A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites

Swift, Russell P.; Rajaram, Krithika; Liu, Hans; Dziedzic, Amanda; Jedlicka, Anne; Roberts, Aleah D.; Matthews, Krista Ann; Jhun, Hugo; Bumpus, Namandjé N.; Tewari, Shivendra G.; Wallqvist, Anders; Prigge, Sean T.

doi:10.1371/journal.ppat.1008316

Cited by 41 publications

(109 citation statements)

References 73 publications

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“…To investigate the role and essentiality of carbon metabolism within the organelle of P. falciparum parasites, we attempted to delete the genes encoding oTPT (PF3D7_0508300) and iTPT (PF3D7_0530200) using Cas9-mediated genome editing ( Ghorbal et al, 2014 ; Wagner et al, 2014 ). Deletions were generated in the PfMev apicoplast bypass line, which is engineered to convert supplemented mevalonate into isoprenoid precursors in the parasite cytosol, allowing these parasites to survive disruption of the apicoplast organelle ( Swift et al, 2020 ). We confirmed the deletion of oTPT and iTPT, and the absence of parental parasites using genotyping PCR reactions ( Figures 2a and 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…We were unable to amplify a gene from the apicoplast genome ( sufB ) in the PfMev Δ otpt and PfMev Δ itpt parasite lines, indicating that the organellar genome had been lost in both lines ( Figure 2b ; Gisselberg et al, 2013 ). To visualize the apicoplast, super-folder green (SFG) was appended to the first 55 amino acids of the P. falciparum ACP (api-SFG), constituting the signal and transit peptide to direct trafficking to the apicoplast, as previously described ( Swift et al, 2020 ). Disruption of the apicoplast was confirmed via live epifluorescence microscopy, which showed multiple vesicles throughout the cell instead of the discrete intact structure that is typically observed in wild type parasites ( Figure 2c ; Gisselberg et al, 2013 ; Yeh and DeRisi, 2011 ).…”

Section: Resultsmentioning

confidence: 99%

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The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

Swift

Rajaram

Keutcha

et al. 2020

eLife

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The apicoplast of Plasmodium falciparum parasites is believed to rely on the import of three-carbon phosphate compounds for use in organelle anabolic pathways, in addition to the generation of energy and reducing power within the organelle. We generated a series of genetic deletions in an apicoplast metabolic bypass line to determine which genes involved in apicoplast carbon metabolism are required for blood-stage parasite survival and organelle maintenance. We found that pyruvate kinase II (PyrKII) is essential for organelle maintenance, but that production of pyruvate by PyrKII is not responsible for this phenomenon. Enzymatic characterization of PyrKII revealed activity against all NDPs and dNDPs tested, suggesting that it may be capable of generating a broad range of nucleotide triphosphates. Conditional mislocalization of PyrKII resulted in decreased transcript levels within the apicoplast that preceded organelle disruption, suggesting that PyrKII is required for organelle maintenance due to its role in nucleotide triphosphate generation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

Swift

Rajaram

Keutcha

et al. 2020

eLife

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“…The Cas9 enzyme and the guide RNA were expressed from a modified version of pUF1-Cas9 ( 51 ). Plasmid pUF1-Cas9 (11,096 bp) was modified in several steps to accommodate the insertion of the guide RNA expression cassette found in pRS-LacZ ( 52 ). In the first step, the promoter driving Cas9 expression (Hsp86) was removed using XhoI and AflII and replaced with an adaptamer formed from 5′-phosphorylated primers pCas.XhoBtg.F and pCas.XhoBtg.R ( Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…The orientation with the U6 5′ regulatory element adjacent to Cas9 was chosen with the expectation that this region would act as a 3′ UTR for Cas9 (it contains the 3′ UTR of the ribosomal L18 protein, PF3D7_1341200). Finally, the plasmid was digested with BtgZI to remove the preguide RNA and the LacZ expression cassette from pRS-LacZ ( 52 ) was excised with BsaI and inserted into this site. The final pCasG-LacZ plasmid (10,886) is smaller than the original pUF1-Cas9 plasmid and contains a guide RNA expression cassette that can be used for blue/white colony screening after insertion of a guide RNA sequence with BsaI ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

Rajaram

Liu

Prigge

2020

mSphere

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One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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“…Slow antiparasitic activity is thought to be a fundamental limitation of DOX and other antibiotics that target apicoplast maintenance. We report that slightly higher [8][9][10] µM DOX concentrations kill parasites with first-cycle activity that blocks apicoplast biogenesis.…”

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confidence: 90%

Doxycycline has Distinct Apicoplast-Specific Mechanisms of Antimalarial Activity

Guo

Nalder

Okada

et al. 2020

Preprint

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Doxycycline (DOX) is a key antimalarial drug that is thought to kill Plasmodium falciparum parasites by blocking protein translation in the essential apicoplast organelle. Although parasite resistance to DOX has not emerged, clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 μM, the DOX concentration readily achieved in human plasma at current dosing. Slow antiparasitic activity is thought to be a fundamental limitation of DOX and other antibiotics that target apicoplast maintenance. We report that slightly higher 8-10 μM DOX concentrations kill parasites with first-cycle activity that blocks apicoplast biogenesis. This faster activity is rescued by exogenous isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific target. Exogenous iron rescues first- but not second-cycle activity by 1 or 10 μM DOX, revealing that first-cycle activity involves a metal-dependent mechanism that is distinct from the canonical delayed-death mechanism. These results provide a new paradigm for understanding fundamental antiparasitic mechanisms of DOX and suggest the possibility of repurposing DOX as a faster-acting antimalarial at higher dosing, which remains well tolerated. Multiple mechanisms of DOX activity would be expected to limit parasite resistance.

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A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites

Cited by 41 publications

References 73 publications

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

Doxycycline has Distinct Apicoplast-Specific Mechanisms of Antimalarial Activity

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