Identification Of Proteins Associated With Virulence By ITRAQ-based Quantitative Proteomic Analysis Of Excretory-secretory Antigens From Toxoplasma Gondii

Toxoplasma gondii is an important opportunistic parasite,being highly prevalent in both human and animals with a worldwide distribution.Most infections caused by T.gondii are asymptomatic in humans,but they are more harmful for immunocompromised patients and pregnant women.T.gondii was initially considered derived from the three major lineages,Type Ⅰ,Ⅱ,and Ⅲ,which were performed in North America and Europe.Among them,Type Ⅰ isolates are non-cystogenic and considered as the most virulent,while Type Ⅱ and Type Ⅲ are cystogenic and avirulent.Recent years,researchers have found that genotype Chinese 1(ToxoDB#9)is dominantly distributed in mainland China.Although it is well known that all non-cystogenic isolates show high virulence,there are marked genetic and virulence discrepancy among themselves.However,the key reasons for the differences have not been fully clarified.Excretory-secretory antigens(ESA)are soluble antigens produced by T.gondii tachyzoites in the course of the invasion.ESA is a protein complex containing three main protein components,which are considered to be microneme proteins,rhoptry proteins and dense granule proteins.ESA of T.gondii are not only crucial for the invasion process of T.gondii,but also closely related with the strong humoral and cellular immune response of host by inducing protective immunity against infection effect.However,the intense immune reaction can also cause severe immune pathological damage within the body.TNF-α is an important class of Thl-type cytokines that mediate the inflammatory response.Previous studies have confirmed that the induction of TNF-αis also involved in the pathogenesis of toxoplasmosis.Both in vitro and in vivo,experimental results show that TNF-α is an important cytokine inhibiting the proliferation of Toxoplasma gondii.ESA is vital for the invasion proceed and the interaction with the host immune system in toxoplasmosis.Hence,targeted studies of ESA has great value for revealing important proteins associated with virulence within non-cystogenic isolates.In this study,we have selected RH strain and TgCtwh3 strain of T.gondii as our research object.RH strain is the most common isolate belonging to type Ⅰ genotype.TgCtwh3 strain is the most representative non-cystogenic isolate in Chinese 1 genotype.In this research,we reported that ESA of RH and TgCtwh3 triggered distinct levels of TNF-α production as well as macrophage M1 polarization.ESA of both strains could induce TNF-α production,and the TNF-α level triggered by ESA of RH strain was higher.Real-time quantitative PCR results showed that ESA of RH could induce a macrophage M1 polarization with iNOS and IL-6 mRNA significantly up-regulated,but TgCtwh3 ESA did not show such effect.Then iTRAQ was employed to identify differentially expressed ESA proteins in the two strains.We used Gene Ontology(GO)analysis to provide functional comment for the differentially expressed proteins.The results showed that 27 differentially expressed proteins which containing 11 microneme associated proteins,7 rhoptrie proteins,3 dense granule proteins and 6 surface proteins from parasites’surface and secretory organelle were successfully identified,Among them,24 proteins were higher expressed in RH strain,compared with TgCtwh3 strain.Moreover,Gene Ontology analysis illustrated these differentially expressed proteins which were highly abundant in RH devote to the divergence in the virulence between RH and TgCtwh3.They are mainly involved in the process of adhesion and cell invasion.Besides,correlation analysis for the differentially expressed proteins found that 14 secretory proteins were highly interrelated with other proteins,containing 6 microneme associated proteins,3 rhoptry proteins,and 4 surface proteins as well as one dense granule protein called GRA12.Then,we choose five candidate genes for the differentially expressed proteins,and carried out a verification at transcriptional level.The protein abundance degrees of these five candidate genes were notable higher in the RH ESA than that in TgCtwh3 ESA with over 3-fold change in relative abundance,and more than two unique peptides.Consistent with those of the iTRAQ results,the qPCR outcomes suggested that these proteins observed as differentially expressed were regulated at transcriptional level.In this study,we selected one important node protein MIC3 for further functional analysis.Through in vitro experiments,we confirmed that MIC3 could arouse a TNF-α secretory response in a dose-dependent manner.In addition,a high dose of MIC3 could trigger a macrophage M1 polarization.In this study,we confirmed that MIC3 not only play an important role in the invasion of T.gondii,but also induce a TNF-α secretory response.