Improved Proteomic Approach for the Discovery of Potential Vaccine Targets in Trypanosoma cruzi

Abstract

Chagas disease, caused by Trypanosoma cruzi, is a devastating parasitic infection affecting millions of people. Although many efforts have been made for the development of immunotherapies, there is no available vaccine against this deadly infection. One major hurdle for the rational approach to develop a T. cruzi vaccine is the limited information about the proteins produced by different phylogenetic lineages, strains, and stages of the parasite. Here, we have adapted a 1D nanoHPLC system to perform online 2D LC-MS/MS, using the autosampler to inject the eluting salt solutions in the first dimension separation. The application of this methodology for the proteomic analysis of the infective trypomastigote stage of T. cruzi led to the identification of 1,448 non-redundant proteins. Furthermore, about 14% of the identified sequences comprise surface proteins, most of them glycosylphosphatidylinositol (GPI)-anchored and related to parasite pathogenesis. Immunoinformatic analysis revealed thousands of potential peptides with high affinity for major histocompatibility complex (MHC) class I and II presentation. The high diversity of proteins expressed on trypomastigote surface may have many implications for host-cell invasion and immunoevasion mechanisms triggered by the parasite. Finally, we performed a rational approach to filter potential epitopes that could be further tested and validated for development of a Chagas disease vaccine.

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

“…After cooling to room temperature, cysteine residues were alkylated by adding 2 μL of 400 mM iodoacetamide and incubating at 37° C in the dark for 1 h. Proteins were digested overnight at 37° C by adding 140 μL of 3.5 ng/μL trypsin dissolved in a buffer containing 50 mM NH 4 HCO 3 and 1.3 mM CaCl 2 . The digested peptides were loaded onto C18 microspin columns; centrifuged according to the manufacturer’s instructions; the column was equilibrated by flushing twice with 100 μL of 100 % methanol and twice with 100 μL of 0.1 % trifluoroacetic acid (TFA), then the sample was loaded and the column was washed three times with 100 μL of 5 % acetonitrile (ACN)/0.1 % TFA before eluting the peptides in 100 μL 80% ACN/0.1% TFA [41, 42]. After drying in a SpeedVac, the samples were resuspended in 20 μL 0.1% formic acid and stored at −20° C. Samples were subjected to LC-MS/MS analysis on an NanoAcquity UPLC (Waters) connected to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific).…”

Intraspecies Variation in Trypanosoma cruzi GPI-Mucins: Biological Activities and Differential Expression of α-Galactosyl Residues

“…11 The major subfamily of TcMUC (i.e., TcMUC II [844 gene members]) is mainly expressed in the mammalian trypomastigote stage. 12,13 It consists of a highly antigenic coat, with variations that account for interstrain features, such as virulence and immunomodulatory properties.…”

Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins

“…Cumulative expression data indicate that this dichotomy has a functional correlate, as transition between developmental forms leads to the expression of different, non-overlapping set of mucin genes (22,28,29). Briefly, TcSMUG products, and particularly the TcSMUG S ones, provide the backbone for the 35-50 kDa mucins (also known as Gp35/50 mucins) expressed on the surface of developmental forms found within the triatomines, i.e.…”