Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Rayner M.L Queiroz,Sébastien Charneau,Samuel C Mandacaru,Veit Schwämmle,Beatriz D Lima,Peter Roepstorff,Carlos A.O Ricart

doi:10.1074/mcp.m114.040329

Abstract

Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets.

Highlights

From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910 –900 Brazil; §Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark
The infection is caused by the protozoan Trypanosoma cruzi, a single-cell parasite that undergoes differentiation events throughout its life cycle to adapt to a variety of environments inside mammalian hosts and triatomine bug vectors
Amastigogenesis occurs when metacyclic trypomastigotes turn into amastigotes inside mammalian nucleated host cells or after infection by tissue-derived bloodstream trypomastigotes, when the so-called second amastigogenesis is observed, inside host cells [15]

Summary

Introduction

As well as host-cell infection, involves the intracellular release of Ca2ϩ in the coordination of molecular events involved in those processes. Amastigogenesis occurs when metacyclic trypomastigotes turn into amastigotes inside mammalian nucleated host cells (first amastigogenesis) or after infection by tissue-derived bloodstream trypomastigotes, when the so-called second amastigogenesis is observed, inside host cells [15]. This process, which is not completely understood at the molecular level, takes place preferably inside host cells, extracellular amastigotes may be observed during the infection’s acute phase [16]

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