ABSTRACT: The methylerythritol 4-phosphate (MEP) pathway, essential for producing isoprenoids in Plasmodium parasites. The key isoprenoids produced by the MEP pathway are farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP), vital for protein isoprenylation, ubiquinone, and dolichol biosynthesis. In vitro studies have demonstrated that prenols, like farnesol (FOH) and geranylgeraniol (GGOH), can temporarily circumvent the MEP pathway, rescuing parasites from MEP inhibitors (Crispim et al., 2024). It was identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. We decided to explore the biological role of PolK through proteomics. To this end, we compared proteomic profiles between transgenic P. falciparum parasites preserving the PfPolK gene (control) to parasites in which the gene was excised as explained elsewhere (Crispim et al., 2024). Proteomic analysis was performed with three replicates of each sample (parasites preserving or not PfPolK) and it was identified 442 proteins in control parasites compared to 439 in PfPolK knockout parasites. Of these, 24 proteins were only present in parasites lacking PfPolK compared to the control, which had 27 exclusive proteins found. Pyruvate kinase II (PF3D7_1037100) was exclusively detected in parasites lacking PfPolK, and not in control parasites. Paired t-test between groups using Benjamini-Hochberg-based FDR correction (FDR < 0.05) led to the identification of regulated proteins. Molecular deletion of PfPolK upregulated the expression of 37 proteins and downregulated 12, compared to control parasites. Most regulated proteins are enzymes, especially those related to the following GO terms: carbohydrate metabolic process (GO:0005975), nucleobase-containing small molecule metabolic process (GO:0055086), cellular amino acid metabolic process (GO:0006520), carbohydrate derivative metabolic process (GO:1901135), tRNA metabolic process (GO:0006399), protein catabolic process (GO:0030163). The excision of PfPolK upregulated the expression of proliferating cell nuclear antigen 1 (PCNA, PF3D7_1361900), several tRNA ligases (e.g.: leucine--tRNA ligase, PF3D7_0622800; cysteine--tRNA ligase, PF3D7_1015200), as well as several enzymes related to oxidative stress defense (e.g.: glutathione S-transferase, PF3D7_1419300 thioredoxin 1, PF3D7_1457200 ) and proteostasis (e.g.: heat shock protein 110, PF3D7_0708800; Hsp70/Hsp90 organizing protein, PF3D7_1434300), together with some enzymes related to protein catabolism (e.g.: plasmepsin III, PF3D7_1408100; aminopeptidase P, PF3D7_1454400). The excision of PfPolK make no detectable or downregulated Ras-related protein Rab-2 (PF3D7_1231100) and merozoite surface protein 1 and 2 (PF3D7_0930300 and PF3D7_0206800, respectively) which are prenylated/glycosylated proteins which may been affected of some dysregulation of the isoprenoid pathway. Contradictorily, parasites lacking PfPolK showed an upregulation of HAD-2 (PF3D7_1226300) along with several glycolytic enzymes or glucose metabolism-related enzymes, including hexokinase (PF3D7_0624000), fructose-bisphosphate aldolase (PF3D7_1444800), enolase (PF3D7_1015900), and the glycolytic pyruvate kinase type I (PF3D7_0626800) and inositol-3-phosphate synthase (PF3D7_0511800). REFERENCES • Crispim M, Verdaguer IB, Hernández A, Kronenberger T, Fenollar À, Yamaguchi LF, Alberione MP, Ramirez M, de Oliveira SS, Katzin AM, Izquierdo L. Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites. PLoS Pathog. 2024 Jan 26;20(1):e1011557. doi: 10.1371/journal.ppat.1011557. PMID: 38277417; PMCID: PMC10849223.
Ignasi Bofill Verdaguer