The Opportunistic Pathogen Toxoplasma gondii Deploys a Diverse Legion of Invasion and Survival Proteins

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 ϳ100 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 posttranslational 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.

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

“…However, additional genes selected for tagging either never showed YFP expression (55.m04618, 8.m00176, 8.m00178 80.m02161, and 50.m00008) or displayed a pattern that resembled that of mistargeting or retention (20.m03958 and 49.m00054) in a subapical location, which is presumably a staging ground for the apical invasion organelles (Table 1). Similar results have been seen with some exogenously GFP-tagged genes encoding secretory proteins (49). These gene products do not appear to have a feature in common, e.g., a transmembrane anchor, which could account for the failure of the fusion protein to reach the invasion organelles.…”

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

“…In this case, the observations would be more consistent with an elevation in proteolytic activity in the ESA rather than an effect on susceptibility of the substrate to proteolysis. Two proteases have been reported to be present in the ESA, SUB1 and a putative metalloproteinase encoded by the predicted gene TwinScan 4000 (Ts4000) (44). SUB1 is a glycosylphosphatidylinositol-anchored serine protease of the subtilase family, whereas TS4000 is a member of the insulinase family.…”

Expression Quantitative Trait Locus Mapping of Toxoplasma Genes Reveals Multiple Mechanisms for Strain-Specific Differences in Gene Expression

“…In fact, five of the six cis-mapping genes with signal peptides were already known to localize to this group of secretory organelles. GRA7 is found in the dense granules (14); ROP18 (17,30), toxofilin (9,28), and ROP8 (16) are found in the rhoptries; and 46.m01601 was found in stimulated secretion products containing primarily dense granule and microneme proteins (formerly called TgTwinsan_2661 [41]). Moreover, GRA7, ROP18, and ROP8 can all be found in the host cell at different times postinvasion (14,16,30).…”