Research Of The Pathogenesis Of Toxoplasma Gondii Rhoptry Protein16

Toxoplasma gondii is an obligate intracellular parasitic protozoa, almost all of the warm-blooded animals can be infected. In recent years, as urban development, population increase, pets team continues to expand, coupled to ignore factors such as food hygiene, infected with Toxoplasma gondii a potential risk of extreme rise. Pregnant women infected with Toxoplasma gondii, usually can give this infection to her fetus, if the infection occurs early in embryonic development can lead to mental retardation, eye and brain disease, multiple organ disease and other serious consequences, affecting the development of the fetus, causing miscarriage,fetal malformations, stillbirth, premature birth, birth defects and other congenital toxoplasmosis. The survey shows that China’s congenital Toxoplasma infection rates reach22%in birth defects in children, the mentally handicapped and the incidence of psychiatric patients may be bound up with Toxoplasma infection.In the process of Toxoplasma gondii invate the host cells, rhoptry proteins play an important role. So far, only two Toxoplasma gondii rhoptry proteins can invade the host cell nucleus, they are rhoptry protein16(ROP16) and PP2C-Hn. ROP16can quickly invade to the host cell nucleus, it is concerned with virulence of Toxoplasma gondii. It has proven ROP16can affect host cell signal transduction and activator of transcription factor (STAT) signaling pathway and thus interfere with the host cell proliferation, differentiation, and apoptosis. However, the specific function of the protein into the nucleus play is not known.Our hypothesis are deal with①since the ROP16can invade into the host nucleus, it can affect the normal physiological functions of the host cell and the nucleus;②since the tropism of Toxoplasma gondii on brain cells, then the intrusion Toxoplasma of the brain cells can affect the normal physiological function of proliferation, differentiation, and apoptosis in these cells, likely, ROP16invade into the nucleus, then it could affect the nucleus function.③since T. gondii infection of brain cells, may affect early brain development. We aim to proceed from the ROP16physiological function of basic research to explore the protein interaction with the human body to obtain the downstream target gene information to look for factors that are associated with growth and development, and ROP16and interaction of protein binding to its function impact, reveal the formation of toxoplasmosis and Toxoplasma infection can cause birth defects and mental retardation mechanism.In this paper, the studies will focus on confirming the interaction between ROP16and nuclear protein in the body, the main contents and results are as follows:(1) The construction of the cells which expressed ROP16stably:The internationally recognized T. gondii virulent strain RH strains were injected into the abdomen of C57mice for the formation of ascites, the total RNA from the ascites was extracted, we got the purpose gene ROP16by RT-PCR amplification, and constructed the eukaryotic expression vector PEXPR-IBA-105IP-ROP16. We transfected the eukaryotic expression vector into293T cells by lip2000, the cells would be screened by puromycin, then the cells which expressed ROP16would be cloned, western-blot identified the cloned293T-ROP16cells successfully.(2) Screening human body nucleoproteins which interacted with ROP16:The293T-ROP16cells which expressed ROP16successfully were cultured and immune precipitation technology was used to capture the proteins which would be interacted with ROP16in host, combining MALDITOF/TOF tandem mass spectrometer assay to identify the captured proteins, we got7credibility nucleoprotein.(3) The immunoprecipitation technique confirmed that ROP16and five of the seven candidate nucleoprotein (Histone H2B, Histone H4, lamin B1,hnRNP A2/B1,STMN1) interaction,we confirmed two proteins could interact with ROP16,they were LMNB1and HIST1H2B.(4) Confirmation of the ROP16-interacting proteins:Using two-way co-immunoprecipitation confirmed that ROP16between LMNB1and HIST1H2B combined, confocal laser confirmed that ROP16and laminBl were co-located in the cell membrane, and ROP16and histoneH2B were co-located in the nucleus.(5) The influence of ROP16on nerve cells growth and differentiation:We constructed the virus vector PWPI-ROP16, and packaged the virus in293T cells. We infected human brain cells u-87MG and SH-SY5Y by the packaged virus, real time PCR confirmed that the specific marker of astroglia in u-87MG cells and neuron which infected with ROP16had downregulation.(6) TUNEL method and flow cytometry detected the function of ROP16in the u-87MG and SH-SY5Y cells proliferation and apoptosis. Both u-87MG and SH-SY5Y cells which expressed ROP16stably and the u-87MG and SH-SY5Y cells transfected with idling plasmid as negative control had no difference in proliferation, but in apoptosis,the former was more distinct than the latter.(7) The function of ROP16after interacting’with LMNB1:With the application of retinoic acid (RA), SH-SY5Y cells were induced to differentiate into neurons, ROP16made the neurons have some changes, including increasing the number of nucleoli, the nuclear membrane vesiculated, a large number of clustered arrangement of microtubules compared to the negative control under the electron microscope.Conclusion:ROP16interacted with lamin B1and histone H2B respectively. ROP16inhibit the growth of u-87MG cells and the differentiation of SH-SY5Y cells. ROP16stimulates u-87MG and SH-SY5Y cells apoptosis. The ROP16and LMNB1interactions may influence the function of the LMNB1maintaining nuclear membrane, so that the neuronal cells are abnormal in morphogenesis. Part Two Research of interaction between TFPI-2and PSAPTissue factor pathway inhibitor-2(TFPI-2), also named as placenta protein-5(PP5) and matrix associated serine protease inhibitor (MSPI), is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily. The mature protein contains a short acidic amino-terminal region, three tandem Kunitz domains and a carboxyl-terminal tail highly enriched in basic amino acids. Several studies revealed that hTFPI-2could inhibit several proteases activity including trypsin, plasmin, chymotrypsin, plasma kallikrein, cathepsin G and a wide variety of matrix metalloproteases(MMPs). Therefore, hTFPI-2could inhibit invasive and metastatic ability of many tumors such as prostate cancer, lung cancer, fibrosarcoma, melanoma and gliomas, et al. Furthermore, the rupture of atherosclerosis plaque can also be inhibited by hTFPI-2. But the biological functions of TFPI-2are still unclear. Especially, the interactions of TFPI-2with other proteins haven’t been revealed well.We used the yeast two-hybrid approach to find TFPI-2-interacting proteins. These efforts resulted in the identification of several proteins as TFPI-2’s interaction partners, one of which is Prosaposin (PSAP). Studies have showed that PSAP is a multi-functional glycoprotein with versatile neurotrophic activities. It was identified initially as the precursor of sphingolipid activator proteins, saposins A-D, required for enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. Mature saposins are generated from prosaposin through proteolytic processing to distribute to lysosomes, while PS AP is found as a secreted protein in various body fluids such as human milk, serum, cerebrospinal fluid and seminal plasma and also in neuronal and other plasma membranes.In this paper, the studies will focus on confirming the endogenous interaction between TFPI-2and PSAP, as follow:1) With Co-immunoprecipitation and co-localization experiments, we confirmed the endogenous interaction between TFPI-2and PSAP.2) Through cell invasion and immigration assays, we confirmed that PSAP can promote HT1080cells invasion and migration and TFPI-2can inhibit this process. Besides, enzymatic activity assay indicated that PSAP can enhance the activity of MMP-2in HT1080in a concentration-dependent manner and TFPI-2can also block this enhancement.3) We identified thatPSAP had no influence on the expression of MMP-2and MMP-9by Western-Blot and real-time RCR.Conclusion:TFPI-2can interact with PSAP endogenously and inhibit PSAP function of promoting HT1080cells invasion and migration.