The Research On The Phosphoproteome Of Host Cell Infected With Toxoplasma Gondii

1. Background and ObjectiveToxoplasma gondii, an obligate intracellular parasite which can infected almost any nucleated cell type and has a worldwide distribution, infects approximately 30% of the human population worldwide[4]. Although most human infections are asymptomatic, the infection can be fatal in immunosuppressed individuals and unborn fetus, and can also cause most severe pathology like disseminated congenital infections and neurological complications. During acute T.gondii infection, tachyzoites progress through the lytic cycle including host cell infection,, replication within the host cell and egress into the extracellular environment for a new host cell invasion[5].The detailed mechanism of parasite’s interaction with the host cell is still far from understand clearly. Mountainous evidence shows phosphorylation as critical event in the pathogen-host interaction.Phosphorylation is one of the most important protein modifications and can affect the activity of almost 1/3 of total protein. Phospho-form modification can alter the protein between phosphorylation and dephosphorylation, from one to another with the acquisition or lose of activity. Although the researches about protein phosphorylation occurred in the host-pathogen interaction have been undergoing for decades and got some great achievements, the comprehensive and systematic research was still needed to know the full picture of the whole story. It is in these loci that we decided to develop our research about the phosphoproteome of host cell infected by T.gondii.We’ve observed two groups of HFFs infected by T.gondii in the same ratio but for different time (one is for 2hrs, the other is for 6hrs) compared with an uninfected group to find out the different events and cell signaling involved in the early-infection and later-infection.2. Methods1) Labeling the HFFsHFFs were cultured in different SILAC medium for 6 generations and labeled with L-arginine and L-lysine,13C6 L-Arginine-HCL and 13C6 L-Lysine-2HCL,13C6 15N2 L-Lysine-2HCL and 13C615N4 L-Arginine-HCL, respectively. We named the three groups with their different molecular weight, which are (L-arginine, L-lysine)as the Light group, (13C6 L-Arginine-HCL,13C6 L-Lysine-2HCL)as the Mediate group and (13C615N2 L-Lysine-2HCL,13C615N4 L-Arginine-HCL)as the heavy group. After 6 generations of cell culturing, more than 90% protein of the HFFs were labeled, and we’ve got 6 dishes (d=15cm) of HFFs of each group.2) Sample PreparationTachyzoites were added into the H and M groups evenly in ratio of 10:1.For M group, the invasion time was controlled to be 2hrs, and for H group the invasion time was 6 hrs. L group was without T.gondii invasion. After that, different groups of cell were collected separately with cell-scrapers.3) Enrichment of phosphopeptide and LC-MS/MS analysisTotal protein of three groups was mixed in ratio of 1:1:1, followed by trypsin digestion, then the peptides were pre-separated the mixed with SCX to gain different fractions. The different fractions were added into TiO2 column for phosphopeptide enrichment. Then the NanoLC Platform system coupled to the Triple TOF 5600 system were used for LC-MS/MS analysis.4) Database searching and bioinformatics analysisTwo databases were chose for the protein identification of our research, they were the IPI human (91464 sequences) and ToxoDB-release-9.0. After peptide and protein identification, the data set was analyzed with different bioinformatics tools like DAVID, MAS v3.0, STRING v9.1, Cytoscape, et.al for further information.3. Results1) Identification of phosphoproteomes of HFFsFor host cells, we identified total 5593 peptides and 3415 of them were phosophorylated, with 2020 phoshorylation sites that matched to 967 host cell phosphoproteins. Considering the high frequency of localized pSerine, pThreonine and pTyrosine, we observed the phosphorylation sites localized in our data and found pS was the most abundant phosphorylated residue with 1869 phosphorylation sites localized on it, and followed by pT with 149 phosphorylated sites localized on it, but for pY only 2 phosphorylation sites were identified in our data.2) Comparative Proteomic analysisAs a total of 450 phosphopeptides were identified in the comparison between M and L group, among which the phosphorylation of 267 phosphopeptides were up-regulated and 183 were down-regulated, matched to 301 phosphoproteins; 354 phosphopeptides were identified in the comparsion between H and L group, among which the phosphorylation of 193 phosphopeptides were up-regulated and 161 were down-regulated, matched to 249 phosphoproteins; 425 phosphopeptides were identified in the comparison between H and M group, among which the phosphorylation of 181 phosphopeptides were up-regulated and 244 were down-regulated, matched to 288 phosphoproteins.3) Bioinformatics analysis of the data setThe significant regulated phosphopeptides mentioned above were defined as peptides involved in "early invasion phase" (M vs. L), "later invasion phase" (H vs. L), and "mid-late invasion phase" (H vs. M). These data were selected for GO analysis, COG analysis and KEGG analysis. The results turned out that the phopshorylation of the peptides involved in the three invasion phases may be differently regulated and showed some differences in their COG, GO, and KEGG analysis results.4. Conclusion and DiscussionLarge-scale phosphoproteomic data sets of HFFs infected by T.gondii for different time have been obtained in this research, it for the first time reveals the phosphoregulation of the host cell caused by T.gondii invasion, and may provide clues how the host cell resist T.gondii’s invasion or be adapted to T.gondii’s parasitism. We observed phosphoproteins among control group (L),2hrs invasion group (M),and 6hrs invasion group(H),and found out 450,249 and 288 significant regulated phosphoproteins respectively. The COG、GO、KEGG analysis of these significant regulated phosphoproteins showed different invasion phases emphasize on different cellular processes, which is compared with later invasion phase the early invasion phase emphasize more on anti-apoptosis, and compared with early invasion phase the later invasion phase emphasized more on the recombination of cytoskeleton.