| 1. |
- Brugat, Thibaut, et al.
(författare)
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Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection
- 2017
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Ingår i: Nature Microbiology. - : Macmillan Publishers Ltd.. - 2058-5276. ; 2:4
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Tidskriftsartikel (refereegranskat)abstract
- Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting chronic infections(1-5), creating an infectious reservoir to sustain transmission(1,6). It is widely accepted that the maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation(7). However, genes involved in this process have been identified in only two of five human-infecting species: Plasmodium falciparum and Plasmodium knowlesi. Furthermore, little is understood about the early events in the establishment of chronic infection in these species. Using a rodent model we demonstrate that from the infecting population, only a minority of parasites, expressing one of several clusters of virulence-associated pir genes, establishes a chronic infection. This process occurs in different species of parasites and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintenance of chronic P. falciparum infections(7-9). Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Because pir genes are common to most, if not all, species of Plasmodium(10), this process may be a common way of regulating the establishment of chronic infections.
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| 2. |
- Bushell, Ellen, et al.
(författare)
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Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes
- 2017
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Ingår i: Cell. - : Cell Press. - 0092-8674 .- 1097-4172. ; 170:2, s. 260-272.e1-e4
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Tidskriftsartikel (refereegranskat)abstract
- The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.
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| 3. |
- Böhme, Ulrike, et al.
(författare)
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Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals
- 2018
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 28:4, s. 547-560
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Tidskriftsartikel (refereegranskat)abstract
- Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.
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| 4. |
- Frénal, Karine, et al.
(författare)
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Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion
- 2013
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Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 14:8, s. 895-911
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Tidskriftsartikel (refereegranskat)abstract
- The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.
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| 5. |
- Gomes, Ana Rita, et al.
(författare)
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A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite
- 2015
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Ingår i: Cell Host and Microbe. - : Cell Press. - 1931-3128 .- 1934-6069. ; 17:3, s. 404-413
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Tidskriftsartikel (refereegranskat)abstract
- The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.
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| 6. |
- Hillier, Charles, et al.
(författare)
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Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality
- 2019
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 28:6, s. 1635-1647
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Tidskriftsartikel (refereegranskat)abstract
- Malaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies > 20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets.
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| 7. |
- Hopp, Christine S, et al.
(författare)
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Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium
- 2016
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Ingår i: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 18:11, s. 1625-1641
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Tidskriftsartikel (refereegranskat)abstract
- As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post‐translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co‐localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non‐infectious to mice. Importantly, genetic complementation of the DHHC3‐ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation.
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| 8. |
- Ishizaki, Takahiro, et al.
(författare)
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CRISPR/Cas9 and genetic screens in malaria parasites : small genomes, big impact
- 2022
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Ingår i: Biochemical Society Transactions. - : Portland Press. - 0300-5127 .- 1470-8752. ; 50:3, s. 1069-1079
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Forskningsöversikt (refereegranskat)abstract
- The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.
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| 9. |
- Marr, Edward J, et al.
(författare)
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An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi
- 2020
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Ingår i: Wellcome open research. - : Wellcome open research. - 2398-502X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.
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| 10. |
- Ning, Jue, et al.
(författare)
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Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates
- 2013
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory Press (CSHL). - 0890-9369 .- 1549-5477. ; 27:10, s. 1198-1215
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Tidskriftsartikel (refereegranskat)abstract
- Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.
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