An Investigation of the Arginine Methylome in Toxoplasma gondii with additional Observations on MAGI, a Cyst Wall Protein

Abstract

Parasites of the Apicomplexa phylum, such as Toxoplasma gondii , have complex life cycles involving multiple stages with distinct biology and morphologies. Posttranslational modifications (PTM), such as phosphorylation, acetylation and glycosylation, regulate numerous cellular processes, playing a role in every aspect of cell biology. PTM can occur on proteins at any time throughout their existence and through alterations of target protein activity, localization, protein-protein interactions, among other functions, dramatically increase proteome diversity and complexity. In addition, PTM can be induced or removed upon changes in cellular environment and state. Thus, PTM are likely to be key regulators of developmental transitions, biology and pathogenesis of apicomplexan parasites.;Arginine methylation, is a common posttranslational modification found on nuclear and cytoplasmic proteins, proposed to have roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite Toxoplasma gondii has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The T. gondii genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in T. gondii, we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of T. gondii contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Further supporting interplay of PTMs, a number of kinases are also arginine methylated. Since PRMT1 is thought to be a major PRMT in T. gondii, an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in T. gondii. We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators (e.g. AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in T. gondii, as a potential regulator of a large number of processes including RNA biology and transcription.;The data we obtained suggests that MMA is an abundant, dynamic PTM in T. gondii that regulates RNA biology and likely plays a role in transcription. We posit that MMA addition to T. gondii proteins is in large part catalyzed by PRMT1, these data are significant in light that most TgPRMT isoforms still only have activity types based on bioinformatic predictions. Our approach allows us to survey all MMA substrates, confirming many previously detected methyl marks and discovering new ones, while at the same time assessing the likelihood of highly confident TgPRMT1 candidates. Further the use of gene enrichment analysis has allowed us to look beyond methylation at potential crosstalk with other PTMs that are commonly found in T. gondii proteome, thus bringing us closer to understanding how the complement of PTMs interact with each other to determine protein function and parasite biology.;In a separate series of experiments, I examined the composition of the cyst wall of T. gondii. Cyst wall biology is still poorly understood despite the clinical and biological importance of this structure for latency and transmission. In order to study the cyst wall, we screened hybridoma libraries for monoclonal antibodies that reacted with the cyst wall. One of these mAbs (bB6) detected a cyst wall protein and was further characterized using immunoprecipitation followed by SDS-PAGE and mass spectrometry to identify the antigen it recognized. This mAb (bB6) was found to detect matrix antigen 1 (MAG1). We subsequently characterized the role of MAG1 by generating a mag1 knockout strain and examining it with regard to its effect on cyst wall structure, function, morphology (in vitro and in vivo), parasite growth, infectivity (in vitro and in vivo), cyst formation and brain histopathology in mouse. Survival studies in mice infected with WT T gondii compared to the DeltaMAG1 strain suggested a virulence phenotype in the latter, with the DeltaMAG1 strain infected mice having significantly reduced mortality and both fewer and smaller cysts in the brain. Our work has shown a reliable workflow for novel cyst wall protein discovery and in the case of cyst wall/matrix protein MAG1, we have demonstrated an in vivo tissue cyst phenotype, have produced mag1 disrupted and complement mutants in addition to a robust anti-MAG1 monoclonal antibody that has already proven useful to the parasitology community.