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Journal of Proteomics Sep 2020Passage of malaria parasites through mosquitoes involves multiple developmental transitions, from gametocytes that are ingested with the blood meal, through to...
Passage of malaria parasites through mosquitoes involves multiple developmental transitions, from gametocytes that are ingested with the blood meal, through to sporozoites that are transmitted by insect bite to the host. During the transformation from gametocyte to oocyst, the parasite forms a unique transient organelle named the crystalloid, which is involved in sporozoite formation. In Plasmodium berghei, a complex of six LCCL domain-containing proteins (LAPs) reside in the crystalloid and are required for its biogenesis. However, little else is known about the molecular mechanisms that underlie the crystalloid's role in sporogony. In this study, we have used transgenic parasites stably expressing LAP3 fused to GFP, combined with GFP affinity pulldown and high accuracy mass spectrometry, to identify an extended LAP interactome of some fifty proteins. We show that many of these are targeted to the crystalloid, including members of two protein families with CPW-WPC and pleckstrin homology-like domains, respectively. Our findings indicate that the LAPs are part of an intricate protein complex, the formation of which facilitates both crystalloid targeting and biogenesis. SIGNIFICANCE: Reducing malaria parasite transmission by mosquitoes is a key component of malaria eradication and control strategies. This study sheds important new light on the molecular composition of the crystalloid, an enigmatic parasite organelle that is essential for sporozoite formation and transmission from the insect to the vertebrate host. Our findings provide new mechanistic insight into how proteins are delivered to the crystalloid, and indicate that the molecular mechanisms that underlie crystalloid function are complex, involving several protein families unique to Plasmodium and closely related organisms. The new crystalloid proteins identified will form a useful starting point for studies aimed at unravelling how the crystalloid organelle facilitates sporogony and transmission.
Topics: Animals; Crystalloid Solutions; Humans; Malaria; Organelles; Plasmodium berghei; Protozoan Proteins
PubMed: 32736136
DOI: 10.1016/j.jprot.2020.103925 -
Molecular & Cellular Proteomics : MCP Dec 2020, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus In red blood cells of the vertebrate...
, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus In red blood cells of the vertebrate host, multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.
Topics: Animals; Erythrocytes; Female; Gametogenesis; Germ Cells; Life Cycle Stages; Male; Mice; Plasmodium berghei; Proteome; Proteomics; Protozoan Proteins; Subcellular Fractions; Transport Vesicles
PubMed: 32883804
DOI: 10.1074/mcp.RA120.002212 -
Infection and Immunity Jan 2023In the acidic lysosome-like digestive vacuole, parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the...
In the acidic lysosome-like digestive vacuole, parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of liver stages.
Topics: Mice; Animals; Malaria; Mice, Inbred BALB C; Plasmodium berghei; Sporozoites; Liver; Heme; DNA
PubMed: 36622216
DOI: 10.1128/iai.00304-22 -
Emerging Microbes & Infections Dec 2022The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in...
The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the parasite growth, and also impaired the transmission cycle of and in . Our findings indicate that the deletion of MCA-2 hampers the development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology.
Topics: Animals; Gametogenesis; Humans; Malaria; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 35264080
DOI: 10.1080/22221751.2022.2052357 -
Nucleic Acids Research Jan 2020The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered...
The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.
Topics: Animals; Anopheles; Argonaute Proteins; Female; Genes, Reporter; Genetic Engineering; Green Fluorescent Proteins; Life Cycle Stages; Mice; Mice, Inbred C57BL; Mosquito Vectors; Organisms, Genetically Modified; Perforin; Plasmodium berghei; Protozoan Proteins; RNA Interference; RNA, Small Interfering; Transgenes
PubMed: 31680162
DOI: 10.1093/nar/gkz927 -
BioMed Research International 2023Emergence of resistance to antimalarial drugs presents a major drawback in efforts to control malaria. To address this problem, there is an urgent and continuous need...
BACKGROUND
Emergence of resistance to antimalarial drugs presents a major drawback in efforts to control malaria. To address this problem, there is an urgent and continuous need for the development of new and effective antimalarial agents. (L.) link extract has exhibited antiplasmodial activity in many pharmacological studies. To our knowledge, data on its antimalarial efficacy is still very limited. A recent study demonstrated that polar extracts from the plant roots inhibit proliferation in a mouse model. This study further describes the efficacy and safety of a methanolic root extract of the plant as an antimalarial agent by demonstrating its effect on hematological, biochemical, and histological parameters of -infected BALB/c mice.
METHODS
Rane's test, a curative approach, was used to evaluate the antimalarial efficacy of methanolic root extract in -infected BALB/c mice. The effect of the extract on both hematological and biochemical parameters was evaluated using automated analyzers. Kidney, liver, lung, spleen, and brain tissues were harvested from euthanized mice and examined for changes in organ architecture.
RESULTS
This study demonstrates that methanolic root extract of significantly inhibited parasitemia in BALB/c mice ( < 0.01). Infected mice that were treated with the extract depicted a significantly low level of total leucocytes ( < 0.01), red blood cell distribution width ( < 0.01), and a significantly high hemoglobin concentration ( < 0.001) compared to the infected animals that were administered with the vehicle only. The infected animals that were treated with the extract exhibited a significantly low level of urea, creatinine, bilirubin, and alkaline phosphatase ( < 0.05), compared to the infected animals that were given the vehicle only. The level of sodium, potassium and chloride ions, lymphocytes, granulocytes, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, total protein, albumin, aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total platelets, mean platelet volume (MPV), and platelet distribution width of the infected animals treated with the extract was not significantly different from those of the infected animals that were given the vehicle only ( > 0.05). The extract alleviated organ pathological changes in the infected mice. The extract did not induce any remarkable adverse effect on the growth, hematological, and biochemical parameters of uninfected animals ( > 0.05). In addition, administration of the extract did not alter the gross appearance and histological architecture of the organs, implying that the extract was well tolerated in mice.
CONCLUSIONS
methanolic root extract exhibited good antimalarial activity against and may be safe in mice.
Topics: Mice; Animals; Antimalarials; Plasmodium berghei; Senna Plant; Mice, Inbred BALB C; Alkaline Phosphatase; Plant Extracts
PubMed: 37583959
DOI: 10.1155/2023/8296195 -
Malaria Journal Jul 2020Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These...
BACKGROUND
Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65.
METHODS
C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer.
RESULTS
Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia.
CONCLUSION
Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.
Topics: Acute Lung Injury; Animals; Bronchoalveolar Lavage Fluid; Female; Heme Oxygenase-1; Leukocyte Count; Lung; Malaria; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Plasmodium berghei; Protective Agents; Thrombocytopenia
PubMed: 32611348
DOI: 10.1186/s12936-020-03305-6 -
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 -
Pathogens and Global Health Mar 2015Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation... (Review)
Review
Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation to a range of rodent and mosquito species established P. berghei as a malaria model parasite. The introduction of stable transfection technology, permitted classical reverse genetics strategies and thus systematic functional profiling of the gene repertoire. In the past 10 years following the publication of the P. berghei genome sequence, many new tools for experimental genetics approaches have been developed and existing ones have been improved. The infection of mice is the principal limitation towards a genome-wide repository of mutant parasite lines. In the past few years, there have been some promising and most welcome developments that allow rapid selection and isolation of recombinant parasites while simultaneously minimising animal usage. Here, we provide an overview of all the currently available tools and methods.
Topics: Animals; Anopheles; Disease Models, Animal; Genetic Vectors; Genome-Wide Association Study; Host-Parasite Interactions; Life Cycle Stages; Malaria; Mice; Molecular Sequence Data; Organisms, Genetically Modified; Plasmodium berghei; Rats; Transfection
PubMed: 25789828
DOI: 10.1179/2047773215Y.0000000006 -
Molecular Microbiology Jul 2016Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including...
Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.
Topics: Animals; Antimalarials; Culicidae; Drug Resistance, Multiple; Female; Life Cycle Stages; Malaria; Malaria, Falciparum; Male; Membrane Transport Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Multidrug Resistance-Associated Proteins; Oocytes; Plasmodium berghei; Plasmodium falciparum; Sporozoites
PubMed: 26991313
DOI: 10.1111/mmi.13373