title: Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition.
abstract: Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min-1mg protein-1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min-1mg protein-1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min-1mg protein-1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Cinthia Bernardes
authors.initials: CB
authors.lastname: Gomes
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Franklin Souza
authors.initials: FS
authors.lastname: -Silva
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Karen Dos Santos
authors.initials: KD
authors.lastname: Charret
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Bernardo Acácio Santini
authors.initials: BA
authors.lastname: Pereira
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunoparasitologia, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Léa Cysne
authors.initials: LC
authors.lastname: Finkelstein
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Raquel
authors.initials: R
authors.lastname: Santos-de-Souza
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Luzia Monteiro
authors.initials: LM
authors.lastname: de Castro Côrtes
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Ultraestrutura Celular, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Mirian Claudia Souza
authors.initials: MC
authors.lastname: Pereira
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Ultraestrutura Celular, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil.
authors.forename: Francisco Odêncio
authors.initials: FO
authors.lastname: Rodrigues de Oliveira
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro, CEP: 21040-360, Brazil. Electronic address: calves@ioc.fiocruz.br.
authors.forename: Carlos Roberto
authors.initials: CR
authors.lastname: Alves
mesh_terms: D000818:Animals; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D057056:Cysteine Proteases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007892:Leishmania braziliensis / Q000201:enzymology* / Q000254:growth & development; D059748:Proteolysis / Q000187:drug effects; D013696:Temperature
chemical_list: D015853:Cysteine Proteinase Inhibitors; D002403:Cathepsins; D057056:Cysteine Proteases
keywords: Cathepsin L; Cysteine proteinases B; Leishmania (Viannia) braziliensis; Trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane; Z-Phe-Arg 7-amido-4-methylcoumarin hydrochloride; Z-Phe-Phe-fluoromethyl ketoneand