Selection linked integration (SLI) for endogenous gene tagging and knock sideways in Plasmodium falciparum parasites

Birnbaum, Jakob; Flemming, Sven; Reichard, Nick; Soares, Alexandra Blancke; Mesén-Ramírez, Paolo; Jonscher, Ernst; Bergmann, Bärbel; Spielmann, Tobias

doi:10.1038/protex.2017.022

Cited by 4 publications

(4 citation statements)

References 8 publications

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“…To study the biosynthesis of DGs and the movement of DG proteins through the secretory system, we fused the gene encoding the well-established DG marker RESA [22] with the gene encoding mNG. This fusion was introduced into 3D7 parasites and integration into the native RESA locus was selected for using selection-linked integration (SLI) [58] (). Integration of the RESA-mNG gene fusion into the native RESA locus was verified using PCR and production of the expected mNG fusion protein product was verified by immunoblotting ().…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…We aimed to produce a plasmid encoding a fusion of mNeonGreen (mNG) to the 3′ region of RESA (PF3D7_0102200), followed by sequence encoding the T2A skip peptide and a neomycin resistance marker, to allow for selection of parasites with integrated plasmid using G418 [58,59]. First, the gene encoding GFP was removed from the plasmid pRESA-GFP [60] by digestion with PstI and MluI and replaced with a 705 bp insert encoding mNG that was amplified using primers CVO550 and CVO551 (Table S1, available in the online version of this article) and digested with PstI and MluI for insertion into the pRESA-GFP backbone, producing plasmid pTV001.…”

Section: Plasmidsmentioning

confidence: 99%

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Timing of dense granule biogenesis in asexual malaria parasites

Vallintine,

van Ooij

2023

Microbiology

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Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles – micronemes, rhoptries and dense granules (DGs) – whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

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Timing of dense granule biogenesis in asexual malaria parasites

Vallintine,

van Ooij

2023

Microbiology

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“…1A). We used a pKIC-ter plasmid for inducing selection-linked integration (SLI) (17). SLI allows the expression of a target protein to be controlled by an endogenous promoter, which is important for LLPS studies, as slight modifications of the expression level of target proteins easily influence LLPS.…”

Section: Pfk9 Condensates Appeared In Pf Parasites Under Low-glucose ...mentioning

confidence: 99%

Glycolytic enzymes form membrane-less condensates in the malaria parasitePlasmodium falciparumby sensing glucose levels

Ishii,

Sakura,

Inaoka

et al. 2024

Preprint

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Recent studies have shown that liquid-liquid phase separation (LLPS) in cells can regulate essential cellular events, including metabolic processes. Glycolytic bodies (G-bodies) are biomolecular condensates formed through the LLPS of glycolytic enzymes, and they accelerate glycolysis to overcome hypoxic stress in several organisms. Although the asexual blood stage (ABS) of the human malaria parasitePlasmodium falciparumhighly depends on glycolysis for energy production, there have been no reports of the formation of such G-bodies throughout the parasite’s lifecycle. Using fluorescence tagging and live imaging, we found that G-body-like condensates containing phosphofructokinase 9 (PFK 9) and phosphoglycerate kinase (PGK) were formed in the parasite cells after long-term culture under conditions of low glucose. These G-body-like structures appeared stable, but membrane staining and osmotic stress experiments suggested that the observed condensates were not associated with lipid membrane. Further microscopic observations and mathematical analyses of high signal-to-noise ratio images indicated that small condensates were formed transiently first, and these then gradually grew and stabilized in the cytosol. These results suggested that the formation of glycolytic enzyme condensates may be an important cellular response for adapting to blood sugar level oscillations in the host and maintaining the parasite’s multiplication in the ABS.Significance statementGlycolytic bodies (G-bodies), which are biomolecular condensates formed through the liquid-liquid phase separation of glycolytic enzymes, can accelerate glycolysis to produce energy and overcome hypoxic stress. The parasites that cause malaria depend on glycolysis for energy production, but there have been no reports that these parasites form G-bodies. We demonstrated that membrane-less G-body-like structures formed in media containing low levels of glucose. Small condensates appeared first and over time, the condensates became larger and more stable. The formation of glycolytic enzyme condensates may be important for the malaria parasite to adapt to fluctuating blood sugar levels in the host. These results further our understanding of the cellular mechanisms for the survival of malaria parasites.

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A genetic system to study Plasmodium falciparum protein function

et al. 2017

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Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

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Selection linked integration (SLI) for endogenous gene tagging and knock sideways in Plasmodium falciparum parasites

Cited by 4 publications

References 8 publications

Timing of dense granule biogenesis in asexual malaria parasites

Timing of dense granule biogenesis in asexual malaria parasites

Glycolytic enzymes form membrane-less condensates in the malaria parasitePlasmodium falciparumby sensing glucose levels

A genetic system to study Plasmodium falciparum protein function

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