Abstract
Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.
Highlights
Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans
We analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics)
Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed ϳ100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis
Summary
Reagents and Chemicals—Porcine trypsin (sequencing grade) was from Promega (Madison, WI). Peptide monoisotopic masses were used to search against Toxoplasma NCBI data base via the MS-Fit algorithm (prospector.ucsf.edu) or to search Toxoplasma protein/EST/genome data bases via an in-house installed Mascot program [35]. Protein identification was performed by searching the T. gondii protein subset of NCBI “nr,” the amino acid TgTwinScan gene predictions, and the clustered EST data bases using the TurboSequest algorithm [36] in the Bioworks 3.1 software package (Thermo Finnigan). Ortholog Identification—To identify orthologs to ESA proteins, we followed the approach used to generate the TIGR orthologous gene
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