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• W4365458461 abstract "Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO’s for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism. Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO’s for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism. The fatty acid (FA) composition of lipid bilayers can profoundly influence the biophysical properties (fluidity, curvature, microdomain organization) and biological functions (secretion, signal transduction, adaptation to stress) of cellular membranes (1de Carvalho C.C.C.R. Caramujo M.J. The various roles of fatty acids.Molecules. 2018; 23: E2583Crossref PubMed Scopus (302) Google Scholar, 2Ibarguren M. López D.J. Escribá P.V. The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health.Biochim. Biophys. Acta. 2014; 1838: 1518-1528Crossref PubMed Scopus (221) Google Scholar, 3de Mendoza D. Pilon M. Control of membrane lipid homeostasis by lipid-bilayer associated sensors: a mechanism conserved from bacteria to humans.Prog. Lipid Res. 2019; 76100996Crossref PubMed Scopus (36) Google Scholar). As such, regulation of the FA pools used for synthesis and maintenance of biological membranes is a critical homeostatic function of all cells (4Wang B. Tontonoz P. Phospholipid remodeling in physiology and disease.Annu. Rev. Physiol. 2019; 81: 165-188Crossref PubMed Scopus (168) Google Scholar). While these regulatory processes have been well-studied in mammals and yeast (5Glatz J.F.C. Nabben M. Luiken J.J.F.P. CD36 (SR-B2) as master regulator of cellular fatty acid homeostasis.Curr. Opin. Lipidol. 2022; 33: 103-111Crossref PubMed Scopus (17) Google Scholar, 6Tehlivets O. Scheuringer K. Kohlwein S.D. Fatty acid synthesis and elongation in yeast.Biochim. Biophys. Acta. 2007; 1771: 255-270Crossref PubMed Scopus (353) Google Scholar, 7Gimeno R.E. Fatty acid transport proteins.Curr. Opin. Lipidol. 2007; 18: 271-276Crossref PubMed Scopus (134) Google Scholar), the mechanisms governing FA and lipid homeostasis in pathogenic protozoa are poorly understood. Trypanosoma cruzi is a member of a diverse group of flagellated protozoan parasites that include parasitic organisms responsible for globally relevant human diseases such as leishmaniasis (Leishmania spp.), sleeping sickness (Trypanosoma brucei). and Chagas disease (T. cruzi). Throughout its complex life cycle, T. cruzi colonizes hematophagous triatomine insects and mammalian hosts and switches between actively dividing and nondividing forms in each setting. Survival in these disparate environments requires both morphological and metabolic adaptation (8de Souza W. de Carvalho T.M.U. Barrias E.S. Review on Trypanosoma cruzi: host cell interaction.Int. J. Cell Biol. 2010; 2010295394Crossref PubMed Scopus (178) Google Scholar). A critical feature of adaptation in T. cruzi is the ability to alter membrane lipid composition and the fatty acyl moieties of the abundant glycosylphosphatidylinositols that anchor arrays of glycoproteins and glycolipids to the parasite surface (9McConville M.J. Ferguson M.A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes.Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (806) Google Scholar, 10Lee S.H. Stephens J.L. Englund P.T. A fatty-acid synthesis mechanism specialized for parasitism.Nat. Rev. Microbiol. 2007; 5: 287-297Crossref PubMed Scopus (99) Google Scholar). The biological consequences of FA remodeling during development are best documented for surface glycosylphosphatidylinositols-anchored mucins, where changes in FA chain length and/or degree of saturation can activate or dampen the proinflammatory activity of these molecules (11Almeida I.C. Camargo M.M. Procópio D.O. Silva L.S. Mehlert A. Tracassos L.R. et al.Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents.EMBO J. 2000; 19: 1476-1485Crossref PubMed Scopus (206) Google Scholar). It is currently unknown how T. cruzi regulates the composition or abundance of FAs needed for lipid synthesis (12Booth L.-A. Smith T.K. Lipid metabolism in Trypanosoma cruzi: a review.Mol. Biochem. Parasitol. 2020; 240111324Crossref PubMed Scopus (13) Google Scholar), but similar to other cell types, this protozoan can synthesize long-chain FA (LCFA) endogenously (10Lee S.H. Stephens J.L. Englund P.T. A fatty-acid synthesis mechanism specialized for parasitism.Nat. Rev. Microbiol. 2007; 5: 287-297Crossref PubMed Scopus (99) Google Scholar, 13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and to take up FAs from the environment (14Pereira M.G. Visbal G. Costa T.F.R. Frases S. de Souza W. Atella G. et al.Trypanosoma cruzi epimastigotes store cholesteryl esters in lipid droplets after cholesterol endocytosis.Mol. Biochem. Parasitol. 2018; 224: 6-16Crossref PubMed Scopus (15) Google Scholar). Bulk synthesis of LCFA in most eukaryotes is achieved using a cytosolic type-I fatty acid synthase (15Leibundgut M. Maier T. Jenni S. Ban N. The multienzyme architecture of eukaryotic fatty acid synthases.Curr. Opin. Struct. Biol. 2008; 18: 714-725Crossref PubMed Scopus (137) Google Scholar) that is absent in trypanosomatids. Some eukaryotes, such as plants (16Ohlrogge J. Browse J. Lipid biosynthesis.Plant Cell. 1995; 7: 957-970Crossref PubMed Google Scholar, 17He M. Qin C.-X. Wang X. Ding N.-Z. Plant unsaturated fatty acids: biosynthesis and regulation.Front. Plant Sci. 2020; 11: 390Crossref PubMed Scopus (106) Google Scholar, 18Harwood J.L. Recent advances in the biosynthesis of plant fatty acids.Biochim. Biophys. Acta. 1996; 1301: 7-56Crossref PubMed Scopus (401) Google Scholar) and apicomplexan parasites like Plasmodium (19Mazumdar J. Striepen B. Make it or take it: fatty acid metabolism of apicomplexan parasites.Eukaryot. Cell. 2007; 6: 1727-1735Crossref PubMed Scopus (102) Google Scholar, 20Tarun A.S. Vaughan A.M. Kappe S.H.I. Redefining the role of de novo fatty acid synthesis in Plasmodium parasites.Trends Parasitol. 2009; 25: 545-550Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 21Shears M.J. Botté C.Y. McFadden G.I. Fatty acid metabolism in the Plasmodium apicoplast: drugs, doubts and knockouts.Mol. Biochem. Parasitol. 2015; 199: 34-50Crossref PubMed Scopus (63) Google Scholar) have instead a type-II fatty acid synthase (FAS-II) system localized in plastids. A mitochondrial FAS-II system is present in trypanosomes (12Booth L.-A. Smith T.K. Lipid metabolism in Trypanosoma cruzi: a review.Mol. Biochem. Parasitol. 2020; 240111324Crossref PubMed Scopus (13) Google Scholar, 13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), which has a more restricted role in cells as a nutrient-sensitive coordinator of mitochondrial oxidative phosphorylation (22Nowinski S.M. Van Vranken J.G. Dove K.K. Rutter J. Impact of mitochondrial fatty acid synthesis on mitochondrial biogenesis.Curr. Biol. 2018; 28: R1212-R1219Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and contributes only about 10% of total FA activity in these parasites (23Ramakrishnan S. Serricchio M. Striepen B. Bütikofer P. Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans.Prog. Lipid Res. 2013; 52: 488-512Crossref PubMed Scopus (112) Google Scholar, 24Stephens J.L. Lee S.H. Paul K.S. Englund P.T. Mitochondrial fatty acid synthesis in Trypanosoma brucei.J. Biol. Chem. 2007; 282: 4427-4436Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Short-chain FA (SCFA) intermediates generated by FAS-II can also be diverted to synthesize other molecules, such as lipoic acid, a critical cofactor needed for the stabilization, and activity of a subset of mitochondrial enzymes (24Stephens J.L. Lee S.H. Paul K.S. Englund P.T. Mitochondrial fatty acid synthesis in Trypanosoma brucei.J. Biol. Chem. 2007; 282: 4427-4436Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Vacchina P. Lambruschi D.A. Uttaro A.D. Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy.Exp. Parasitol. 2018; 186: 17-23Crossref PubMed Scopus (6) Google Scholar). Besides having a mitochondrial FAS-II system, trypanosomatids have develop a specialized microsomal FA elongase (ELO) system to synthesize bulk LCFA (10Lee S.H. Stephens J.L. Englund P.T. A fatty-acid synthesis mechanism specialized for parasitism.Nat. Rev. Microbiol. 2007; 5: 287-297Crossref PubMed Scopus (99) Google Scholar, 13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). First described in T. brucei (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the trypanosomatid ELO system consists of individual FA ELO enzymes (ELOs 1-5 in T. cruzi (26Livore V.I. Tripodi K.E.J. Uttaro A.D. Elongation of polyunsaturated fatty acids in trypanosomatids.FEBS J. 2007; 274: 264-274Crossref PubMed Scopus (32) Google Scholar)), which exhibit distinct specificities for fatty acyl-CoA substrates based on the carbon-chain length and/or degree of unsaturation (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). ELOs 1-4 are numbered according to their relative position in a sequential pathway (ELO1, ELO2, etc.), where the product of one ELO becomes the substrate for the following enzyme (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Studies using isolated membranes indicate that the ELO enzymes can also function independently to elongate exogenous fatty acyl substrates of the appropriate chain length (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). ELOs utilize malonyl-CoA as a two-carbon donor to extend fatty acyl-CoA substrates in a four-step cycle involving: (1) a β-ketoacyl-CoA synthase (the “ELO”) responsible for condensing a preexisting fatty acyl-CoA with malonyl-CoA, this is the first and rate-limiting step in each elongation cycle and guides substrate specificity; (2) a β-ketoacyl-CoA reductase; (3) β-hydroxyacyl-CoA dehydratase; and (4) a trans-2-enoyl-CoA reductase (10Lee S.H. Stephens J.L. Englund P.T. A fatty-acid synthesis mechanism specialized for parasitism.Nat. Rev. Microbiol. 2007; 5: 287-297Crossref PubMed Scopus (99) Google Scholar, 13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The ELO1 cycle elongates short-chain fatty acyl-CoA substrates (C4, C6, and C8) to generate C10. Still, unlike type-I fatty acid synthase, ELO1 cannot initiate FA synthesis using acetyl-CoA; instead it uses butyryl-CoA as the main substrate (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The source of butyryl-CoA for this reaction remains unknown. ELO2 extends C10 and C12 substrates to generate C14 and ELO3 elongates C14 to generate C16 and C18 fatty acids. ELO4 produces very LCFAs (up to 26 carbons in length) (27Livore V.I. Uttaro A.D. Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.Parasitol. Res. 2015; 114: 265-271Crossref PubMed Scopus (5) Google Scholar) and ELO5 acts as a polyunsaturated FA ELO (26Livore V.I. Tripodi K.E.J. Uttaro A.D. Elongation of polyunsaturated fatty acids in trypanosomatids.FEBS J. 2007; 274: 264-274Crossref PubMed Scopus (32) Google Scholar). Apart from the initial characterization of the microsomal ELO pathway in T. brucei (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and the demonstration of functional ELOs in T. cruzi (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 26Livore V.I. Tripodi K.E.J. Uttaro A.D. Elongation of polyunsaturated fatty acids in trypanosomatids.FEBS J. 2007; 274: 264-274Crossref PubMed Scopus (32) Google Scholar, 28Paul K.S. Jiang D. Morita Y.S. Englund P.T. Fatty acid synthesis in African trypanosomes: a solution to the myristate mystery.Trends Parasitol. 2001; 17: 381-387Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and Leishmania (10Lee S.H. Stephens J.L. Englund P.T. A fatty-acid synthesis mechanism specialized for parasitism.Nat. Rev. Microbiol. 2007; 5: 287-297Crossref PubMed Scopus (99) Google Scholar, 13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 26Livore V.I. Tripodi K.E.J. Uttaro A.D. Elongation of polyunsaturated fatty acids in trypanosomatids.FEBS J. 2007; 274: 264-274Crossref PubMed Scopus (32) Google Scholar), the relative contribution of this pathway toward lipidome maintenance in trypanosomatids has not been determined. In this study, we examined the role of the ELO system in supporting lipid homeostasis in axenic T. cruzi epimastigotes (EPI). As 16- and 18-carbon FA species represent ∼60% of total FA species in this organism (27Livore V.I. Uttaro A.D. Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.Parasitol. Res. 2015; 114: 265-271Crossref PubMed Scopus (5) Google Scholar, 29Esteves M.G. Gonzales-Perdomo M. Alviano C.S. Angluster J. Goldenberg S. Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi.FEMS Microbiol. Lett. 1989; 50: 31-34Crossref PubMed Scopus (20) Google Scholar, 30Leon W. Monteiro A.M. Alviano C.S. Esteves M.J. Angluster J. Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi.Acta Trop. 1989; 46: 131-136Crossref PubMed Scopus (9) Google Scholar), we focused our analysis on the ELO pathway enzymes, ELOs 1-3, which are expected to produce C16 and C18 from SCFA substrates in a processive pathway (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Unbiased lipidomic analyses of loss-of-function mutants, Δelo1, Δelo2, and Δelo3, revealed that both ELO2 and ELO3 contribute significantly to maintaining of global lipidomic profiles in T. cruzi. Despite marked lipidome remodeling in Δelo2 and Δelo3 EPI, lipidomic changes were well-tolerated with little impact on the growth of these mutants in culture. In contrast, ELO1 was found to be dispensable for LCFA synthesis and global lipidome maintenance. Instead, ELO1 expression was required to support mitochondrial metabolism and proliferation. Further analysis revealed that loss of ELO1 function was associated with decreased protein lipoylation, which was rescued by octanoic acid (C8:0) supplementation or genetic complementation with functional elo1. Together, these findings highlight the modular nature of the trypanosomatid ELO pathway and reveal an unexpected role for ELO1 in supporting aspects of mitochondrial metabolism in T. cruzi EPI, including impact on the generation of SCFA precursors for lipoic acid synthesis, a function generally attributed to mitochondrial FAS-II. The T. cruzi FA ELO genes, elo1 (TcCLB.506661.30/TcCLB.511245.130) elo2 (TcCLB.506661.20/TcCLB.511245.140), and elo3 (TcCLB.506661.10/TcCLB.511245.150) (Fig. 1A), were individually targeted for disruption in axenic T. cruzi EPI using CRISPR/Cas9 and integration of homology-directed repair cassettes (Fig. 1B). Cloning of transfected EPI during initial drug selection post-transfection facilitated recovery of parasites with insertions in both alleles with the successful generation of three independent ELO KOs, Δelo1, Δelo2, and Δelo3, as confirmed by Southern blot (Fig. 1C) and PCR (Fig. 1D). Genetic complementation of each Δelo mutant was achieved with a modified pTREX expression vector encoding a C-terminal GFP-tagged copy of the relevant full-length elo gene on the mutant background (Fig. 1E). Consistent with the reported microsomal localization of the ELO pathway enzymes in T. brucei (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), all ELO-GFP proteins localized to the endoplasmic reticulum (ER) in T. cruzi EPI as confirmed by colocalization with the ER chaperone BiP (31Bangs J.D. Uyetake L. Brickman M.J. Balber A.E. Boothroyd J.C. Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote.J. Cell Sci. 1993; 105: 1101-1113Crossref PubMed Google Scholar) (Fig. 1E). As the main system used for bulk LCFA synthesis in trypanosomatids, the ELO pathway is assumed to play a critical role in membrane lipid synthesis and remodeling in this group of organisms. Still, the contribution of this pathway in shaping the lipidome in these organisms has yet to be determined experimentally. To evaluate the impact of disruption of ELO pathway enzymes on the lipid composition of T. cruzi parasites, we performed label-free, quantitative ultra-HPLC (UHPLC) coupled to high-resolution tandem MS (UHPLC-HR-MS/MS) of lipids extracted from WT, Δelo1, Δelo2, Δelo3 and genetically complemented EPI lines. A total of 1133 lipid species were identified after manual curation of LipidSearch-assigned IDs (ThermoFisher) (Table S1), the majority of which were found to be significantly altered in abundance (p < 0.05; one-way ANOVA) across all mutants as compared to WT EPI (Fig. 2A). Two-dimensional principal component analysis (PCA) identified overall trends in the lipidome data (Fig. 2B), where the Δelo3 mutant emerged as the distinct outlier, well-separated from the other parasite lines along both PC1 and PC2 (Fig. 2B). The Δelo1 EPI lipidome was the least divergent from WT and Δelo2 separated from both of these lines along PC2 (Fig. 2B). When broken down according to major lipid subclass (Fig. S1) PCA clustering patterns were found to be similar to that observed for the total lipidome (Fig. 2B), indicating that global lipidomic differences between of WT and Δelo mutant T. cruzi EPI are likely not driven by specific lipid subclasses. Volcano plots displaying pairwise comparisons of WT and individual Δelo mutants (Fig. 2C) and heatmap data highlighting the 50 lipids that changed most dramatically in the mutants compared to WT EPI (Fig. 2D) demonstrate that lipidomic changes are most prominent in Δelo2 and Δelo3 mutants. Consistent with this conclusion, 232 and 149 lipids were found to be changed ≥2- or ≤0.5-fold change (p-value < 0.05) in Δelo2 and Δelo3, respectively (Table S2). In contrast, comparatively few lipids (48) exhibited ≥2- or ≤0.5-fold change in abundance in the Δelo1 mutant when compared to WT EPI (Fig. 2, C and D) (Table S2). Consistent with the anticipated function of ELO3 in extending 14-carbon FAs to C16 and C18 species, we noted a marked reduction in C18:0- and C18:1-containing lipids in Δelo3 EPI and a concomitant increase in lipids containing C14:0, C14:1, C16:0, and C16:1 in this mutant (Fig. 2, D–F). A similar trend was observed for Δelo2 EPI, where lipids containing C10:0 and C12:0 were elevated, and lipids containing C14:0 and C16:0 FA were present at decreased levels (Fig. 2, D–F). Given that most lipid alterations observed in the Δelo mutants were restored in the genetically complemented lines (Fig. S2), we conclude that the observed lipidomic changes are due to the loss of specific ELO enzyme function. Combined, these data provide the first demonstration that ELO2 and ELO3 play important roles in lipid in T. cruzi but that ELO1 contributes little to LCFA synthesis and global lipidomic profiles in this organism. In addition to de novo synthesis of FAs, T. cruzi takes up FA and lipids from the environment that can be incorporated into their own lipid pools (14Pereira M.G. Visbal G. Costa T.F.R. Frases S. de Souza W. Atella G. et al.Trypanosoma cruzi epimastigotes store cholesteryl esters in lipid droplets after cholesterol endocytosis.Mol. Biochem. Parasitol. 2018; 224: 6-16Crossref PubMed Scopus (15) Google Scholar, 32Pereira M.G. Visbal G. Salgado L.T. Vidal J.C. Godinho J.L.P. De Cicco N.N.T. et al.Trypanosoma cruzi epimastigotes are able to manage internal cholesterol levels under nutritional lipid stress conditions.PLoS One. 2015; 10e0128949Crossref Scopus (15) Google Scholar, 33Pereira M.G. Nakayasu E.S. Sant’Anna C. De Cicco N.N.T. Atella G.C. de Souza W. et al.Trypanosoma cruzi epimastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions.PLoS One. 2011; 6e22359Crossref Scopus (34) Google Scholar, 34Einicker-Lamas M. Nascimento M.T.C. Masuda C.A. Oliveira M.M. Caruso-Neves C. Trypanosoma cruzi epimastigotes: regulation of myo-inositol transport by effectors of protein kinases A and C.Exp. Parasitol. 2007; 117: 171-177Crossref PubMed Scopus (16) Google Scholar, 35Wainszelbaum M.J. Belaunzarán M.L. Lammel E.M. Florin-Christensen M. Florin.Christensen J. Isola E.L.D. Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.Biochem. J. 2003; 375: 705-712Crossref PubMed Google Scholar). The observation that ELO2- and ELO3-deficient EPI experience global lipidomic changes, characterized by a decrease in lipids containing 18-carbon FA side chains, despite continuous culture in medium containing 10% serum, suggests that serum lipids/exogenous FAs are insufficient to overcome the loss of ELO activity. To examine the possibility that T. cruzi Δelo mutants may experience defects in their ability to take up or utilize exogenous FA for lipid synthesis, we performed metabolic tracing studies using [1-14C]-labeled FAs of varying chain lengths (C4:0, C14:0, C16:0, and C18:0). Lipids extracted from 14C-FA-labeled parasites were resolved using reversed-phase (RP) TLC to identify major lipid classes into which label was incorporated (Fig. 3, A and B). Although differences in 14C-label labeling intensity were observed with the different 14C-FA provided, label was incorporated into neutral (Fig. 3A) and polar (Fig. 3B) lipids across all parasite lines (Fig. 3, A and B). A notable exception was the Δelo1 mutant, which failed to incorporate 14C-butyric acid (C4:0) carbons into parasite neutral and polar lipids (Fig. 3, A and B). To more directly assess the impact of specific ELO-deficiencies on FA elongation in intact parasites, FA methyl esters (FAMEs) generated from total lipids extracted from each parasite line following 14C-FA labeling were resolved by TLC (Fig. 3C). As expected, carbons from exogenous [1-14C]-FA were detected in LCFA (C14:0, C16:0, and C18:0) in all parasite lines, except for Δelo1, which failed to significantly incorporate 14C from labeled butyric acid into LCFA species (Fig. 3C). Given the relatively faint signal in WT parasites obtained when 14C-butyric acid was used as a metabolic tracer (Fig. 3, A–C), additional labeling experiments were performed to optimize the detection of labeled lipids following incubation with 14C-butyric acid (Fig. 3D). Results show significantly reduced incorporation of 14C from butyric acid into LCFAs that is unique to the Δelo1 mutant but was fully restored in genetically complemented Δelo1::elo1-gfp parasites (Fig. 3D). Faint bands for Δelo1 mutant, suggest that some butyric acid is being elongated presumably by a less efficient activity of ELO2. Given that Δelo1 EPIs readily elongate 14C-myristic acid (C14:0) and 14C-palmitic acid (C16:0) substrates (Fig. 3C), it is unlikely that impaired import of FAs explains the inability of this mutant to elongate 14C-butyric acid. Together, these results confirm the function of T. cruzi ELO1 as a SCFA ELO that utilizes butyric acid (butyryl-CoA) as a substrate for elongation (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Moreover, the unimpeded ability of Δelo1 EPI to elongate C14:0 and C16:0 substrates, presumably through the actions of ELO2 and ELO3, is consistent with a modular ELO system, in which individual ELOs can function independently of the other pathway enzymes (13Lee S.H. Stephens J.L. Paul K.S. Englund P.T. Fatty acid synthesis by ELOs in trypanosomes.Cell. 2006; 126: 691-699Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). It is worth noting the reduced labeling of neutral and polar lipids Fig. 3, A and B) and FAMEs (Fig. 3, C and D) in the Δelo2 mutant when 14C-butyric acid was provided as substrate. These findings indicate that the products of ELO1 (≤10 carbons length), which are generally not found in the neutral and polar lipids of T. cruzi EPI (Table S1), need to be elongated further, before incorporation into these lipids. Given that carbons from 14C-butyric acid were detectable in polar lipids (Fig. 3B) and C16, C18 FAMEs (Fig. 3D) in the Δelo2 mutant, albeit at a lower intensity than in WT or Δelo3 EPI, point to partial redundancy in the system, possibly through extended capabilities of ELO1 and/or ELO3. Functional overlap and redundancy within the ELO pathway may explain why disruption of ELO2 or ELO3, individually, fails to promote more profound lipidome perturbation in the KO parasite lines. To evaluate the biological impact of ELO gene disruption in T. cruzi, we first examined the growth kinetics of Δelo1, Δelo2, and Δelo3 EPI in liver infusion tryptose (LIT) medium containing 10% serum (Fig. 4). Strikingly, ELO1-deficient T. cruzi EPI exhibit marked growth impairment in the log-phase (doubling time [DT] of 60 ± 12 h) compared to WT parasites (DT = 31 ± 7 h). Δelo2 EPIs exhibit a mild growth defect (DT = 41 ± 9 h), whereas the Δelo3 mutant replicates at rates similar to WT (Fig. 4). The reduced rates of growth observed for Δelo1 and Δelo2 EPIs do not involve changes in the parasite cell cycle, as the proportion of parasites associated with different phases of the cell cycle (G1-S-G2/M) remained unaltered in each mutant as compared to WT (Fig. S3). The growth deficits observed for Δelo1 and Δelo2 mutants were rescued following stable expression of ELO1-GFP or ELO2-GFP in the respective mutant lines (Fig. 4). As expected, growth of genetically" @default.
• W4365458461 created "2023-04-15" @default.
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• W4365458461 date "2023-06-01" @default.
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• W4365458461 title "Fatty acid elongases 1-3 have distinct roles in mitochondrial function, growth, and lipid homeostasis in Trypanosoma cruzi" @default.
• W4365458461 cites W1495212988 @default.
• W4365458461 cites W1751804109 @default.
• W4365458461 cites W1939117002 @default.
• W4365458461 cites W1943700220 @default.
• W4365458461 cites W1944304173 @default.
• W4365458461 cites W1964318956 @default.
• W4365458461 cites W1967908209 @default.
• W4365458461 cites W1968069269 @default.
• W4365458461 cites W1971772314 @default.
• W4365458461 cites W1973182554 @default.
• W4365458461 cites W1973926329 @default.
• W4365458461 cites W1977742263 @default.
• W4365458461 cites W1979824753 @default.
• W4365458461 cites W1984053258 @default.
• W4365458461 cites W1985799174 @default.
• W4365458461 cites W1989189766 @default.
• W4365458461 cites W1989299822 @default.
• W4365458461 cites W2000800638 @default.
• W4365458461 cites W2004785282 @default.
• W4365458461 cites W2008158746 @default.
• W4365458461 cites W2027974246 @default.
• W4365458461 cites W2031846075 @default.
• W4365458461 cites W2034357086 @default.
• W4365458461 cites W2042450476 @default.
• W4365458461 cites W2048450131 @default.
• W4365458461 cites W2053233170 @default.
• W4365458461 cites W2065464302 @default.
• W4365458461 cites W2070201590 @default.
• W4365458461 cites W2079947769 @default.
• W4365458461 cites W2086825927 @default.
• W4365458461 cites W2120350838 @default.
• W4365458461 cites W2126909426 @default.
• W4365458461 cites W2127012454 @default.
• W4365458461 cites W2139700394 @default.
• W4365458461 cites W2147024725 @default.
• W4365458461 cites W2167279371 @default.
• W4365458461 cites W2588347323 @default.
• W4365458461 cites W2593533643 @default.
• W4365458461 cites W2734782517 @default.
• W4365458461 cites W2772709248 @default.
• W4365458461 cites W2775883868 @default.
• W4365458461 cites W2793770483 @default.
• W4365458461 cites W2875867225 @default.
• W4365458461 cites W2884729353 @default.
• W4365458461 cites W2896352304 @default.
• W4365458461 cites W2897073365 @default.
• W4365458461 cites W2899384460 @default.
• W4365458461 cites W2969298955 @default.
• W4365458461 cites W2974361058 @default.
• W4365458461 cites W3017912976 @default.
• W4365458461 cites W3087523347 @default.
• W4365458461 cites W3210740488 @default.
• W4365458461 cites W4225324600 @default.
• W4365458461 cites W4232708467 @default.
• W4365458461 cites W4283026018 @default.
• W4365458461 cites W597398767 @default.
• W4365458461 doi "https://doi.org/10.1016/j.jbc.2023.104715" @default.
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