Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Faculty of Biology M 5 Lomonosov Moscow State University Moscow Russia

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czech Republic

Martsinovsky Institute of Medical Parasitology Tropical and Vector Borne Diseases Sechenov University Moscow Russia

Zobrazit více v PubMed

Maslov DA, Opperdoes FR, Kostygov AY, et al. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019;146(1):1–27. PubMed

Lukeš J, Butenko A, Hashimi H, et al. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol. 2018;34(6):466–480. PubMed

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

WHO . Leishmaniasis. Accessed on 4 March, 2021. https://www.who.int/en/news-room/fact-sheets/detail/leishmaniasis .

Gillespie PM, Beaumier CM, Strych U, et al. Status of vaccine research and development of vaccines for leishmaniasis. Vaccine. 2016;34(26):2992–2995. PubMed

Ghorbani M, Farhoudi R. Leishmaniasis in humans: drug or vaccine therapy? Drug Des Devel Ther. 2018;12:25–40. PubMed PMC

Dostálová A, Volf P. Leishmania development in sand flies: parasite-vector interactions overview. Parasit Vectors. 2012;5(1):276. PubMed PMC

Dvorák V, Shaw JJ, Volf P. Parasite biology: the vectors. In: Bruschi F, Gradoni L, editors. The leishmaniases: old neglected tropical diseases. Cham, Switzerland: Springer; 2018. p. 31–77.

Rossi M, Fasel N. How to master the host immune system? Leishmania parasites have the solutions!. Int Immunol. 2018;30(3):103–111. PubMed PMC

Gossage SM, Rogers ME, Bates PA. Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle. Int J Parasitol. 2003;33(10):1027–1034. PubMed PMC

Bates PA, Rogers ME. New insights into the developmental biology and transmission mechanisms of Leishmania. Curr Mol Med. 2004;4(6):601–609. PubMed

Sádlová J, Volf P. Peritrophic matrix of phlebotomus duboscqi and its kinetics during Leishmania major development. Cell Tissue Res. 2009;337(2):313–325. PubMed PMC

Lukeš J, Skalický T, Týč J, et al. Evolution of parasitism in kinetoplastid flagellates. Mol Biochem Parasitol. 2014;195(2):115–122. PubMed

Ghosh S, Banerjee P, Sarkar A, et al. Coinfection of Leptomonas seymouri and leishmania donovani in Indian leishmaniasis. J Clin Microbiol. 2012;50(8):2774–2778. PubMed PMC

Singh N, Chikara S, Sundar S. SOLiD sequencing of genomes of clinical isolates of Leishmania donovani from India confirm Leptomonas co-infection and raise some key questions. PLoS One. 2013;8(2):e55738. PubMed PMC

Srivastava P, Prajapati VK, Vanaerschot M, et al. Detection of Leptomonas sp. parasites in clinical isolates of kala-azar patients from India. Infect Genet Evol. 2010;10(7):1145–1150. PubMed PMC

Bhattarai NR, Das ML, Rijal S, et al. Natural infection of Phlebotomus argentipes with Leishmania and other trypanosomatids in a visceral leishmaniasis endemic region of nepal. Trans R Soc Trop Med Hyg. 2009;103(11):1087–1092. PubMed

Maruyama SR, De Santana AKM, Takamiya NT, et al. Non- Leishmania parasite in fatal visceral Leishmaniasis–like disease, brazil. Emerg Infect Dis. 2019;25(11):2088–2092. PubMed PMC

De Sa MF, De Sa CM, Veronese MA, et al. Morphologic and biochemical characterization of Crithidia brasiliensis sp.n. J Protozool. 1980;27(3):253–257. PubMed

McGhee RB. The infection of avian embryos with Crithidia species and Leishmania tarentola. J Infect Dis. 1959;105(1):18–25. PubMed

Ishemgulova A, Butenko A, Kortišová L, et al. Molecular mechanisms of thermal resistance of the insect trypanosomatid Crithidia thermophila. PLoS One. 2017;12(3):e0174165. PubMed PMC

Flegontov P, Butenko A, Firsov S, et al. Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania. Sci Rep. 2016;6(1):23704. PubMed PMC

Butenko A, Kostygov AY, Sádlová J, et al. Comparative genomics of Leishmania (Mundinia). BMC Genomics. 2019;20(1):726. PubMed PMC

Sloan MA, Brooks K, Otto TD, et al. Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania. PLoS Genet. 2019;15(11):e1008452. PubMed PMC

Škodová-Sveráková I, Záhonová K, Bučková B, et al. Catalase and ascorbate peroxidase in euglenozoan protists. Pathogens. 2020;9(4):317. PubMed PMC

Kraeva N, Horáková E, Kostygov A, et al. Catalase in Leishmaniinae: with me or against me? Infect Genet Evol. 2017;50:121–127. PubMed

Bianchi C, Kostygov AY, Kraeva N, et al. An enigmatic catalase of Blastocrithidia. Mol Biochem Parasitol. 2019;232:111199. PubMed

Khan YA, Andrews NW, Mittra B. ROS regulate differentiation of visceralizing Leishmania species into the virulent amastigote form. Parasitol Open. 2018;4:e19. PubMed PMC

Mittra B, Cortez M, Haydock A, et al. Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J Exp Med. 2013;210(2):401–416. PubMed PMC

Horáková E, Faktorová D, Kraeva N, et al. Catalase compromises the development of the insect and mammalian stages of Trypanosoma brucei. FEBS J. 2020;287(5):964–977. PubMed

Freire ACG, Alves CL, Goes GR, et al. Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi. Parasitology. 2017;144(11):1498–1510. PubMed

Opperdoes FR, Butenko A, Flegontov P, et al. Comparative metabolism of free-living bodo saltans and parasitic trypanosomatids. J Eukaryot Microbiol. 2016;63(5):657–678. PubMed

Duncan SM, Myburgh E, Alves-Ferreira EV, et al. DiCre-based inducible disruption of Leishmania genes. Methods Mol Biol. 2019;1971:211–224. PubMed

Duncan SM, Jones NG, Mottram JC. Recent advances in Leishmania reverse genetics: manipulating a manipulative parasite. Mol Biochem Parasitol. 2017;216:30–38. PubMed

Podešvová L, Huang H, Yurchenko V. Inducible protein stabilization system in Leishmania mexicana. Mol Biochem Parasitol. 2017;214:62–64. PubMed

Madeira Da Silva L, Owens KL, Murta SM, et al. Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins. Proc Natl Acad Sci U S A. 2009;106(18):7583–7588. PubMed PMC

Clayton CE. Gene expression in kinetoplastids. Curr Opin Microbiol. 2016;32:46–51. PubMed

Boucher N, Wu Y, Dumas C, et al. A common mechanism of stage-regulated gene expression in Leishmania mediated by a conserved 3′-untranslated region element. J Biol Chem. 2002;277(22):19511–19520. PubMed

Mishra KK, Holzer TR, Moore LL, et al. A negative regulatory element controls mRNA abundance of the Leishmania mexicana paraflagellar rod gene pfr2. Eukaryot Cell. 2003;2(5):1009–1017. PubMed PMC

McNicoll F, Müller M, Cloutier S, et al. Distinct 3′-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania. J Biol Chem. 2005;280(42):35238–35246. PubMed

Dillon LA, Okrah K, Hughitt VK, et al. Transcriptomic profiling of gene expression and RNA processing during Leishmania major differentiation. Nucleic Acids Res. 2015;43(14):6799–6813. PubMed PMC

Ishemgulova A, Kraeva N, Faktorová D, et al. T7 polymerase-driven transcription is downregulated in metacyclic promastigotes and amastigotes of Leishmania mexicana. Folia Parasitol. 2016;63:016. PubMed

Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc. 2012;2012(2):199–204. PubMed

Bates PA, Tetley L. Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH. Exp Parasitol. 1993;76(4):412–423. PubMed

Volf P, Volfová V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36(Suppl 1):S1–9. PubMed

Benson DA, Cavanaugh M, Clark K, et al. GenBank. Nucleic Acids Res. 2018;46(D1):D41–D7. PubMed PMC

Aslett M, Aurrecoechea C, Berriman M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38(suppl_1):D457–62. PubMed PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–780. PubMed PMC

Marchler-Bauer A, Derbyshire MK, Gonzales NR, et al. CDD: nCBI’s conserved domain database. Nucleic Acids Res. 2015;43(D1):D222–6. PubMed PMC

Clayton C, Adams M, Almeida R, et al. Genetic nomenclature for Trypanosoma and Leishmania. Mol Biochem Parasitol. 1998;97(1–2):221–224. PubMed

Dean S, Sunter J, Wheeler RJ, et al. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5(1):140197. PubMed PMC

Merritt C, Stuart K. Identification of essential and non-essential protein kinases by a fusion PCR method for efficient production of transgenic Trypanosoma brucei. Mol Biochem Parasitol. 2013;190(1):44–49. PubMed PMC

Kraeva N, Ishemgulova A, Lukeš J, et al. Tetracycline-inducible gene expression system in Leishmania mexicana. Mol Biochem Parasitol. 2014;198(1):11–13. PubMed

Koren S, Walenz BP, Berlin K, et al. Canu: scalable and accurate long-read assembly via adaptive k -mer weighting and repeat separation. Genome Res. 2017;27(5):722–736. PubMed PMC

Slater GS, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6(1):31. PubMed PMC

Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410. PubMed

Li H, Durbin R. Fast and accurate long-read alignment with Burrows–wheeler transform. Bioinformatics. 2010;26(5):589–595. PubMed PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–2120. PubMed PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012;9(4):357–359. PubMed PMC

Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841–842. PubMed PMC

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with dESeq2. Genome Biol. 2014;15(12):550. PubMed PMC

Ishemgulova A, Kraeva N, Hlavacova J, et al. A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts. PLoS Negl Trop Dis. 2017;11(7):e0005782. PubMed PMC

Záhonová K, Füssy Z, Oborník M, et al. RuBisCO in non-photosynthetic alga Euglena longa: divergent features, transcriptomic analysis and regulation of complex formation. PLoS One. 2016;11(7):e0158790. PubMed PMC

Räz B, Iten M, Grether-Buhler Y, et al. The alamar blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Trop. 1997;68(2):139–147. PubMed

Mikus J, Steverding D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye alamar Blue. Parasitol Int. 2000;48(3):265–269. PubMed

Myšková J, Votýpka J, Volf P. Leishmania in sand flies: comparison of quantitative Polymerase Chain Reaction with other techniques to determine the intensity of infection. J Med Entomol. 2008;45(1):133–138. PubMed

Grybchuk D, Macedo DH, Kleschenko Y, et al. The first non-LRV RNA virus in Leishmania. Viruses. 2020;12(2):168. PubMed PMC

Leštinová T, Vlková M, Votýpka J, et al. Phlebotomus papatasi exposure cross-protects mice against leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate. Acta Trop. 2015;144:9–18. PubMed

Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–682. PubMed PMC

Sádlová J, Price HP, Smith BA, et al. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, phlebotomus papatasi. Cell Microbiol. 2010;12(12):1765–1779. PubMed PMC

Rogers ME, Chance ML, Bates PA. The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitology. 2002;124(5):495–507. PubMed

Fiebig M, Kelly S, Gluenz E. Comparative life cycle transcriptomics revises Leishmania mexicana genome annotation and links a chromosome duplication with parasitism of vertebrates. PLoS Pathog. 2015;11(10):e1005186. PubMed PMC

Nugent PG, Karsani SA, Wait R, et al. Proteomic analysis of Leishmania mexicana differentiation. Mol Biochem Parasitol. 2004;136(1):51–62. PubMed

Fong D, Chang KP. Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages. Proc Natl Acad Sci U S A. 1981;78(12):7624–7628. PubMed PMC

Fong D, Wallach M, Keithly J, et al. Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana. Proc Natl Acad Sci U S A. 1984;81(18):5782–5786. PubMed PMC

Fong D, Chang KP. Changes in tubulin mRNAs during differentiation of a parasitic protozoan Leishmania mexicana. Ann N Y Acad Sci. 1986;466:129–131. PubMed

Jackson AP, Vaughan S, Gull K. Comparative genomics and concerted evolution of β-tubulin paralogs in Leishmania spp. BMC Genomics. 2006;7(1):137. PubMed PMC

Ramírez CA, Requena JM, Puerta CJ. Alpha tubulin genes from Leishmania braziliensis: genomic organization, gene structure and insights on their expression. BMC Genomics. 2013;14(1):454. PubMed PMC

Ishemgulova A, Hlavacova J, Majerova K, et al. CRISPR/Cas9 in Leishmania mexicana: a case study of LmxBTN1. PLoS One. 2018;13(2):e0192723. PubMed PMC

Donnelly MLL, Luke G, Mehrotra A, et al. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol. 2001;82(5):1013–1025. PubMed

Atkins JF, Wills NM, Loughran G, et al. A case for “StopGo”: reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go). RNA. 2007;13(6):803–810. PubMed PMC

Kim JH, Lee SR, Li LH, et al. High cleavage efficiency of a 2A peptide derived from Porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011;6(4):e18556. PubMed PMC

Tran KD, Rodriguez-Contreras D, Vieira DP, et al. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes. J Biol Chem. 2013;288(31):22721–22733. PubMed PMC

Tran KD, Vieira DP, Sanchez MA, et al. Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages. PLoS One. 2015;10(8):e0134432. PubMed PMC

Bates PA. Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology. 1994;108(1):1–9. PubMed

Kraeva N, Leštinová T, Ishemgulova A, et al. LmxM.22.0250-encoded dual specificity protein/lipid phosphatase impairs Leishmania mexicana virulence in vitro. Pathogens. 2019;8(4):241. PubMed PMC

Glorieux C, Calderon PB. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach. Biol Chem. 2017;398:1095–1108. PubMed

Purdue PE, Castro SM, Protopopov V, et al. Targeting of human catalase to peroxisomes is dependent upon a novel C-terminal peroxisomal targeting sequence. Ann N Y Acad Sci. 1996;804(1 Peroxisomes):775–776. PubMed

Edwards C, Lloyd D. Subcellular fractionation by differential and zonal centrifugation of the trypanosomatid Crithidia fasciculata. J Gen Microbiol. 1977;100(2):339–346. PubMed

Poulin R, Randhawa HS. Evolution of parasitism along convergent lines: from ecology to genomics. Parasitology. 2015;142(S1):S6–S15. PubMed PMC

Butenko A, Hammond M, Field MC, et al. Reductionist pathways for parasitism in euglenozoans? Expanded datasets provide new insights. Trends Parasitol. 2021;37(2):100–116. PubMed

Jackson AP, Otto TD, Aslett M, et al. Kinetoplastid phylogenomics reveals the evolutionary innovations associated with the origins of parasitism. Curr Biol. 2016;26(2):161–172. PubMed PMC

Harkins KM, Schwartz RS, Cartwright RA, et al. Phylogenomic reconstruction supports supercontinent origins for Leishmania. Infect Genet Evol. 2016;38:101–109. PubMed

Kraeva N, Butenko A, Hlaváčová J, et al. Leptomonas seymouri: adaptations to the dixenous life cycle analyzed by genome sequencing, transcriptome profiling and co-infection with leishmania donovani. PLoS Pathog. 2015;11(8):11:e1005127. PubMed PMC

Jirků M, Yurchenko V, Lukeš J, et al. New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae. J Eukaryot Microbiol. 2012;59(6):537–547. PubMed

Kostygov AY, Yurchenko V. Revised classification of the subfamily Leishmaniinae (Trypanosomatidae). Folia Parasitol. 2017;64:020. PubMed

Garcia-Estrada C, Perez-Pertejo Y, Ordonez D, et al. Characterization of the 5′ region of the Leishmania infantum LORIEN/MAT2 gene cluster and role of LORIEN flanking regions in post-transcriptional regulation. Biochimie. 2008;90(9):1325–1336. PubMed

Charest H, Zhang WW, Matlashewski G. The developmental expression of Leishmania donovani A2 amastigote-specific genes is post-transcriptionally mediated and involves elements located in the 3ʹ-untranslated region. J Biol Chem. 1996;271(29):17081–17090. PubMed

Ryan MD, King AM, Thomas GP. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol. 1991;72(11):2727–2732. PubMed

Koh HG, Kang NK, Kim EK, et al. Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides. J Biotechnol. 2018;278:39–47. PubMed

Garcia ES, Castro DP, Figueiredo MB, et al. Immune homeostasis to microorganisms in the guts of triatomines (Reduviidae)–a review. Mem Inst Oswaldo Cruz. 2010;105(5):605–610. PubMed

Diaz-Albiter H, Mitford R, Genta FA, et al. Reactive oxygen species scavenging by catalase is important for female Lutzomyia longipalpis fecundity and mortality. PLoS One. 2011;6(3):e17486. PubMed PMC

Diaz-Albiter H, Sant’Anna MR, Genta FA, et al. Reactive oxygen species-mediated immunity against Leishmania mexicana and Serratia marcescens in the sand phlebotomine fly Lutzomyia longipalpis. J Biol Chem. 2012;287(28):23995–24003. PubMed PMC

Da Silva R, Sacks DL. Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation. Infect Immun. 1987;55(11):2802–2806. PubMed PMC

Mallinson DJ, Coombs GH. Interaction of Leishmania metacyclics with macrophages. Int J Parasitol. 1989;19(6):647–656. PubMed

Giraud E, Martin O, Yakob L, et al. Quantifying Leishmania metacyclic promastigotes from individual sandfly bites reveals the efficiency of vector transmission. Commun Biol. 2019;2(1):84. PubMed PMC

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector

Leishmania guyanensis M4147 as a new LRV1-bearing model parasite: Phosphatidate phosphatase 2-like protein controls cell cycle progression and intracellular lipid content

Comparative Analysis of Three Trypanosomatid Catalases of Different Origin

Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana

Analyses of Leishmania-LRV Co-Phylogenetic Patterns and Evolutionary Variability of Viral Proteins