Figure 3

De novo biosynthesis of medium-long polyprenols (POH) and dolichols (DOH) in P. falciparum. (a) Scheme used for metabolic labeling with [1-¹³C]glucose or [3-¹³C]IPP in highly synchronous ring stage cultures. Parasites were recovered at schizont stage for LC-HRMS analysis. A representative Giemsa-stained smear is shown and scale bar indicates 2 µm. The fate of ¹³C through the MEP pathway for [1-¹³C]glucose is showed as a half-black circle to indicate ¹³C abundance, which is 50% of the initial one. A black triangle depicts the localization of the ¹³C atom in the exogenously supplied [3-¹³C]IPP. (b) A representative mass spectrum of the standards polyprenol 17 (m/z [M + NH₄]⁺ = 1,193.1145), dolichol 17 (m/z [M + NH₄]⁺ = 1,195.1208) and metabolites detected in P. falciparum schizont stage is shown in the two upper rows. Distribution of the ¹³C isotopologues observed for native polyisoprenoid alcohols due to the natural abundance of ¹³C is indicated by brackets. Metabolically labeled polyprenol and dolichol are shown in the two lower panels and brackets indicate the m/z shift observed as a Gaussian distribution of ¹³C-enriched polyprenol and dolichol isotopologues. (c) Mass spectra of dolichol 18 (m/z [M + NH₄]⁺ = 1,263.1868) from uninfected human RBCs cultured in the absence or presence of [1-¹³C]glucose under the same conditions as P. falciparum showing lack of de novo dolichol biosynthesis. Data presented are representative of three independent biological replicates. The figure was generated using CorelDraw 2018 (https://www.coreldraw.com/en/) and Adobe Photoshop 21.2.0 (https://www.adobe.com/products/photoshop.html).