BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

Department of Biochemistry Faculty of Natural Sciences Comenius University Bratislava Slovakia

Department of Parasitology Faculty of Science Charles University BIOCEV Vestec Czechia

Division of Infectious Diseases Department of Medicine University of Alberta Edmonton Canada

e Duve Institute Université catholique de Louvain Brussels Belgium

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czechia

Zobrazit více v PubMed

Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, Lukeš J. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol. 2021;11:200407. doi: 10.1098/rsob.200407. PubMed DOI PMC

Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol. 2018;34(6):466–480. doi: 10.1016/j.pt.2018.03.002. PubMed DOI

Stuart K, Brun R, Croft S, Fairlamb A, Gürtler RE, McKerrow J, Reed S, Tarleton R. Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest. 2008;118(4):1301–1310. doi: 10.1172/JCI33945. PubMed DOI PMC

Bruschi F, Gradoni L. The leishmaniases: old neglected tropical diseases. Cham: Springer; 2018.

Büscher P, Cecchi G, Jamonneau V, Priotto G. Human African trypanosomiasis. Lancet. 2017;390(10110):2397–2409. doi: 10.1016/S0140-6736(17)31510-6. PubMed DOI

Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019;146(1):1–27. doi: 10.1017/S0031182018000951. PubMed DOI

Billington K, Halliday C, Madden R, Dyer P, Barker AR, Moreira-Leite FF, Carrington M, Vaughan S, Hertz-Fowler C, Dean S, et al. Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei. Nat Microbiol. 2023;8(3):533–547. doi: 10.1038/s41564-022-01295-6. PubMed DOI PMC

Horn D. Genome-scale RNAi screens in African trypanosomes. Trends Parasitol. 2022;38(2):160–173. doi: 10.1016/j.pt.2021.09.002. PubMed DOI

van Hellemond JJ, Bakker BM, Tielens AG. Energy metabolism and its compartmentation in Trypanosoma brucei. Adv Microb Physiol. 2005;50:199–226. doi: 10.1016/S0065-2911(05)50005-5. PubMed DOI

Opperdoes FR, Coombs GH. Metabolism of Leishmania: proven and predicted. Trends Parasitol. 2007;23(4):149–158. doi: 10.1016/j.pt.2007.02.004. PubMed DOI

Opperdoes F, Michels PA. The metabolic repertoire of Leishmania and implications for drug discovery. In: Myler P, Fasel N, editors. Leishmania: after the genome. Norfolk: Caister Academic Press; 2008. pp. 123–158.

El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, Aggarwal G, Caler E, Renauld H, Worthey EA, Hertz-Fowler C, et al. Comparative genomics of trypanosomatid parasitic protozoa. Science. 2005;309(5733):404–409. doi: 10.1126/science.1112181. PubMed DOI

Bartholomeu DC, Teixeira SMR, Cruz AK. Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives. Mem Inst Oswaldo Cruz. 2021;116:e200634. doi: 10.1590/0074-02760200634. PubMed DOI PMC

Cantacessi C, Dantas-Torres F, Nolan MJ, Otranto D. The past, present, and future of Leishmania genomics and transcriptomics. Trends Parasitol. 2015;31(3):100–108. doi: 10.1016/j.pt.2014.12.012. PubMed DOI PMC

Shanmugasundram A, Starns D, Böhme U, Amos B, Wilkinson PA, Harb OS, Warrenfeltz S, Kissinger JC, McDowell MA, Roos DS, et al. TriTrypDB: An integrated functional genomics resource for kinetoplastida. PLoS Negl Trop Dis. 2023;17(1):e0011058. doi: 10.1371/journal.pntd.0011058. PubMed DOI PMC

Moloney NM, Barylyuk K, Tromer E, Crook OM, Breckels LM, Lilley KS, Waller RF, MacGregor P. Mapping diversity in African trypanosomes using high resolution spatial proteomics. Nat Commun. 2023;14(1):4401. doi: 10.1038/s41467-023-40125-z. PubMed DOI PMC

Kostygov AY, Albanaz ATS, Butenko A, Gerasimov ES, Lukes J, Yurchenko V: Phylogenetic framework to explore trait evolution in Trypanosomatidae. Trends Parasitol. 2024, 40(2):96–99. PubMed

Yurchenko V, Butenko A, Kostygov AY. Genomics of Trypanosomatidae: where we stand and what needs to be done? Pathogens. 2021;10(9):1124. doi: 10.3390/pathogens10091124. PubMed DOI PMC

Albanaz ATS, Carrington M, Frolov AO, Ganyukova AI, Gerasimov ES, Kostygov AY, Lukeš J, Malysheva MN, Votýpka J, Zakharova A, et al. Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae. BMC Genomics. 2023;24(1):471. doi: 10.1186/s12864-023-09591-z. PubMed DOI PMC

Škodová-Sveráková I, Verner Z, Skalický T, Votýpka J, Horváth A, Lukeš J. Lineage-specific activities of a multipotent mitochondrion of trypanosomatid flagellates. Mol Microbiol. 2015;96(1):55–67. doi: 10.1111/mmi.12920. PubMed DOI

Albanaz ATS, Gerasimov ES, Shaw JJ, Sádlová J, Lukeš J, Volf P, Opperdoes FR, Kostygov AY, Butenko A, Yurchenko V. Genome analysis of Endotrypanum and Porcisia spp., closest phylogenetic relatives of Leishmania, highlights the role of amastins in shaping pathogenicity. Genes. 2021;12(3):444. doi: 10.3390/genes12030444. PubMed DOI PMC

Bílý T, Sheikh S, Mallet A, Bastin P, Pérez-Morga D, Lukeš J, Hashimi H. Ultrastructural changes of the mitochondrion during the life cycle of Trypanosoma brucei. J Eukaryot Microbiol. 2021;68(3):e12846. doi: 10.1111/jeu.12846. PubMed DOI

Čermáková P, Maďarová A, Baráth P, Bellová J, Yurchenko V, Horváth A. Differences in mitochondrial NADH dehydrogenase activities in trypanosomatids. Parasitology. 2021;148(10):1161–1170. doi: 10.1017/S0031182020002425. PubMed DOI PMC

Butenko A, Hammond M, Field MC, Ginger ML, Yurchenko V, Lukeš J. Reductionist pathways for parasitism in euglenozoans? Expanded datasets provide new insights. Trends Parasitol. 2021;37(2):100–116. doi: 10.1016/j.pt.2020.10.001. PubMed DOI

Lukeš J, Tesařová M, Yurchenko V, Votýpka J. Characterization of a new cosmopolitan genus of trypanosomatid parasites, Obscuromonas gen. nov (Blastocrithidiinae subfam. nov) Eur J Protistol. 2021;79:125778. doi: 10.1016/j.ejop.2021.125778. PubMed DOI

Záhonová K, Kostygov A, Ševčíková T, Yurchenko V, Eliáš M. An unprecedented non-canonical nuclear genetic code with all three termination codons reassigned as sense codons. Curr Biol. 2016;26(17):2364–2369. doi: 10.1016/j.cub.2016.06.064. PubMed DOI

Kachale A, Pavlíková Z, Nenarokova A, Roithová A, Durante IM, Miletínová P, Záhonová K, Nenarokov S, Votýpka J, Horáková E, et al. Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment. Nature. 2023;613(7945):751–758. doi: 10.1038/s41586-022-05584-2. PubMed DOI

Baranov PV, Atkins JF. No stopping with a short-stem transfer RNA. Nature. 2023;613(7945):631–632. doi: 10.1038/d41586-022-04585-5. PubMed DOI

Andrade-Alviárez D, Bonive-Boscan AD, Cáceres AJ, Quiñones W, Gualdrón-López M, Ginger ML, Michels PAM. Delineating transitions during the evolution of specialised peroxisomes: glycosome formation in kinetoplastid and diplonemid protists. Front Cell Dev Biol. 2022;10:979269. doi: 10.3389/fcell.2022.979269. PubMed DOI PMC

Haanstra JR, González-Marcano EB, Gualdrón-López M, Michels PA. Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites. Biochim Biophys Acta. 2016;1863(5):1038–1048. doi: 10.1016/j.bbamcr.2015.09.015. PubMed DOI

Opperdoes FR, Borst P. Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 1977;80(2):360–364. doi: 10.1016/0014-5793(77)80476-6. PubMed DOI

Opperdoes FR, Michels PA. The glycosomes of the Kinetoplastida. Biochimie. 1993;75(3–4):231–234. doi: 10.1016/0300-9084(93)90081-3. PubMed DOI

Opperdoes FR, Szikora JP. In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes. Mol Biochem Parasitol. 2006;147(2):193–206. doi: 10.1016/j.molbiopara.2006.02.010. PubMed DOI

Esteve MI, Cazzulo JJ. The 6-phosphogluconate dehydrogenase from Trypanosoma cruzi: the absence of two inter-subunit salt bridges as a reason for enzyme instability. Mol Biochem Parasitol. 2004;133(2):197–207. doi: 10.1016/j.molbiopara.2003.10.007. PubMed DOI

Swinkels BW, Gibson WC, Osinga KA, Kramer R, Veeneman GH, van Boom JH, Borst P. Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei. EMBO J. 1986;5(6):1291–1298. doi: 10.1002/j.1460-2075.1986.tb04358.x. PubMed DOI PMC

Carrero-Lérida J, Pérez-Moreno G, Castillo-Acosta VM, Ruiz-Pérez LM, González-Pacanowska D. Intracellular location of the early steps of the isoprenoid biosynthetic pathway in the trypanosomatids Leishmania major and Trypanosoma brucei. Int J Parasitol. 2009;39(3):307–314. doi: 10.1016/j.ijpara.2008.08.012. PubMed DOI

Cull B, Prado Godinho JL, Fernandes Rodrigues JC, Frank B, Schurigt U, Williams RA, Coombs GH, Mottram JC. Glycosome turnover in Leishmania major is mediated by autophagy. Autophagy. 2014;10(12):2143–2157. doi: 10.4161/auto.36438. PubMed DOI PMC

Galland N, Demeure F, Hannaert V, Verplaetse E, Vertommen D, Van der Smissen P, Courtoy PJ, Michels PA. Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei. Biochim Biophys Acta. 2007;1773(4):521–535. doi: 10.1016/j.bbamcr.2007.01.006. PubMed DOI

Crowe LP, Morris MT. Glycosome heterogeneity in kinetoplastids. Biochem Soc Trans. 2021;49(1):29–39. doi: 10.1042/BST20190517. PubMed DOI PMC

Lukeš J, Skalický T, Týč J, Votýpka J, Yurchenko V. Evolution of parasitism in kinetoplastid flagellates. Mol Biochem Parasitol. 2014;195(2):115–122. doi: 10.1016/j.molbiopara.2014.05.007. PubMed DOI

Kraeva N, Horáková E, Kostygov A, Kořený L, Butenko A, Yurchenko V, Lukeš J. Catalase in Leishmaniinae: with me or against me? Infect Genet Evol. 2017;50:121–127. doi: 10.1016/j.meegid.2016.06.054. PubMed DOI

Bianchi C, Kostygov AY, Kraeva N, Záhonová K, Horáková E, Sobotka R, Lukeš J, Yurchenko V. An enigmatic catalase of Blastocrithidia. Mol Biochem Parasitol. 2019;232:111199. doi: 10.1016/j.molbiopara.2019.111199. PubMed DOI

Chmelová L, Bianchi C, Albanaz ATS, Režnarová J, Wheeler R, Kostygov AY, Kraeva N, Yurchenko V. Comparative analysis of three trypanosomatid catalases of different origin. Antioxidants. 2021;11(1):46. doi: 10.3390/antiox11010046. PubMed DOI PMC

Docampo R, de Souza W, Miranda K, Rohloff P, Moreno SN. Acidocalcisomes - conserved from bacteria to man. Nat Rev Microbiol. 2005;3(3):251–261. doi: 10.1038/nrmicro1097. PubMed DOI

Docampo R, Moreno SN. Acidocalcisomes. Cell Calcium. 2011;50(2):113–119. doi: 10.1016/j.ceca.2011.05.012. PubMed DOI PMC

Docampo R, Huang G. Acidocalcisomes of eukaryotes. Curr Opin Cell Biol. 2016;41:66–72. doi: 10.1016/j.ceb.2016.04.007. PubMed DOI PMC

Vercesi AE, Moreno SN, Docampo R. Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei. Biochem J. 1994;304:227–233. doi: 10.1042/bj3040227. PubMed DOI PMC

Docampo R, Scott DA, Vercesi AE, Moreno SN. Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi. Biochem J. 1995;310:1005–1012. doi: 10.1042/bj3101005. PubMed DOI PMC

Moreno SN, Zhong L. Acidocalcisomes in Toxoplasma gondii tachyzoites. Biochem J. 1996;313:655–659. doi: 10.1042/bj3130655. PubMed DOI PMC

Luo S, Marchesini N, Moreno SN, Docampo R. A plant-like vacuolar H+-pyrophosphatase in Plasmodium falciparum. FEBS Lett. 1999;460(2):217–220. doi: 10.1016/S0014-5793(99)01353-8. PubMed DOI

Ruiz FA, Marchesini N, Seufferheld M. Govindjee, Docampo R: The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes. J Biol Chem. 2001;276(49):46196–46203. doi: 10.1074/jbc.M105268200. PubMed DOI

Pusnik M, Schmidt O, Perry AJ, Oeljeklaus S, Niemann M, Warscheid B, Lithgow T, Meisinger C, Schneider A. Mitochondrial preprotein translocase of trypanosomatids has a bacterial origin. Curr Biol. 2011;21(20):1738–1743. doi: 10.1016/j.cub.2011.08.060. PubMed DOI

Villafraz O, Biran M, Pineda E, Plazolles N, Cahoreau E, Ornitz Oliveira Souza R, Thonnus M, Allmann S, Tetaud E, Rivière L et al: Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline. PLoS Pathog. 2021, 17(3):e1009204. PubMed PMC

van Hellemond JJ, Opperdoes FR, Tielens AG. The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei. Biochem Soc Trans. 2005;33:967–971. doi: 10.1042/BST0330967. PubMed DOI

Tielens AG, van Hellemond JJ. Surprising variety in energy metabolism within Trypanosomatidae. Trends Parasitol. 2009;25(10):482–490. doi: 10.1016/j.pt.2009.07.007. PubMed DOI

Martin WF, Tielens AGM, Mentel M. Mitochondria and anaerobic energy metabolism in eukaryotes: biochemistry and evolution. Düsseldorf: De Gruyter; 2021.

Kaufer A, Barratt J, Stark D, Ellis J. The complete coding region of the maxicircle as a superior phylogenetic marker for exploring evolutionary relationships between members of the Leishmaniinae. Infect Genet Evol. 2019;70:90–100. doi: 10.1016/j.meegid.2019.02.002. PubMed DOI

Gerasimov ES, Zamyatnina KA, Matveeva NS, Rudenskaya YA, Kraeva N, Kolesnikov AA, Yurchenko V. Common structural patterns in the maxicircle divergent region of Trypanosomatidae. Pathogens. 2020;9(2):100. doi: 10.3390/pathogens9020100. PubMed DOI PMC

Acestor N, Zíková A, Dalley RA, Anupama A, Panigrahi AK, Stuart KD: Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form. Mol Cell Proteomics. 2011, 10(9):M110 006908. PubMed PMC

Panigrahi AK, Ziková A, Dalley RA, Acestor N, Ogata Y, Anupama A, Myler PJ, Stuart KD. Mitochondrial complexes in Trypanosoma brucei: a novel complex and a unique oxidoreductase complex. Mol Cell Proteomics. 2008;7(3):534–545. doi: 10.1074/mcp.M700430-MCP200. PubMed DOI

Duarte M, Ferreira C, Khandpur GK, Flohr T, Zimmermann J, Castro H, Herrmann JM, Morgan B, Tomás AM. Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I. Proc Natl Acad Sci U S A. 2021;118(42):e2103803118. doi: 10.1073/pnas.2103803118. PubMed DOI PMC

Čermáková P, Verner Z, Man P, Lukeš J, Horváth A. Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida) FEBS J. 2007;274(12):3150–3158. doi: 10.1111/j.1742-4658.2007.05847.x. PubMed DOI

Hierro-Yap C, Šubrtová K, Gahura O, Panicucci B, Dewar C, Chinopoulos C, Schnaufer A, Zíková A. Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei. J Biol Chem. 2021;296:100357. doi: 10.1016/j.jbc.2021.100357. PubMed DOI PMC

Verner Z, Škodová I, Poláková S, Ďurišová-Benkovičová V, Horváth A, Lukeš J. Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclic Trypanosoma brucei. Parasitology. 2013;140(3):328–337. doi: 10.1017/S003118201200162X. PubMed DOI

van Hellemond JJ, Simons B, Millenaar FF, Tielens AG. A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp. J Eukaryot Microbiol. 1998;45(4):426–430. doi: 10.1111/j.1550-7408.1998.tb05094.x. PubMed DOI

Chaudhuri M, Ott RD, Hill GC. Trypanosome alternative oxidase: from molecule to function. Trends Parasitol. 2006;22(10):484–491. doi: 10.1016/j.pt.2006.08.007. PubMed DOI

Angerer H, Nasiri HR, Niedergesass V, Kerscher S, Schwalbe H, Brandt U. Tracing the tail of ubiquinone in mitochondrial complex I. Biochim Biophys Acta. 2012;1817(10):1776–1784. doi: 10.1016/j.bbabio.2012.03.021. PubMed DOI

Lai DH, Poropat E, Pravia C, Landoni M, Couto AS, Rojo FG, Fuchs AG, Dubin M, Elingold I, Rodríguez JB, et al. Solanesyl diphosphate synthase, an enzyme of the ubiquinone synthetic pathway, is required throughout the life cycle of Trypanosoma brucei. Eukaryot Cell. 2014;13(2):320–328. doi: 10.1128/EC.00271-13. PubMed DOI PMC

Opperdoes FR, Butenko A, Flegontov P, Yurchenko V, Lukeš J. Comparative metabolism of free-living Bodo saltans and parasitic trypanosomatids. J Eukaryot Microbiol. 2016;63(5):657–678. doi: 10.1111/jeu.12315. PubMed DOI

Mitchell GC, Baker JH, Sleigh MA. Feeding of a freshwater flagellate, Bodo saltans, on diverse bacteria. J Protozool. 1988;35(2):219–222. doi: 10.1111/j.1550-7408.1988.tb04327.x. DOI

Stairs CW, Eme L, Muñoz-Gómez SA, Cohen A, Dellaire G, Shepherd JN, Fawcett JP, Roger AJ. Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis. Elife. 2018;7:e34292. doi: 10.7554/eLife.34292. PubMed DOI PMC

Besteiro S, Biran M, Biteau N, Coustou V, Baltz T, Canioni P, Bringaud F. Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme NADH-dependent fumarate reductase. J Biol Chem. 2002;277(41):38001–38012. doi: 10.1074/jbc.M201759200. PubMed DOI

Hernandez FR, Turrens JF. Rotenone at high concentrations inhibits NADH-fumarate reductase and the mitochondrial respiratory chain of Trypanosoma brucei and T. cruzi. Mol Biochem Parasitol. 1998;93(1):135–137. doi: 10.1016/S0166-6851(98)00015-2. PubMed DOI

Coustou V, Biran M, Besteiro S, Riviere L, Baltz T, Franconi JM, Bringaud F. Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei. J Biol Chem. 2006;281(37):26832–26846. doi: 10.1074/jbc.M601377200. PubMed DOI

Coustou V, Besteiro S, Riviere L, Biran M, Biteau N, Franconi JM, Boshart M, Baltz T, Bringaud F. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei. J Biol Chem. 2005;280(17):16559–16570. doi: 10.1074/jbc.M500343200. PubMed DOI

van Grinsven KW, van Den Abbeele J, van den Bossche P, van Hellemond JJ, Tielens AG. Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms. Eukaryot Cell. 2009;8(8):1307–1311. doi: 10.1128/EC.00091-09. PubMed DOI PMC

Opperdoes FR, Butenko A, Zakharova A, Gerasimov ES, Zimmer SL, Lukeš J, Yurchenko V. The remarkable metabolism of Vickermania ingenoplastis: genomic predictions. Pathogens. 2021;10(1):68. doi: 10.3390/pathogens10010068. PubMed DOI PMC

Redman CA, Coombs GH. The products and pathways of glucose catabolism in Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum. J Eukaryot Microbiol. 1997;44(1):46–51. doi: 10.1111/j.1550-7408.1997.tb05690.x. DOI

Walsh CT. Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. J Biol Chem. 1989;264(5):2393–2396. doi: 10.1016/S0021-9258(19)81624-1. PubMed DOI

Abendroth J, Choi R, Wall A, Clifton MC, Lukacs CM, Staker BL, Van Voorhis W, Myler P, Lorimer DD, Edwards TE. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia. Acta Crystallogr F Struct Biol Commun. 2015;71(Pt 5):566–571. doi: 10.1107/S2053230X15001831. PubMed DOI PMC

Hofer A. Targeting the nucleotide metabolism of Trypanosoma brucei and other trypanosomatids. FEMS Microbiol Rev. 2023;47(3):fuad020. doi: 10.1093/femsre/fuad020. PubMed DOI PMC

Balaña-Fouce R, Calvo-Álvarez E, Álvarez-Velilla R, Prada CF, Pérez-Pertejo Y, Reguera RM. Role of trypanosomatid's arginase in polyamine biosynthesis and pathogenesis. Mol Biochem Parasitol. 2012;181(2):85–93. doi: 10.1016/j.molbiopara.2011.10.007. PubMed DOI

Kostygov AY, Yurchenko V. Revised classification of the subfamily Leishmaniinae (Trypanosomatidae) Folia Parasitol. 2017;64:020. doi: 10.14411/fp.2017.020. PubMed DOI

Williams RA, Kelly SM, Mottram JC, Coombs GH. 3-Mercaptopyruvate sulfurtransferase of Leishmania contains an unusual C-terminal extension and is involved in thioredoxin and antioxidant metabolism. J Biol Chem. 2003;278(3):1480–1486. doi: 10.1074/jbc.M209395200. PubMed DOI

Singh K, Singh KP, Equbal A, Suman SS, Zaidi A, Garg G, Pandey K, Das P, Ali V. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani. Biochimie. 2016;131:29–44. doi: 10.1016/j.biochi.2016.09.004. PubMed DOI

Williams RA, Westrop GD, Coombs GH. Two pathways for cysteine biosynthesis in Leishmania major. Biochem J. 2009;420(3):451–462. doi: 10.1042/BJ20082441. PubMed DOI

Marchese L, Nascimento JF, Damasceno FS, Bringaud F, Michels PAM, Silber AM. The uptake and metabolism of amino acids, and their unique role in the biology of pathogenic trypanosomatids. Pathogens. 2018;7(2):36. doi: 10.3390/pathogens7020036. PubMed DOI PMC

Barderi P, Campetella O, Frasch AC, Santome JA, Hellman U, Pettersson U, Cazzulo JJ. The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression. Biochem J. 1998;330:951–958. doi: 10.1042/bj3300951. PubMed DOI PMC

Mantilla BS, Paes LS, Pral EM, Martil DE, Thiemann OH, Fernandez-Silva P, Bastos EL, Silber AM. Role of Δ1-pyrroline-5-carboxylate dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi. J Biol Chem. 2015;290(12):7767–7790. doi: 10.1074/jbc.M114.574525. PubMed DOI PMC

Marchese L, Olavarria K, Mantilla BS, Avila CC, Souza ROO, Damasceno FS, Elias MC, Silber AM. Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools. Biochem J. 2020;477(10):1827–1845. doi: 10.1042/BCJ20200232. PubMed DOI

Kikuchi G, Motokawa Y, Yoshida T, Hiraga K. Glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia. Proc Jpn Acad Ser B Phys Biol Sci. 2008;84(7):246–263. doi: 10.2183/pjab.84.246. PubMed DOI PMC

Hai Y, Dugery RJ, Healy D, Christianson DW. Formiminoglutamase from Trypanosoma cruzi is an arginase-like manganese metalloenzyme. Biochemistry. 2013;52(51):9294–9309. doi: 10.1021/bi401352h. PubMed DOI PMC

Silber AM, Colli W, Ulrich H, Alves MJ, Pereira CA. Amino acid metabolic routes in Trypanosoma cruzi: possible therapeutic targets against Chagas' disease. Curr Drug Targets Infect Disord. 2005;5(1):53–64. doi: 10.2174/1568005053174636. PubMed DOI

Ginger ML, Chance ML, Sadler IH, Goad LJ. The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana. J Biol Chem. 2001;276(15):11674–11682. doi: 10.1074/jbc.M006850200. PubMed DOI

Marciano D, Santana M, Mantilla BS, Silber AM, Marino-Buslje C, Nowicki C. Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major. Mol Biochem Parasitol. 2010;173(2):170–174. doi: 10.1016/j.molbiopara.2010.06.004. PubMed DOI

Berger BJ, Dai WW, Wang H, Stark RE, Cerami A. Aromatic amino acid transamination and methionine recycling in trypanosomatids. Proc Natl Acad Sci U S A. 1996;93(9):4126–4130. doi: 10.1073/pnas.93.9.4126. PubMed DOI PMC

Nosei C, Avila JL. Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp. Comp Biochem Physiol B. 1985;81(3):701–704. doi: 10.1016/0305-0491(85)90390-6. PubMed DOI

Capelluto DG, Hellman U, Cazzulo JJ, Cannata JJ. Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi. Eur J Biochem. 2000;267(3):712–719. doi: 10.1046/j.1432-1327.2000.01047.x. PubMed DOI

Capelluto DG, Hellman U, Cazzulo JJ, Cannata JJ. Purification and partial characterization of three isoforms of serine hydroxymethyltransferase from Crithidia fasciculata. Mol Biochem Parasitol. 1999;98(2):187–201. doi: 10.1016/S0166-6851(98)00166-2. PubMed DOI

El Sawalhy A, Seed JR, Hall JE, El Attar H. Increased excretion of aromatic amino acid catabolites in animals infected with Trypanosoma brucei evansi. J Parasitol. 1998;84(3):469–473. doi: 10.2307/3284707. PubMed DOI

El Sawalhy A, Seed JR, El Attar H, Hall JE. Catabolism of tryptophan by Trypanosoma evansi. J Eukaryot Microbiol. 1995;42(6):684–690. doi: 10.1111/j.1550-7408.1995.tb01616.x. PubMed DOI

Pyrih J, Hammond M, Alves A, Dean S, Sunter JD, Wheeler RJ, Gull K, Lukeš J. Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology. Cell Rep. 2023;42(9):113083. doi: 10.1016/j.celrep.2023.113083. PubMed DOI

Cunningham ML, Beverley SM. Pteridine salvage throughout the Leishmania infectious cycle: implications for antifolate chemotherapy. Mol Biochem Parasitol. 2001;113(2):199–213. doi: 10.1016/S0166-6851(01)00213-4. PubMed DOI

Vickers TJ, Beverley SM. Folate metabolic pathways in Leishmania. Essays Biochem. 2011;51:63–80. doi: 10.1042/bse0510063. PubMed DOI PMC

Dewar S, Sienkiewicz N, Ong HB, Wall RJ, Horn D, Fairlamb AH. The role of folate transport in antifolate drug action in Trypanosoma brucei. J Biol Chem. 2016;291(47):24768–24778. doi: 10.1074/jbc.M116.750422. PubMed DOI PMC

Dole VS, Myler PJ, Stuart KD, Madhubala R. Expression of biopterin transporter (BT1) protein in Leishmania. FEMS Microbiol Lett. 2002;208(1):89–91. doi: 10.1111/j.1574-6968.2002.tb11065.x. PubMed DOI

Ravooru N, Paul OS, Nagendra HG, Sathyanarayanan N. Data enabled prediction analysis assigns folate/biopterin transporter (BT1) family to 36 hypothetical membrane proteins in Leishmania donovani. Bioinformation. 2019;15(10):697–708. doi: 10.6026/97320630015697. PubMed DOI PMC

Smithson DC, Lee J, Shelat AA, Phillips MA, Guy RK. Discovery of potent and selective inhibitors of Trypanosoma brucei ornithine decarboxylase. J Biol Chem. 2010;285(22):16771–16781. doi: 10.1074/jbc.M109.081588. PubMed DOI PMC

Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, et al. ATP synthesis and storage. Purinergic Signal. 2012;8(3):343–357. doi: 10.1007/s11302-012-9305-8. PubMed DOI PMC

Pereira CA, Bouvier LA. Cámara Md, Miranda MR: Singular features of trypanosomatids' phosphotransferases involved in cell energy management. Enzyme Res. 2011;2011:576483. doi: 10.4061/2011/576483. PubMed DOI PMC

Voncken F, Gao F, Wadforth C, Harley M, Colasante C. The phosphoarginine energy-buffering system of Trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations. PLoS One. 2013;8(6):e65908. doi: 10.1371/journal.pone.0065908. PubMed DOI PMC

Pereira CA, Alonso GD, Ivaldi S, Silber A, Alves MJ, Bouvier LA, Flawia MM, Torres HN. Arginine metabolism in Trypanosoma cruzi is coupled to parasite stage and replication. FEBS Lett. 2002;526(1–3):111–114. doi: 10.1016/S0014-5793(02)03157-5. PubMed DOI

Wilson ZN, Gilroy CA, Boitz JM, Ullman B, Yates PA. Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani. J Biol Chem. 2012;287(16):12759–12770. doi: 10.1074/jbc.M112.346502. PubMed DOI PMC

Vertommen D, Van Roy J, Szikora JP, Rider MH, Michels PA, Opperdoes FR. Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol. 2008;158(2):189–201. doi: 10.1016/j.molbiopara.2007.12.008. PubMed DOI

Mikkola S. Nucleotide sugars in chemistry and biology. Molecules. 2020;25(23):5755. doi: 10.3390/molecules25235755. PubMed DOI PMC

Turnock DC, Ferguson MA. Sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Eukaryot Cell. 2007;6(8):1450–1463. doi: 10.1128/EC.00175-07. PubMed DOI PMC

Bandini G, Mariño K, Guther ML, Wernimont AK, Kuettel S, Qiu W, Afzal S, Kelner A, Hui R, Ferguson MA. Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase. Mol Microbiol. 2012;85(3):513–534. doi: 10.1111/j.1365-2958.2012.08124.x. PubMed DOI PMC

Matveyev AV, Alves JM, Serrano MG, Lee V, Lara AM, Barton WA, Costa-Martins AG, Beverley SM, Camargo EP, Teixeira MM, Buck GA. The evolutionary loss of RNAi key determinants in kinetoplastids as a multiple sporadic phenomenon. J Mol Evol. 2017;84(2–3):104–115. doi: 10.1007/s00239-017-9780-1. PubMed DOI PMC

Lye LF, Owens K, Shi H, Murta SM, Vieira AC, Turco SJ, Tschudi C, Ullu E, Beverley SM. Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog. 2010;6(10):e1001161. doi: 10.1371/journal.ppat.1001161. PubMed DOI PMC

Leroux M, Luquain-Costaz C, Lawton P, Azzouz-Maache S, Delton I. Fatty acid composition and metabolism in Leishmania parasite species: potential biomarkers or drug targets for leishmaniasis? Int J Mol Sci. 2023;24(5):4702. doi: 10.3390/ijms24054702. PubMed DOI PMC

Smith TK, Bütikofer P. Lipid metabolism in Trypanosoma brucei. Mol Biochem Parasitol. 2010;172(2):66–79. doi: 10.1016/j.molbiopara.2010.04.001. PubMed DOI PMC

Michels PA, Bringaud F, Herman M, Hannaert V. Metabolic functions of glycosomes in trypanosomatids. Biochim Biophys Acta. 2006;1763(12):1463–1477. doi: 10.1016/j.bbamcr.2006.08.019. PubMed DOI

Berman JD, Gallalee JV, Best JM, Hill T. Uptake, distribution, and oxidation of fatty acids by Leishmania mexicana amastigotes. J Parasitol. 1987;73(3):555–560. doi: 10.2307/3282136. PubMed DOI

Lee SH, Stephens JL, Englund PT. A fatty-acid synthesis mechanism specialized for parasitism. Nat Rev Microbiol. 2007;5(4):287–297. doi: 10.1038/nrmicro1617. PubMed DOI

Livore VI, Tripodi KE, Uttaro AD. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS J. 2007;274(1):264–274. doi: 10.1111/j.1742-4658.2006.05581.x. PubMed DOI

Tripodi KE, Buttigliero LV, Altabe SG, Uttaro AD. Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan. FEBS J. 2006;273(2):271–280. doi: 10.1111/j.1742-4658.2005.05049.x. PubMed DOI

Parreira de Aquino G, Mendes Gomes MA, Kopke Salinas R, Laranjeira-Silva MF. Lipid and fatty acid metabolism in trypanosomatids. Microb Cell. 2021;8(11):262–275. doi: 10.15698/mic2021.11.764. PubMed DOI PMC

Lepesheva GI, Villalta F, Waterman MR. Targeting Trypanosoma cruzi sterol 14alpha-demethylase (CYP51) Adv Parasitol. 2011;75:65–87. doi: 10.1016/B978-0-12-385863-4.00004-6. PubMed DOI PMC

Yu X, Cojocaru V, Mustafa G, Salo-Ahen OM, Lepesheva GI, Wade RC. Dynamics of CYP51: implications for function and inhibitor design. J Mol Recognit. 2015;28(2):59–73. doi: 10.1002/jmr.2412. PubMed DOI PMC

Wilkinson SR, Prathalingam SR, Taylor MC, Horn D, Kelly JM. Vitamin C biosynthesis in trypanosomes: a role for the glycosome. Proc Natl Acad Sci U S A. 2005;102(33):11645–11650. doi: 10.1073/pnas.0504251102. PubMed DOI PMC

Tripodi KE, Menendez Bravo SM, Cricco JA. Role of heme and heme-proteins in trypanosomatid essential metabolic pathways. Enzyme Res. 2011;2011:873230. doi: 10.4061/2011/873230. PubMed DOI PMC

Cenci U, Moog D, Curtis BA, Tanifuji G, Eme L, Lukes J, Archibald JM. Heme pathway evolution in kinetoplastid protists. BMC Evol Biol. 2016;16(1):109. doi: 10.1186/s12862-016-0664-6. PubMed DOI PMC

Miguel DC, Flannery AR, Mittra B, Andrews NW. Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence. Infect Immun. 2013;81(10):3620–3626. doi: 10.1128/IAI.00687-13. PubMed DOI PMC

Huynh C, Yuan X, Miguel DC, Renberg RL, Protchenko O, Philpott CC, Hamza I, Andrews NW. Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1. PLoS Pathog. 2012;8(7):e1002795. doi: 10.1371/journal.ppat.1002795. PubMed DOI PMC

Renberg RL, Yuan X, Samuel TK, Miguel DC, Hamza I, Andrews NW, Flannery AR. The heme transport capacity of LHR1 determines the extent of virulence in Leishmania amazonensis. PLoS Negl Trop Dis. 2015;9(5):e0003804. doi: 10.1371/journal.pntd.0003804. PubMed DOI PMC

Kořený L, Oborník M, Lukeš J. Make it, take it, or leave it: heme metabolism of parasites. PLoS Pathog. 2013;9(1):e1003088. doi: 10.1371/journal.ppat.1003088. PubMed DOI PMC

Yurchenko V, Kostygov A, Havlová J, Grybchuk-Ieremenko A, Ševčíková T, Lukeš J, Ševčík J, Votýpka J. Diversity of trypanosomatids in cockroaches and the description of Herpetomonas tarakana sp. n. J Eukaryot Microbiol. 2016;63(2):198–209. doi: 10.1111/jeu.12268. PubMed DOI

Hamilton PT, Votýpka J, Dostalova A, Yurchenko V, Bird NH, Lukeš J, Lemaitre B, Perlman SJ. Infection dynamics and immune response in a newly described Drosophila-trypanosomatid association. mBio. 2015;6(5):e01356–01315. doi: 10.1128/mBio.01356-15. PubMed DOI PMC

Durante IM, Butenko A, Rašková V, Charyyeva A, Svobodová M, Yurchenko V, Hashimi H, Lukeš J. Large-scale phylogenetic analysis of trypanosomatid adenylate cyclases reveals associations with extracellular lifestyle and host-pathogen interplay. Genome Biol Evol. 2020;12(12):2403–2416. doi: 10.1093/gbe/evaa226. PubMed DOI PMC

Verplaetse E, Rigden DJ, Michels PA. Identification, characterization and essentiality of the unusual peroxin 13 from Trypanosoma brucei. Biochim Biophys Acta. 2009;1793(3):516–527. doi: 10.1016/j.bbamcr.2008.12.020. PubMed DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Opperdoes FR, Lemey P: Phylogenetic analysis using protein sequences. In: The phylogenetic handbook A practical approach to phylogenetic analysis and hypothesis testing Edited by Lemey P, Salemy M, Vandamme A-M, 2 edn. Cambridge: Cambridge University Press; 2009: 310-338.

Bairoch A, Boeckmann B, Ferro S, Gasteiger E. Swiss-Prot: juggling between evolution and stability. Brief Bioinform. 2004;5(1):39–55. doi: 10.1093/bib/5.1.39. PubMed DOI

Blum M, Chang HY, Chuguransky S, Grego T, Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T, Qureshi M, Raj S, et al. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res. 2021;49(D1):D344–D354. doi: 10.1093/nar/gkaa977. PubMed DOI PMC

Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ, et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021;49(D1):D412–D419. doi: 10.1093/nar/gkaa913. PubMed DOI PMC

Butenko A, Opperdoes FR, Flegontova O, Horak A, Hampl V, Keeling P, Gawryluk RMR, Tikhonenkov D, Flegontov P, Lukeš J. Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids. BMC Biol. 2020;18(1):23. doi: 10.1186/s12915-020-0754-1. PubMed DOI PMC

Armenteros JJA, Salvatore M, Emanuelsson O, Winther O, von Heijne G, Elofsson A, Nielsen H. Detecting sequence signals in targeting peptides using deep learning. Life Sci Alliance. 2019;2(5):e201900429. doi: 10.26508/lsa.201900429. PubMed DOI PMC

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment