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EMBO Reports Jul 2023Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are...
Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.
Topics: Animals; Sporozoites; Ligands; Plasmodium; Culicidae; Liver; Protozoan Proteins; Plasmodium berghei; Mammals
PubMed: 37306042
DOI: 10.15252/embr.202357064 -
Frontiers in Cellular and Infection... 2022Malaria-associated acute respiratory distress syndrome (MA-ARDS) is increasingly gaining recognition as a severe malaria complication because of poor prognostic... (Review)
Review
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is increasingly gaining recognition as a severe malaria complication because of poor prognostic outcomes, high lethality rate, and limited therapeutic interventions. Unfortunately, invasive clinical studies are challenging to conduct and yields insufficient mechanistic insights. These limitations have led to the development of suitable MA-ARDS experimental mouse models. In patients and mice, MA-ARDS is characterized by edematous lung, along with marked infiltration of inflammatory cells and damage of the alveolar-capillary barriers. Although, the pathogenic pathways have yet to be fully understood, the use of different experimental mouse models is fundamental in the identification of mediators of pulmonary vascular damage. In this review, we discuss the current knowledge on endothelial activation, leukocyte recruitment, leukocyte induced-endothelial dysfunction, and other important findings, to better understand the pathogenesis pathways leading to endothelial pulmonary barrier lesions and increased vascular permeability. We also discuss how the advances in imaging techniques can contribute to a better understanding of the lung lesions induced during MA-ARDS, and how it could aid to monitor MA-ARDS severity.
Topics: Animals; Disease Models, Animal; Humans; Lung; Malaria; Mice; Mice, Inbred C57BL; Plasmodium berghei; Respiratory Distress Syndrome
PubMed: 35677654
DOI: 10.3389/fcimb.2022.899581 -
Cellular Microbiology Jan 2017Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an...
Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.
Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites.
Topics: Apicoplasts; Bacteria; DNA Mutational Analysis; Fatty Acids; Gene Knockout Techniques; Genetic Complementation Test; Glycerol-3-Phosphate O-Acyltransferase; Plasmodium berghei; Plasmodium falciparum; Protein Transport
PubMed: 27324409
DOI: 10.1111/cmi.12633 -
BMC Genomics Sep 2017The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the... (Comparative Study)
Comparative Study
BACKGROUND
The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation.
RESULTS
RNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development.
CONCLUSIONS
We characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways.
Topics: Gene Expression Profiling; Nucleotide Motifs; Phylogeny; Plasmodium berghei; RNA, Messenger; Sequence Homology, Nucleic Acid
PubMed: 28923023
DOI: 10.1186/s12864-017-4100-0 -
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 -
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 -
Parasite (Paris, France) Sep 2009Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the... (Review)
Review
Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.
Topics: Animals; Hepatocytes; Humans; Life Cycle Stages; Malaria; Plasmodium; Plasmodium berghei; Plasmodium falciparum; Signal Transduction; Toxoplasma
PubMed: 19839262
DOI: 10.1051/parasite/2009163169 -
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 -
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