Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondiireveals commitment to a host-independent lifestyle

Abstract

BackgroundToxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited.ResultsUsing one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid β-oxidation and the degradation of branched amino acids.ConclusionsThe study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

“…Finally, the hypothesis that tissue cysts of brain-resident parasites actively alter host dopamine to exert behavioral control faces exceptional challenge from the observation that parasites defective in their ability to establish lifelong residency in the brain still result in abnormal cat attraction [51]. Additionally, the expression of the AAH genes is relatively low in both the lytic and chronic asexual stages [24] and is only upregulated in the sexual stages [30] and mass spectrometry has failed to find evidence of these proteins in tachyzoite or bradyzoite stages but identified them in the oocyst [14]. Hence if the AAH gene products are involved in altering dopamine levels in the CNS of infected rodents, they would need to do so based on exceedingly low expression levels, and in a localized region.…”

“…gondii oocysts are highly proteinaceous, composed of >90% protein [6], as well as β 1–3 glucan carbohydrates [11], and acid-fast lipids [12]. Large-scale proteomic analyses have identified 1,031 [13] or 1,304 [14] individual, non-redundant proteins associated with the oocyst. Although the function and localization of many remain unknown, two classes of oocyst wall structural proteins have been identified in other apicomplexans.…”

“…Although T . gondii does not contain clear homologues of Eimeria ’s EmGam proteins, many tyrosine-rich proteins have been identified in both outer oocyst wall and inner sporocyst wall fractions by mass spectrometry [13, 14], although they have not been definitively identified as structural components in the oocyst wall [5]. …”

“…gondii [30], and protein mass spectrometry identifies both tyrosine-rich proteins and the tyrosine hydroxylases AAH1 and AAH2 in the oocyst of T . gondii [13, 14]. In contrast, these hydroxylases are not found in similar mass spectrometric analyses of tachyzoites or bradyzoites [14].…”

“…gondii [13, 14]. In contrast, these hydroxylases are not found in similar mass spectrometric analyses of tachyzoites or bradyzoites [14]. …”