Shotgun proteomics to unravel the complexity of the Leishmaniainfantum exoproteome and the relative abundance of its constituent

Abstract

The exoproteome of some Leishmania species has revealed important insights into host–parasite inter-action, paving the way for the proposal of novel disease-oriented interventions. The focus of the presentinvestigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgunproteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvestedby centrifugation at different time points. Cell integrity was evaluated through the counting of viableparasites using propidium iodide labeling, followed by flow cytometry analysis. The 6 h culture super-natant, operationally defined here as exoproteome, was then conditioned to in solution digestion andthe resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and cat-egorized according to their cellular function. Their relative abundance index (emPAI) allowed inferencethat the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved innucleotide 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 ofnon-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms ofCanine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantumexoproteome. Western blotting experiments revealed that this sub-proteome was useful at discrimi-nating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, themolecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assaysopened up new research avenues related to treatment, prognosis and diagnosis of CVL