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Nature Reviews. Microbiology Jan 2017Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and... (Review)
Review
Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.
Topics: Animals; Cryoelectron Microscopy; Electron Microscope Tomography; Erythrocytes; Host-Parasite Interactions; Humans; Malaria; Merozoites; Plasmodium berghei; Plasmodium falciparum; Sporozoites; Time-Lapse Imaging
PubMed: 27890922
DOI: 10.1038/nrmicro.2016.158 -
Journal of Global Antimicrobial... Mar 2020The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo.
OBJECTIVES
The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo.
METHODS
Pyrimethamine-loaded nanomicelles were prepared and their zeta potential, particle size and polydispersity index were measured. For antiplasmodial assessment, 54 mice were randomly divided into six groups. Four groups were infected intraperitoneally with P. berghei, whereas the two remaining groups did not receive the parasite (negative controls). Three of the P. berghei-infected groups received treatment with either pyrimethamine-loaded nanomicelles (2 mg/kg), pyrimethamine (2 mg/kg) or empty nanomicelles (2 mg/kg); the fourth group remained untreated (positive control). The parasitaemia rate, survival rate and histopathological changes in the liver, spleen and kidneys were examined and were compared with the negative and positive control groups.
RESULTS
The mean parasitaemia rate differed significantly between the nanoformulated pyrimethamine group and each of the other groups (P<0.05). Moreover, the survival rate of mice in the nanoformulated pyrimethamine group (7/9; 78%) was significantly higher compared with each of the other groups (P<0.01). The main histopathological changes, including hepatic necrosis in the liver, lymphoid hypoplasia in the spleen, and tubular nephrosis and perivascular and interstitial lymphocytic infiltration in the kidneys, were considerably lower in the nanoformulated pyrimethamine group than in the pyrimethamine and positive control groups.
CONCLUSION
Pyrimethamine-loaded nanomicelles showed potent antimalarial activity and can be considered as a potential candidate for further examination of their suitability as an antimalarial drug.
Topics: Animals; Antimalarials; Disease Models, Animal; Drug Compounding; Liver; Malaria; Male; Mice; Micelles; Nanoparticles; Parasitemia; Particle Size; Plasmodium berghei; Poloxamer; Pyrimethamine; Random Allocation; Spleen; Survival Analysis; Treatment Outcome
PubMed: 31404680
DOI: 10.1016/j.jgar.2019.08.002 -
International Journal of Antimicrobial... Dec 2023The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes...
BACKGROUND
The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AM) parasites from Plasmodium berghei ANKA (AM), and determine their fitness cost.
METHODS
Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED and ED) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I and I) were determined using the ratio of either ED or ED of AM to AM, respectively. The stability of the AM parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AM and AM with 95% confidence intervals.
RESULTS
ED and ED of AM were 0.61 and 3.43 mg/kg/day respectively. I and I after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AM (24.70% ± 3.60) relative to AM (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%.
CONCLUSION
A moderately stable AMP. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended.
Topics: Animals; Humans; Artemether; Antimalarials; Malaria; Parasites; Plasmodium berghei; Plasmodium falciparum; Drug Resistance
PubMed: 37865152
DOI: 10.1016/j.ijantimicag.2023.107012 -
Advances in Experimental Medicine and... 2016To understand much of the behaviour of microbial pathogens, it is necessary to image living cells, their interactions with each other and with host cells. Species such... (Review)
Review
To understand much of the behaviour of microbial pathogens, it is necessary to image living cells, their interactions with each other and with host cells. Species such as Escherichia coli are difficult subjects to image: they are typically microscopic, colourless and transparent. Traditional cell visualisation techniques such as fluorescent tagging or phase-contrast microscopy give excellent information on cell behaviour in two dimensions, but no information about cells moving in three dimensions. We review the use of digital holographic microscopy for three-dimensional imaging at high speeds, and demonstrate its use for capturing the shape and swimming behaviour of three important model pathogens: E. coli, Plasmodium spp. and Leishmania spp.
Topics: Escherichia coli; Holography; Image Processing, Computer-Assisted; Leishmania mexicana; Microscopy; Movement; Optical Imaging; Plasmodium berghei; Time Factors
PubMed: 27193535
DOI: 10.1007/978-3-319-32189-9_3 -
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 -
Nature Reviews. Microbiology Aug 2016Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular... (Review)
Review
Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host.
Topics: Amino Acid Motifs; Animals; Erythrocytes; Host-Pathogen Interactions; Humans; Immune Evasion; Malaria; Malaria, Falciparum; Plasmodium berghei; Plasmodium falciparum; Protein Sorting Signals; Protein Translocation Systems; Protein Transport; Protozoan Proteins; Transport Vesicles
PubMed: 27374802
DOI: 10.1038/nrmicro.2016.79 -
Parasite Immunology Nov 2020Merozoite surface protein 8 (MSP-8) of Plasmodium parasites plays an important role in erythrocyte invasion and is a potential malaria vaccine candidate.
AIMS
Merozoite surface protein 8 (MSP-8) of Plasmodium parasites plays an important role in erythrocyte invasion and is a potential malaria vaccine candidate.
METHODS AND RESULTS
In this study, virus-like particles (VLPs) expressing MSP-8 of Plasmodium berghei on the surface of influenza virus matrix protein 1 (M1) core protein were generated for vaccine efficacy assessment. Mice were intramuscularly (IM) immunized with MSP-8 VLPs twice and challenge-infected with P. berghei. We found that VLP vaccination elicited higher levels of P. berghei-specific IgG antibody response in the sera, along with blood CD4 and CD8 T-cell response enhancement compared to the naïve control mice. CD4 and CD8 effector memory T-cell and memory B-cell responses in the spleen were found to be higher in VLP-immunized mice compared to control mice. VLP vaccination significantly reduced inflammatory cytokine (IFN-γ) response in the spleen and parasitemia levels in blood compared to naïve control mice.
CONCLUSIONS
These results indicate that MSP-8 containing virus-like particles could be a vaccine candidate for blood-stage vaccine design.
Topics: Animals; Antigens, Protozoan; Female; Immunization; Malaria; Malaria Vaccines; Mice; Mice, Inbred BALB C; Parasitemia; Plasmodium berghei; Protozoan Proteins
PubMed: 32738150
DOI: 10.1111/pim.12781 -
JCI Insight Dec 2019Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing....
Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing. Efforts toward eradication are threatened by the lack of an effective vaccine and a rise in antiparasite drug resistance. Alternative approaches are urgently needed. Repurposing of available, approved drugs with distinct modes of action are being considered as viable and immediate adjuncts to standard antimicrobial treatment. Such strategies may be well suited to the obligatory and clinically silent first phase of Plasmodium infection, where massive parasite replication occurs within hepatocytes in the liver. Here, we report that the widely used antidiabetic drug, metformin, impairs parasite liver stage development of both rodent-infecting Plasmodium berghei and human-infecting P. falciparum parasites. Prophylactic treatment with metformin curtails parasite intracellular growth in vitro. An additional effect was observed in mice with a decrease in the numbers of infected hepatocytes. Moreover, metformin provided in combination with conventional liver- or blood-acting antimalarial drugs further reduced the total burden of P. berghei infection and substantially lessened disease severity in mice. Together, our findings indicate that repurposing of metformin in a prophylactic regimen could be considered for malaria chemoprevention.
Topics: Animals; Antimalarials; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Repositioning; Drug Therapy, Combination; Hepatocytes; Humans; Inhibitory Concentration 50; Liver; Malaria; Male; Mefloquine; Metformin; Mice; Parasite Load; Parasitic Sensitivity Tests; Plasmodium berghei; Plasmodium falciparum; Primaquine; Primary Cell Culture
PubMed: 31852843
DOI: 10.1172/jci.insight.127441 -
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 -
Infection and Immunity Aug 2018Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene...
Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene (PBANKA_060330) from and studied its potential for use as a TBV. This gene, referred to as , encodes a protein of 37 kDa with a signal peptide and multiple transmembrane domains and is preferentially expressed in gametocytes. A recombinant Pbg37 (rPbg37) protein targeting the N-terminal 63 amino acids (amino acids 26 to 88) expressed in bacteria elicited strong antibody responses in mice. Western blotting demonstrated Pbg37 expression in gametocytes, zygotes, and, to a lesser extent, ookinetes and its predominant association with the membranes of gametocytes. Indirect immunofluorescence assay showed an abundant surface localization of Pbg37 on gametes and zygotes but reduced amounts on retorts and ookinetes. Knockout of (Δ) led to a considerable reduction in gametocytemia, which translated into a ~92.1% decrease in the oocyst number in mosquitoes. Deletion of had a more substantial influence on the development and maturation of microgametocytes. As a result, the Δ lines exhibited a higher female/male gametocyte ratio, fewer mature male gametocytes, and defects in the exflagellation of mature microgametocytes. To test the transmission-blocking potential of Pbg37, an ookinete assay showed that the major inhibitory effects of anti-Pbg37 antiserum were on the exflagellation and fertilization processes. Direct feeding of mosquitoes on mice immunized with rPbg37 or a control protein showed that rPbg37-immunized and -infected mice had a significant reduction (49.1%) in oocyst density compared to the controls. The conservation of this gene in warrants further investigations in human malaria parasites.
Topics: Animals; Antibodies, Protozoan; Antibody Formation; Blotting, Western; Disease Models, Animal; Disease Transmission, Infectious; Female; Fluorescent Antibody Technique, Indirect; Gene Deletion; Gene Expression Profiling; Malaria; Malaria Vaccines; Male; Membrane Proteins; Mice, Inbred BALB C; Mosquito Vectors; Parasite Load; Parasitemia; Plasmodium berghei; Protozoan Proteins; Vaccines, Synthetic; Virulence
PubMed: 29866905
DOI: 10.1128/IAI.00785-17