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In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

  • SIMONE S. C. OLIVEIRA (a1), INÊS C. GONÇALVES (a2), VITOR ENNES-VIDAL (a3), ANGELA H. C. S. LOPES (a2), RUBEM F. S. MENNA-BARRETO (a4), CLAUDIA M. D’ÁVILA-LEVY (a3), ANDRÉ L. S. SANTOS (a1) (a5) and MARTA H. BRANQUINHA (a1)...

Summary

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

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Corresponding author

*Corresponding author: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, 21941-902, Rio de Janeiro, Brazil. E-mail: mbranquinha@micro.ufrj.br

References

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Al-Mohammed, H. I., Chance, M. L. and Bates, P. A. (2005). Production and characterization of stable amphotericin-resistant amastigotes and promastigotes of Leishmania mexicana . Antimicrobial Agents and Chemotherapy 49, 32743280.
Andrade, H. M., Murta, S. M., Chapeaurouge, A., Perales, J., Nirdé, P. and Romanha, A. J. (2008). Proteomic analysis of Trypanosoma cruzi resistance to benznidazole. Journal of Proteome Research 7, 23572367.
Atsma, D. E., Bastiaanse, E. M., Jerzewski, A., Van der Valk, L. J. and Van der Laarse, A. (1995). Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition. Circulation Research 76, 10711078.
Beyette, J. R., Emori, Y. and Mykles, D. L. (1997). Immunological analysis of two calpain-like Ca2+- dependent proteinases from lobster striated muscles: relationship to mammalian and Drosophila calpains. Archives of Biochemistry and Biophysics 15, 337341.
Branquinha, M. H., Marinho, F. A., Sangenito, L. S., Oliveira, S. S., Gonçalves, K. C., Ennes-Vidal, V., D'Avila-Levy, C. M. and Santos, A. L. (2013). Calpains: potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids. Current Medicinal Chemistry 20, 31743185.
Brotherton, M. C., Bourassa, S., Leprohon, P., Légaré, D., Poirier, G. G., Droit, A. and Ouellette, M. (2013). Proteomic and genomic analyses of antimony-resistant Leishmania infantum mutant. PLoS ONE 8, e81899.
Caljon, G., De Muylder, G., Durnez, L., Jennes, W., Vanaerschot, M. and Dujardin, J. C. (2016). Alice in microbe's land: adaptations and counter-adaptations of vector-borne parasitic protozoa and their hosts. FEMS Microbiology Review 40, 664685.
Campos, M. C., Castro-Pinto, D. B., Ribeiro, G. A., Berredo-Pinho, M. M., Gomes, L. H., da Silva Bellieny, M. S., Goulart, C. M., Echevarria, A. and Leon, L. L. (2013). P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance. Parasitology Research 112, 23412351.
Cazzulo, J. J., Cazzulo Franke, M. C., Martínez, J. and Franke De Cazzulo, B. M. (1990). Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi . Biochimica et Biophysica Acta 1037, 186191.
Cunha, V., Burkhardt-Medicke, K., Wellner, P., Santos, M. M., Moradas-Ferreira, P., Luckenbach, T. and Ferreira, M. (2017). Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio). Ecotoxicology and Environmental Safety 136, 1423.
d'Avila-Levy, C. M., Altoé, E. C., Uehara, L. A., Branquinha, M. H. and Santos, A. L. (2014). GP63 function in the interaction of trypanosomatids with the invertebrate host: facts and prospects. Subcellular Biochemistry 74, 253270.
Donkor, I. O. (2015). An updated patent review of calpain inhibitors (2012–2014). Expert Opinion on Therapeutic Patents 25, 1731.
Emori, Y. and Saigo, K. (1994). Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila . Journal of Biological Chemistry 269, 2513725142.
Engel, J. C., Torres, C., Hsieh, I., Doyle, P. S. and McKerrow, J. H. (2000). Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi . Journal of Cell Science 113, 13451354.
Ennes-Vidal, V., Menna-Barreto, R. F., Branquinha, M. H., Santos, A. L. S. and D'Avila-Levy, C. M. (2017). Why calpain inhibitors are interesting leading compounds to search for new therapeutic options to treat leishmaniasis? Parasitology 144, 117123.
Ersfeld, K., Barraclough, H. and Gull, K. (2005). Evolutionary relationships and protein domain architecture in an expanded calpain superfamily in kinetoplastid parasites. Journal of Molecular Evolution 61, 742757.
Forster, S., Thumser, A. E., Hood, S. R. and Plant, N. (2012). Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays. PLoS ONE 7, e33253.
Gueiros-Filho, F. J., Viola, J. P., Gomes, F. C., Farina, M., Lins, U., Bertho, A. L., Wirth, D. F. and Lopes, U. G. (1995). Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene. Experimental Parasitology 81, 480490.
Hayes, P., Varga, V., Olego-Fernandez, S., Sunter, J., Ginger, M. L. and Gull, K. (2014). Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology. Journal of Cellular Biology 206, 377384.
Hertz-Fowler, C., Ersfeld, K. and Gull, K. (2001). CAP5·5, a life-cycle-regulated, cytoskeleton-associated protein is a member of a novel family of calpain-related proteins in Trypanosoma brucei . Molecular and Biochemical Parasitology 116, 2534.
Heussen, C. and Dowdle, E. B. (1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Analytical Biochemistry 102, 196202.
Jaskowska, E., Butler, C., Preston, G. and Kelly, S. (2015). Phytomonas: trypanosomatids adapted to plant environments. PLoS Pathogens 11, e1004484.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265275.
Marinho, F. A., Gonçalves, K. C., Oliveira, S. S., Gonçalves, D. S., Matteoli, F. P., Seabra, S. H., Oliveira, A. C., Bellio, M., Oliveira, S. S., Souto-Padrón, T., d'Avila-Levy, C. M., Santos, A. L. and Branquinha, M. H. (2014). The calpain inhibitor MDL28170 induces the expression of apoptotic markers in Leishmania amazonensis promastigotes. PLoS ONE 9, e87659.
Morgan, G. W., Hall, B. S., Denny, P. W., Carrington, M. and Field, M. C. (2002). The kinetoplastida endocytic apparatus. Part I: a dynamic system for nutrition and evasion of host defences. Trends in Parasitology 18, 491496.
Olego-Fernandez, S., Vaughan, S., Shaw, M. K., Gull, K. and Ginger, M. L. (2009). Cell morphogenesis of Trypanosoma brucei requires the paralogous, differentially expressed calpain-related proteins CAP5·5 and CAP5·5V . Protist 60, 576590.
Oliveira, S. S. C., Goncalves, D. S., Garcia-Gomes, A. S., Goncalves, I. C., Seabra, S. H., Menna-Barreto, R. F. S., Lopes, A. H. C. S., d'Avila-Levy, C. M., Santos, A. L. S. and Branquinha, M. H. (2017). Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host. Memórias do Instituto Oswaldo Cruz 112, 3143.
Ono, Y. and Sorimachi, H. (2012). Calpains: an elaborate proteolytic system. Biochimica et Biophysica Acta 1824, 224236.
Porcel, B. M., Denoeud, F., Opperdoes, F., Noel, B., Madoui, M. A., Hammarton, T. C., Field, M. C., da Silva, C., Couloux, A., Poulain, J., Katinka, M., Jabbari, K., Aury, J. M., Campbell, D. A., Cintron, R., Dickens, N. J., Docampo, R., Sturm, N. R., Koumandou, V. L., Fabre, S., Flegontov, P., Lukeš, J., Michaeli, S., Mottram, J. C., Szöőr, B., Zilberstein, D., Bringaud, F., Wincker, P. and Dollet, M. (2014). The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants. PLoS Genetics 10, e1004007.
Rai, S., Bhaskar, E., Goel, S. K., Nath Dwivedi, U., Sundar, S. and Goyal, N. (2013). Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani . PloS ONE 8, e74862.
Rami, A., Ferger, D. and Krieglstein, J. (1997). Blockade of calpain proteolytic activity rescues neurons from glutamate excitotoxicity. Neuroscience Research 27, 9397.
Romeiro, A., Solé-Cava, A., Sousa, M. A., De Souza, W. and Attias, M. (2000). Ultrastructural and biochemical characterization of promastigote and cystic forms of Leptomonas wallacei n. sp. isolated from the intestine of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae). Journal of Eukaryotic Microbiology 47, 208220.
Santos, A. L. S., D´Avila-Levy, C. M., Elias, C. G. R., Vermelho, A. B. and Branquinha, M. H. (2007). Phytomonas serpens: immunological similarities with the human trypanosomatid pathogens. Microbes and Infection 9, 915921.
Vanaerschot, M., Huijben, S., Van den Broeck, F. and Dujardin, J. C. (2014). Drug resistance in vectorborne parasites: multiple actors and scenarios for an evolutionary arms race. FEMS Microbiology Review 38, 4155.
Vergnes, B., Gourbal, B., Girard, I., Sundar, S., Drummelsmith, J. and Ouellette, M. (2007). A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death. Molecular and Cellular Proteomics 6, 88101.
Wang, K. K., Nath, R., Posner, A., Raser, K. J., Buroker-Kilgore, M., Hajimohammadreza, I., Probert, A. W., Marcoux, F. W., Ye, Q., Takano, E., Hatanaka, M., Maki, M., Caner, H., Collins, J. L., Fergus, A., Lee, K. S., Lunney, E. A., Hays, S. J. and Yuen, P. (1996). An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cel-permeable calpain inhibitor and is neuroprotective. Proceedings of the National Academy of Sciences USA 93, 66876692.
Xiong, J., Mao, D. A. and Liu, L. Q. (2015). Research progress on the role of ABC transporters in the drug resistance mechanism of intractable epilepsy. BioMed Research International 2015, 194541.
Yasinzai, M., Khan, M., Nadhman, A. and Shahnaz, G. (2013). Drug resistance in leishmaniasis: current drug-delivery systems and future perspectives. Future Medicinal Chemistry 5, 18771888.
Yong, V., Schmitz, V., Vannier-Santos, M. A., De Lima, A. P., Lalmanach, G., Juliano, L., Gauthier, F. and Scharfstein, J. (2000). Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases. Molecular and Biochemical Parasitology 109, 4759.

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