title: Leishmania (Viannia) braziliensis: influence of successive in vitro cultivation on the expression of promastigote proteinases.
abstract: Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Karina Mastropasqua
authors.initials: KM
authors.lastname: Rebello
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
authors.forename: Bernardo Acácio Santini
authors.initials: BA
authors.lastname: Pereira
authors.forename: Daniela de
authors.initials: Dd
authors.lastname: Pita-Pereira
authors.forename: Milton Ozório
authors.initials: MO
authors.lastname: Moraes
authors.forename: Anna Beatriz Robottom
authors.initials: AB
authors.lastname: Ferreira
authors.forename: Léa
authors.initials: L
authors.lastname: Cysne-Finkelstein
authors.forename: Thomas Dan
authors.initials: TD
authors.lastname: Otto
authors.forename: Luzia Monteiro de Castro
authors.initials: LM
authors.lastname: Côrtes
authors.forename: Gabriel Gomes
authors.initials: GG
authors.lastname: da-Silva
authors.forename: Carlos Roberto
authors.initials: CR
authors.lastname: Alves
mesh_terms: D000818:Animals; D057056:Cysteine Proteases / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015151:Immunoblotting; D007892:Leishmania braziliensis / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012692:Serial Passage; D014774:Virulence; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D037521:Virulence Factors; D057056:Cysteine Proteases