title: Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents.
abstract: The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis. The 6h culture supernatant, operationally defined here as exoproteome, was then conditioned to in solution digestion and the resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and categorized according to their cellular function. Their relative abundance index (emPAI) allowed inference that the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved in nucleotide metabolism and antioxidant activity. Bioinformatic analyses support that approximately 60% of the identified proteins are secreted, of which, 85% possibly reach the extracellular milieu by means of non-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms of Canine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantum exoproteome. Western blotting experiments revealed that this sub-proteome was useful at discriminating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, the molecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assays opened up new research avenues related to treatment, prognosis and diagnosis of CVL.
authors.affiliation: Laboratório de Enzimologia e Proteômica, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Micheline Soares
authors.initials: MS
authors.lastname: Braga
authors.affiliation: Laboratório de Enzimologia e Proteômica, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Leandro Xavier
authors.initials: LX
authors.lastname: Neves
authors.affiliation: Laboratório de Enzimologia e Proteômica, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Jonatan Marques
authors.initials: JM
authors.lastname: Campos
authors.affiliation: Laboratório de Imunopatologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Bruno Mendes
authors.initials: BM
authors.lastname: Roatt
authors.affiliation: Laboratório de Imunopatologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Rodrigo Dian
authors.initials: RD
authors.lastname: de Oliveira Aguiar Soares
authors.affiliation: Laboratório de Imunopatologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Samuel Leôncio
authors.initials: SL
authors.lastname: Braga
authors.affiliation: Laboratório de Pesquisas Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil; Grupo Informática de Biossistemas, Centro de Pesquisas René Rachou - Fiocruz Minas, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Daniela
authors.initials: D
authors.lastname: de Melo Resende
authors.affiliation: Laboratório de Imunopatologia, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil; Laboratório de Pesquisas Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil.
authors.forename: Alexandre Barbosa
authors.initials: AB
authors.lastname: Reis
authors.affiliation: Laboratório de Enzimologia e Proteômica, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil. Electronic address: williamcborges@hotmail.com.
authors.forename: William
authors.initials: W
authors.lastname: Castro-Borges
mesh_terms: D000818:Animals; D015153:Blotting, Western; D006224:Cricetinae; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D018314:Leishmania infantum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007898:Leishmaniasis, Visceral / Q000469:parasitology / Q000662:veterinary*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins
keywords: Exoproteome; Leishmania infantum; Shotgun analysis