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Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents

Author:
Braga, Micheline Soares, Neves, Leandro Xavier, Campos, Jonatan Marques, Roatt, Bruno Mendes, de Oliveira Aguiar Soares, Rodrigo Dian, Braga, Samuel Leôncio, de Melo Resende, Daniela, Reis, Alexandre Barbosa, Castro-Borges, William
Source:
Molecular and biochemical parasitology 2014 v.195 pp. 43-53
ISSN:
0166-6851
Subject:
Leishmania infantum, Western blotting, antioxidant activity, bioinformatics, centrifugation, dogs, flow cytometry, host-parasite relationships, immune response, mass spectrometry, metabolism, parasites, peptides, prognosis, promastigotes, propidium, protein secretion, proteins, proteomics, visceral leishmaniasis
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.
Agid:
5520462