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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

Published online by Cambridge University Press:  17 October 2017

SIMONE S. C. OLIVEIRA ,
INÊS C. GONÇALVES ,
VITOR ENNES-VIDAL ,
ANGELA H. C. S. LOPES ,
RUBEM F. S. MENNA-BARRETO ,
CLAUDIA M. D’ÁVILA-LEVY ,
ANDRÉ L. S. SANTOS  and
MARTA H. BRANQUINHA
SIMONE S. C. OLIVEIRA
Affiliation:
Laboratório de Investigação de Peptidases, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
INÊS C. GONÇALVES
Affiliation:
Laboratório de Bioquímica de Microrganismos, Departamento de Microbiologia Geral, IMPG, UFRJ, Rio de Janeiro, Brazil
VITOR ENNES-VIDAL
Affiliation:
Laboratório de Estudos Integrados em Protozoologia, Coleção de Protozoários, IOC, FIOCRUZ, Rio de Janeiro, Brazil
ANGELA H. C. S. LOPES
Affiliation:
Laboratório de Bioquímica de Microrganismos, Departamento de Microbiologia Geral, IMPG, UFRJ, Rio de Janeiro, Brazil
RUBEM F. S. MENNA-BARRETO
Affiliation:
Laboratório de Biologia Celular, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil
CLAUDIA M. D’ÁVILA-LEVY
Affiliation:
Laboratório de Estudos Integrados em Protozoologia, Coleção de Protozoários, IOC, FIOCRUZ, Rio de Janeiro, Brazil
ANDRÉ L. S. SANTOS
Affiliation:
Laboratório de Investigação de Peptidases, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil Programa de Pós-Graduação em Bioquímica, Instituto de Química, UFRJ, Rio de Janeiro, Brazil
MARTA H. BRANQUINHA*
Affiliation:
Laboratório de Investigação de Peptidases, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
*
*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
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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 µm (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.

Keywords

Phytomonascalpain-like proteinscalpain inhibitorsresistancepeptidases
Type
Research Article
Information
Parasitology , Volume 145 , Issue 3 , March 2018 , pp. 355 - 370
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Copyright
Copyright © Cambridge University Press 2017 

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