Cellular isoprenoid metabolism in Plasmodium parasites depends on the apicoplast

Malaria parasites retain a non-mevalonate/methylerythritol phosphate (MEP) pathway for isoprenoid precursor synthesis in the apicoplast organelle. This pathway synthesizes two 5-carbon isoprene subunits, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), whose sequential condensation is critical for making diverse longer-chain isoprenoids required for a variety of key cellular functions that include protein prenylation, dolichol synthesis for protein glycosylation, and biosynthesis of mitochondrial ubiquinone and heme A (Fig 1). Core enzymes in the MEP pathway are encoded in the nucleus but are trafficked to the apicoplast after translation in the parasite cytoplasm.

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Fig 1. Isoprenoid synthesis and utilization by blood-stage malaria parasites.

Abbreviations: DHAP, dihydroxyacetone phosphate; DMAPP, dimethylallyl pyrophosphate; DOXP, 1-deoxy-D-xylulose 5-phosphate; DXR, DOXP reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FD, ferredoxin; FOS, fosmidomycin; FPP, farnesyl pyrophosphate; FPPS, FPP synthase; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; GGPPS, GGPP synthase; Gly3P, glyceraldehyde-3-phosphate; HMB-PP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl pyrophosphate; IPPI, IPP isomerase; IspG, HMP-PP synthase; IspH, HMB-PP reductase; MEcPP, 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate; MEP, methylerythritol phosphate; MiaA, tRNA dimethylallyltransferase; MiaB, tRNA 2-methylthio-N(6)-dimethylallyladenosine synthase; PEP, phosphoenolpyruvate; PPS, polyprenyl synthase; PYR, pyruvate. Question marks indicate uncertainty.

https://doi.org/10.1371/journal.ppat.1011713.g001

Within the apicoplast, isoprenoid precursor synthesis requires the support of several additional metabolic pathways. The final 2 enzymes in the MEP pathway, IspG and IspH, contain essential Fe-S cluster cofactors that receive electrons from apicoplast-targeted ferredoxin, which also contains an Fe-S cluster. These critical Fe-S cofactors are produced by the apicoplast Suf pathway, which is therefore essential for isoprenoid synthesis [7–10]. One of the Suf pathway proteins, SufB, is encoded on the apicoplast genome, and thus replication, transcription, and translation of the apicoplast genome are expected to be essential for Fe-S cluster provision to the MEP pathway. Substrates for isoprenoid precursor synthesis include glyceraldehyde-3-phosphate, pyruvate, and cytidine triphosphate, whose production and/or import into the apicoplast depend on apicoplast pyruvate kinase II and membrane transporters [11]. Because IPP and DMAPP synthesis requires the integrated function of MEP enzymes and supporting pathways within the apicoplast (Fig 1), disruption of the apicoplast organelle via biochemical and/or genetic perturbations ablates the synthesis of these key isoprenoid precursors [5,6]. Cellular isoprenoid synthesis in Plasmodium therefore requires the apicoplast and the pathways that support and maintain this key organelle.

Apicoplast biogenesis requires isoprenoid precursor synthesis and the pathways that support MEP pathway activity

Prior studies in Plasmodium focused almost exclusively on cellular roles for isoprenoids outside of the apicoplast, since exogenous IPP can rescue parasites from loss of the apicoplast and the attendant ablation of endogenous IPP synthesis. Nevertheless, multiple prior papers reported that MEP pathway inhibitors like fosmidomycin (FOS) blocked apicoplast biogenesis and prevented elongation of the organelle in blood-stage parasites [12–14]. Recent work demonstrated that these defects were due to specific apicoplast dysfunction (rather than a nonspecific phenotype of dying parasites) and confirmed a critical role for isoprenoids in supporting apicoplast biogenesis [15]. The only previously annotated role for isoprenoids in the apicoplast was tRNA prenylation by MiaA, but knockout of this enzyme had no impact on apicoplast biogenesis or parasite viability (Fig 1). However, it was discovered that Plasmodium parasites target a polyprenyl synthase (PPS) enzyme to the apicoplast that synthesizes longer-chain linear isoprenoids required for organelle biogenesis [15]. Indeed, parasites could be rescued from loss of PPS by addition of long-chain decaprenol (C₅₀-OH) but not by GGOH (C₂₀-OH) or shorter polyprenols.

It remains a key unmet challenge to understand why apicoplast biogenesis requires long-chain polyprenols. These compounds can serve multiple functions in plant chloroplasts (e.g., light harvesting and photosynthesis, oxidative stress protection, signaling and defense molecules), but there is little or no evidence for these functions in Plasmodium parasites [1,3,15]. In the absence of other known roles for long-chain linear isoprenoids, one hypothesis is that these lipids tune the structure and flexibility of apicoplast membranes, which is a known or proposed biophysical role for membrane polyprenols in bacteria, plants, and other eukaryotes [16]. The evolutionary pathway by which the Plasmodium apicoplast acquired and retained a PPS enzyme is also unclear. Long-chain isoprenoids like undecaprenyl phosphate serve as the membrane lipid carrier for bacterial peptidoglycans used in cell wall synthesis [16,17]. This pathway (which includes a PPS) was likely present in the ancestral prokaryotic progenitor of the apicoplast, with most enzymatic components being lost during evolution of the current organelle. We hypothesize that PPS may have been retained in the Plasmodium apicoplast as a vestige of this erstwhile peptidoglycan synthesis pathway, due to a separate essential role for long-chain polyprenols in tuning membrane biogenesis.

Due to its reciprocal dependence on isoprenoid precursor synthesis, apicoplast biogenesis also requires organelle functions that support MEP pathway activity, including Suf-mediated Fe-S cluster biogenesis, replication of the apicoplast genome and expression of SufB, pyruvate kinase II activity, and membrane transporters that import the requisite biosynthetic substrates (Fig 1) [4]. Several of these pathways also have separate essential roles in apicoplast biogenesis in addition to and independent of their direct support of MEP pathway activity. In the next section, we explore some of these pathways and how parasite rescue by exogenous IPP has been used to test and understand the role of apicoplast-targeted proteins in organelle biogenesis.

IPP rescue as a tool to probe apicoplast-specific function and organelle biogenesis

The foundational discovery that exogenous IPP could rescue parasites from apicoplast-specific dysfunctions and organelle loss enabled many opportunities to probe the functional properties of the apicoplast and its constituent proteins [5]. Because the apicoplast requires isoprenoid precursors to initiate and complete its biogenesis program [15], 2 general phenotypes have been observed when parasites are rescued by exogenous IPP from chemical or genetic perturbations to the apicoplast. For proteins whose only essential function in blood-stage parasites is to support isoprenoid precursor biosynthesis, including MEP-pathway enzymes (e.g., DXS) and several proteins in the Suf pathway (e.g., SufC) [7,11], IPP bypasses the loss or inhibition of these proteins to rescue parasites and support normal apicoplast elongation and division. In such cases (e.g., FOS treatment, ∆DXS, ∆SufC), however, defective apicoplast biogenesis is observed when IPP supplementation is withheld such that the punctate apicoplast present in rings and trophozoites fails to elongate as parasites progress into schizogeny (Fig 2) [7,15]. For apicoplast-targeted proteins that have essential functions in apicoplast biogenesis that are in addition to or apart from supporting MEP-pathway activity (e.g., ClpC, PPS, SufS), IPP rescues parasite viability from loss of these proteins but the apicoplast fails to elongate and divide such that daughter parasites stably lose the organelle and its genome [7,15,18].

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Fig 2. Apicoplast Fe-S cluster biosynthesis is required for isoprenoid synthesis and apicoplast biogenesis.

(A) Live-cell imaging of ∆SufC NF54 (PfMev) parasites synchronized and cultured 36 hours with or without 50 μM DL-mevalonate (Mev) to stimulate IPP synthesis [7]. (B) Population analysis of parasites imaged under the conditions in (A). A total of 56 parasites were imaged per condition from biological duplicate samples. Apicoplast morphologies were scored as punctate, elongated, or disrupted (dispersed signal) and plotted by population percentage.

https://doi.org/10.1371/journal.ppat.1011713.g002

Current knowledge specifies IPP and coenzyme A (CoA) as the only biosynthetic outputs of the apicoplast that are essential for blood-stage P. falciparum [5,19,20]. Unlike IPP synthesis, however, CoA biosynthesis does not require the apicoplast organelle as a metabolic hub and appears to persist regardless of apicoplast maintenance or loss or targeting of the terminal enzyme to the apicoplast, cytoplasm, or vesicles [19]. Nevertheless, apicoplast biogenesis appears to depend on synthesis of both IPP and CoA [15,19]. Based on these observations, we propose that any essential apicoplast-targeted protein in blood-stage Plasmodium will be required for organelle maintenance due to a critical role in IPP synthesis and/or in other aspects of apicoplast biogenesis apart from IPP synthesis. Future studies of apicoplast functions can test this prediction and extend or revise its validity to different parasite stages in distinct host environments, where additional apicoplast pathways like type II fatty acid synthesis can produce essential outputs [21,22] that may also be required for apicoplast biogenesis.

Acknowledgments

We thank Manoj Duraisingh, Sean Prigge, and Akhil Vaidya for helpful discussions.