Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 114 | JULY 2019
Original Article

Calpains of Leishmania braziliensis: genome analysis, differential expression in metacyclogenesis and effects of MDL28170 [ACCEPTED ARTICLES / PRELIMINARY VERSION]

V Ennes-Vidal1, BS Vitório1, RFS Menna-Barreto2, AN Pitaluga3, SAG Da-Silva4, MH Branquinha5, ALS Santos5, CM d’Avila-Levy1,6,+

1Laboratório de Estudos Integrados em Protozoologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
2Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil 
3Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
4Laboratório de Imunofarmacologia Parasitária, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil 
5Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
6 Present temporary address: de Duve Institute, Université Catholique de Louvain, Brussels, Belgium

DOI: 10.1590/0074-02760190147
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ABSTRACT

Calpain is a multigene family of calcium-dependent cysteine peptidases subjected to a tight on/off regulation, and uncontrolled proteolysis accounts for severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. Here, we characterized calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. 34 calpain-like predicted genes were identified. After domain structure evaluation, RT-qPCR during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in procyclic, (ii) one augmented in metacyclic and (iii) one procyclic-exclusive transcript. An antibody against a consensusconserved peptide reacted with multiple calpain-like proteins in Western blotting, which is consistent with the multigenic family characteristic. Flow cytometry and immunocytochemistry analyses revealed calpain-like molecules mainly in the cytoplasm, to a lesser extent at the plasma membrane, and a faint labeling in the nucleus, which are consistent with calpain localization. Finally, the calpain inhibitor MDL28170 was employed in functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultrastructural alterations conceivable with autophagy and (iv) an enhanced expression of the virulence factor GP63. This report adds new insights about the domain structure, expression and localization of L. braziliensis calpain-like molecules.

V E-V and BSV contributed equally to this work.
+ Corresponding-author: davila.levy@ioc.fiocruz.br
https://orcid.org/0000-0001-8042-4695
Received 26 April 2019
Accepted 29 July 2019

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