mammals
PubMed: 38092766
DOI: 10.1038/s41467-023-43877-w