A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Tang, Yong; Meister, Thomas R.; Walczak, Marta; Pulkoski-Gross, Michael J.; Hari, Sanjay B.; Sauer, Robert T.; Amberg-Johnson, Katherine; Yeh, Ellen

doi:10.1371/journal.pbio.3000136

Cited by 39 publications

(36 citation statements)

References 67 publications

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“…The previously established chemical bypass system has proven to be invaluable to the study of the apicoplast in malaria parasites, having been used to interrogate the essentiality of apicoplast specific genes [15][16][17][18][19], screen for apicoplast-specific drugs [15,[20][21][22][23], and conduct forward genetic screens to elucidate essential apicoplast-specific genes [24]. However, this bypass study are available in GenBank under accession numbers MN822297 and MN822298.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…As a result of its divergent evolution, thousands of genes in the P. falciparum genome remain unannotated (38). We previously designed an unbiased screen that allows for discovery of new genes with essential roles in apicoplast biogenesis (14). Here, we validate a previously uncharacterized gene candidate from our screen, AMR4, and show that it has an essential function in apicoplast biogenesis.…”

Section: Discussionmentioning

confidence: 72%

“…AMR4 is essential and required specifically for apicoplast biogenesis. We previously performed a mutagenesis screen to identify new apicoplast biogenesis genes (14). Loss-of-function mutations in these genes disrupt inheritance of the apicoplast during parasite replication, resulting in organelle loss in daughter cells.…”

Section: Resultsmentioning

confidence: 99%

“…The transit of nucleus-encoded proteins to the apicoplast requires vesicle trafficking from the endoplasmic reticulum (ER) and a unique symbiont-derived ER-associated degradation-like machinery (SELMA) to cross the two outer membranes (7)(8)(9)(10). Additionally, several members of the highly conserved eukaryotic autophagy pathway are unexpectedly required for apicoplast inheritance (11)(12)(13)(14). While the repurposed SELMA and autophagy components hint at an abundance of molecular innovation in apicoplast biogenesis pathways, most of these pathways remain undiscovered.…”

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confidence: 99%

“…To discover new genes required for apicoplast biogenesis and uncover more instances of molecular innovation, we previously conducted a mutagenesis screen to identify mutations that cause apicoplast loss (14). From this screen, we identified a mutation in a gene encoding a CaaX protease and bacteriocin processing (CPBP) family protein (Pf3D7_0913500, S347G).…”

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CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

Meister

Tang

Pulkoski-Gross

et al. 2020

mBio

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Plasmodium parasites and related apicomplexans contain an essential “complex plastid” organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum. Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis. IMPORTANCE Plasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.

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Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei

Mishra,

Varshney,

Mishra

2023

Cell. Mol. Life Sci.

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A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Cited by 39 publications

References 67 publications

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

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

CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei

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