Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

Coleção de Protozários da Fundação Oswaldo Cruz Rio de Janeiro 21040 900 Brazil

Grupo de Biofísica Computacional e Modelagem Molecular Programa de Computação Científica Rio de Janeiro 21040 360 Brazil

Laboratório de Biologia Computacional e Sistemas Instituto Oswaldo Cruz Fundação Oswaldo Cruz Rio de Janeiro 21040 360 Brazil

Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes Instituto de Microbiologia Paulo de Góes Universidade Federal do Rio de Janeiro Rio de Janeiro 21941 902 Brazil

Laboratório de Estudos Integrados em Protozoologia Instituto Oswaldo Cruz Fundação Oswaldo Cruz Rio de Janeiro 21040 360 Brazil

Laboratório de Genômica Funcional e Bioinformática Instituto Oswaldo Cruz Fundação Oswaldo Cruz Rio de Janeiro 21040 360 Brazil

Life Science Research Centre Faculty of Science University of Ostrava 70200 Ostrava Czech Republic

Martsinovsky Institute of Medical Parasitology Tropical and Vector Borne Diseases Sechenov University 119435 Moscow Russia

Programa de Pós Graduação em Bioquímica Instituto de Química Universidade Federal do Rio de Janeiro Rio de Janeiro 21941 902 Brazil

Zobrazit více v PubMed

Ennes-Vidal V., Santos A.L.S., Branquinha M.H., d’Avila-Levy M.H. Proteolytic inhibitors as alternative medicines to treat trypanosomatid-caused diseases: Experience with calpain inhibitors. Mem. Inst. Oswaldo Cruz. 2022;117:e220017. doi: 10.1590/0074-02760220017. PubMed DOI PMC

WHO Leishmaniasis. 8 January 2022. [(accessed on 17 February 2021)]. Available online: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis.

Sacks D.L. Metacyclogenesis in Leishmania promastigotes. Exp. Parasitol. 1989;69:100–103. doi: 10.1016/0014-4894(89)90176-8. PubMed DOI

Teixeira D.E., Benchimol M., Rodrigues J.C., Crepaldi P.H., Pimenta P.F., de Souza W. The cell biology of Leishmania: How to teach using animations. PLoS Pathog. 2013;9:e1003594. doi: 10.1371/journal.ppat.1003594. PubMed DOI PMC

Sangenito L.S., da Silva Santos V., d’Avila-Levy C.M., Branquinha M.H., Santos A.L.S., de Oliveira S.S.C. Leishmaniasis and Chagas Disease—Neglected Tropical Diseases: Treatment Updates. Curr. Top Med. Chem. 2019;19:174–177. doi: 10.2174/156802661903190328155136. PubMed DOI

Maslov D.A., Opperdoes F.R., Kostygov A.Y., Hashimi H., Lukeš J., Yurchenko V. Recent advances in trypanosomatid research: Genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019;146:1–27. doi: 10.1017/S0031182018000951. PubMed DOI

Espinosa O.A., Serrano M.G., Camargo E.P., Teixeira M.M.G., Shaw J.J. An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum. Parasitology. 2018;145:430–442. doi: 10.1017/S0031182016002092. PubMed DOI

Elwasila M. Leishmania tarentolae Wenyon, 1921 from the gecko Tarentolaannularis in the Sudan. Parasitol. Res. 1988;74:591–592. doi: 10.1007/BF00531640. PubMed DOI

Simpson L., Holz G. The status of Leishmania tarentolae/Trypanosoma platydactyli. Parasitol. Today. 1988;4:115–118. doi: 10.1016/0169-4758(88)90043-9. PubMed DOI

Taylor V.M., Muñoz D.L., Cedeño D.L., Vélez I.D., Jones M.A., Robledo S.M. Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents. Parasitol. Res. 2010;126:471–475. doi: 10.1016/j.exppara.2010.05.016. PubMed DOI

Ouellete M., Hettema E., Wüst D., Fase-Fowler F., Borst P. Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania. EMBO J. 1991;10:1009–1016. doi: 10.1002/j.1460-2075.1991.tb08035.x. PubMed DOI PMC

Simpson L., Aphasizhev R., Gao G., Kang X. Mitochondrial proteins and complexes in Leishmania and Trypanosoma involved in U-insertion/deletion RNA editing. RNA. 2004;10:159–170. doi: 10.1261/rna.5170704. PubMed DOI PMC

Basile G., Peticca M. Recombinant protein expression in Leishmania tarentolae. Mol. Biotechnol. 2009;43:273–278. doi: 10.1007/s12033-009-9213-5. PubMed DOI

Breton M., Tremblay M.J., Ouellette M., Papadopoulou B. Live non-pathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis. Infect. Immun. 2005;73:6372–6382. doi: 10.1128/IAI.73.10.6372-6382.2005. PubMed DOI PMC

Raymond F., Boisvert S., Roy G., Ritt J.F., Légaré D., Isnard A., Stanke M., Olivier M., Tremblay M.J., Papadopoulou B., et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res. 2012;40:1131–1147. doi: 10.1093/nar/gkr834. PubMed DOI PMC

d’Avila-Levy C.M., Altoé E.C., Uehara L.A., Branquinha M.H., Santos A.L. GP63 function in the interaction of trypanosomatids with the invertebrate host: Facts and prospects. Subcell. Biochem. 2014;74:253–270. doi: 10.1007/978-94-007-7305-9_11. PubMed DOI

Yao C. Major surface protease of trypanosomatids: One size fi ts all? Infect. Immun. 2010;78:22–31. doi: 10.1128/IAI.00776-09. PubMed DOI PMC

Olivier M., Atayde V.D., Isnard A., Hassani K., Shio M.T. Leishmania virulence factors: Focus on the metalloprotease GP63. Microbes Infect. 2012;14:1377–1389. doi: 10.1016/j.micinf.2012.05.014. PubMed DOI

Soares R.P., Altoé E.C.F., Ennes-Vidal V., da Costa S.M., Rangel E.F., de Souza N.A., da Silva V.C., Volf P., d’Avila-Levy C.M. In Vitro inhibition of Leishmania attachment to sandfly midguts and LL-5 cells by divalent metal chelators, anti-gp63 and phosphoglycans. Protist. 2017;168:326–334. doi: 10.1016/j.protis.2017.03.004. PubMed DOI

Berman H., Henrick K., Nakamura H. Announcing the worldwide Protein Data Bank. Nat. Struct. Biol. 2003;10:980. doi: 10.1038/nsb1203-980. PubMed DOI

Schlagenhauf E., Etges R., Metcalf P. The crystal structure of the Leishmania major surface proteinase leishmanolysin (gp63) Structure. 1998;6:1035–1046. doi: 10.1016/S0969-2126(98)00104-X. PubMed DOI

Sutter A., Antunes D., Silva-Almeida M., Costa M.G.S., Caffarena E.R. Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania(Viannia) braziliensis. Mem. Inst. Oswaldo Cruz. 2017;112:617–625. doi: 10.1590/0074-02760160522. PubMed DOI PMC

Valdivia H.O., Scholte L.L.S., Oliveira G., Gabaldón T., Bartholomeu D.C. The Leishmania metaphylome: A comprehensive survey of Leishmania protein phylogenetic relationships. BMC Genom. 2015;16:887. doi: 10.1186/s12864-015-2091-2. PubMed DOI PMC

Orlando T.C., Rubio M.A.T., Sturm N.R., Campbell D.A., Floeter-Winter L.M. Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: Molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania) Mem. Inst. Oswaldo Cruz. 2002;97:695–701. doi: 10.1590/S0074-02762002000500020. PubMed DOI

Zamarreño F., Herrera F.E., Córsico B., Costabel M.D. Similar structures but different mechanisms: Prediction of FABPs-membrane interaction by electrostatic calculation. Biochim. Biophys. Acta. 2012;1818:1691–1697. doi: 10.1016/j.bbamem.2012.03.003. PubMed DOI

Galassi V.V., Villarreal M.A., Posada V., Montich G.G. Interactions of the fatty acid-binding protein ReP1-NCXSQ with lipid membranes. Influence of the membrane electric field on binding and orientation. Biochim. Biophys. Acta. 2014;1838:910–920. doi: 10.1016/j.bbamem.2013.11.008. PubMed DOI

Talasaz A.H., Nemat-Gorgani M., Liu Y., Ståhl P., Dutton R.W., Ronaghi M., Davis R.W. Prediction of protein orientation upon immobilisation on biological and nonbiological surfaces. Proc. Natl. Acad. Sci. USA. 2006;103:14773–14778. doi: 10.1073/pnas.0605841103. PubMed DOI PMC

Klatt S., Simpson L., Maslov D.A., Konthur Z. Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host. PLoS Negl. Trop. Dis. 2019;13:e0007424. doi: 10.1371/journal.pntd.0007424. PubMed DOI PMC

d’Avila-Levy C.M., Santos A.L.S., Cuervo P., de Jesus J.B., Branquinha M.H. Applications of Zymography (Substrate-SDSPAGE) for Peptidase Screening in a Post-Genomic Era. In: Magdeldin S., editor. Gel Electrophoresis—Advanced Techniques. IntechOpen; London, UK: 2012. DOI

Branquinha M.H., Vermelho A.B., Goldenberg S., Bonaldo M.C. Characterization of proteinases in trypanosomatids. Braz. J. Med. Biol. Res. 1994;27:495–499. PubMed

Rebello K.M., Uehara L.A., Ennes-Vidal V., Garcia-Gomes A.S., Britto C., Azambuja P., Menna-Barreto R.F.S., Santos A.L.S., Branquinha M.H., d’Avila-Levy C.M. Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodniusprolixus. Parasitology. 2019;146:1075–1082. doi: 10.1017/S0031182019000441. PubMed DOI PMC

Aslett M., Aurrecoechea C., Berriman M., Brestelli J., Brunk B.P., Carrington M., Depledge D.P., Fischer S., Gajria B., Gao X., et al. TriTrypDB: A functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38:D457–D462. doi: 10.1093/nar/gkp851. PubMed DOI PMC

Eswar N., Eramian D., Webb B., Shen M.Y., Sali A. Protein structure modeling with MODELLER. Methods Mol. Biol. 2008;426:145–159. doi: 10.1007/978-1-60327-058-8_8. PubMed DOI

Feig M. Local Protein Structure Refinement via Molecular Dynamics Simulations with locPREFMD. J Chem. Inf. Model. 2016;56:1304–1312. doi: 10.1021/acs.jcim.6b00222. PubMed DOI PMC

Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem. Theory Comput. 2015;11:3696–3713. doi: 10.1021/acs.jctc.5b00255. PubMed DOI PMC

Jorgensen W.L., Chandrasekhar J., Madura J.D., Impey R.W., Klein M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935. doi: 10.1063/1.445869. DOI

Ryckaert J.P., Ciccotti G., Berendsen H.J.C. Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 1977;23:327–341. doi: 10.1016/0021-9991(77)90098-5. DOI

Joshi P.B., Sacks D.L., Modi G., McMaster W.R. Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63) Mol. Microbiol. 1998;27:519–530. doi: 10.1046/j.1365-2958.1998.00689.x. PubMed DOI

Boucinha C., Andrade-Neto V.V., Ennes-Vidal V., Branquinha M.H., Santos A.L.S., Torres-Santos E.C., d’Avila-Levy C.M. A Stroll Through the History of Monoxenous Trypanosomatids Infection in Vertebrate Hosts. Front. Cell. Infect. Microbiol. 2022;12:804707. doi: 10.3389/fcimb.2022.804707. PubMed DOI PMC