The Research On The Function Of Host Cell Rho And Rac GTPases In T. Gondii Infection

Toxoplasma gondii is an intracellular protozoon which can infect many types of nucleated cells. It is estimated that approximately one-third of the world’s population is chronically infected. T. gondii infection in the human body, the immune normal population by acute infection quickly converted to latent infection, and thus no significant hazards; and widely spread in immunosuppressed patient’s body, the parasites on the nucleated cells caused widespread inflammation, the main damage to the brain, eyes and lymph nodes, and even cause death; infection in pregnant women, T. gondii can be transmitted through the placenta to the fetus, resulting in miscarriage, stillbirth, stillbirth, birth defects or neonatal toxoplasmosis. The major route of human infection with T. gondiiis the definitive host (cat) through intermediate hosts eating undercooked meat or oocysts excreted by contaminated food.T. gondii infected host will stimulate the body to produce an immune response against T. gondii infection in the early stages of infection, the cellular immune response in the body play a major anti-inflammatory effects. The formation of immune can effectively limit the development of the infection and the formation of the new injury, but generally does not eliminate the infection with T. gondii.M.ost researchers believe that T. gondii has features with insect immunity. Re-infection of the body only in the course of infection fishes having a certain degree of resistance to parasites cleared after the immunity disappears.Toxoplasma keep alive in the environment of the body’s immune response, mainly in the PVM maintained good proliferation and interference with the host cell signal transduction to resistance to infection of the host cells to resist the reaction, but the host immune responsecan only be partially weakened, but not completely eliminated. T. Gondii by the process of induction and inhibition of host immune reaction to maintain a delicate balance, to ensure that the parasitic proliferation within the host cell and have the opportunity to spread. Toxoplasma invasion and parasitic processes have many host cell signaling pathways involvement and extensive changes, changes in cell signaling Toxoplasma invasion, parasitic, proliferation plays an important role and T. gondii-host cells mutual relations.T.gondii infection of the host cell mechanisms have been studied extensively. During invasion of T.gondii, the tachyzoites major pest of intracellular parasitic and multiply rapidly destroy the host cell, the escaping merozoite invasion of neighboring cells, and so forth, to stimulate lymphocytes, infiltration of macrophages, resulting in the organization acute inflammation and necrosis.The T.gondii invading cells process includes:attached to the host cell, extending class cone, into the host cell, parasites move backward motion connection form, secretion Microneme proteins, rhoptry neck proteins secretionrole, parasites slide into the parasitophorous vacuoles in the10s to complete the entry process, relatively slow movement parasites into the cytoplasm, and moved to the nuclear.Toxoplasma invasion of host cells will form parasitophorous vacuole in this process.MJ remove some of the components of the host cell (such as a transmembrane protein), while retaining most of the host cell membrane component to composition to parasitophorous vacuole membrane.GTPases are a large group of enzymes that bind GTP (guanine triphosphate) and catalyze the hydrolysis of GTP to GDP (guanine diphosphate) in the presence of an Mg2+ion. Small GTPases are monomeric GTPases with a molecular weight of20kDa-40kDa and composed of at least five families:Ras,Rho, Rab, Sarl/Arf and Ran.The Rho subfamily is further divided into RhoA, Rac and Cdc42.The most important function of these proteins regulate cytoskeleton and actin reorganization. Other features include the regulation of gene transcription, cell cycle and membrane vesicle transport.The Rho GTPases main function is to regulate actin protein and the reorganization of the cytoskeleton, thereby regulating cell morphology and movement. The group protein activation promote the degradation of the extracellular matrix and on the structure of the cortex and increased cell motility. Rho family proteins in the activation and inactivation plays a crucial role in many basic life activities of eukaryotic cells. T.gondii invasion of host cells in the process along with the structure of the reorganization of the host cell, including host cell centrioles and Golgi complex positioning the parasitophorous vacuole and mitochondria microtubules and lysosomes into the parasitophorous vacuole edge.A group of interferon-inducible large GTPases (IRGs) and a small GTPase, ADP-ribosylation factor-6(ARF6) of the host cell accumulate on the PVM of invading T. gondii. IFN-y-Inducible GTPase (Irga6) contributes to resistance against T. gondii in mice. Irga6is predominantly found in the GDP-bound state in interferon-induced, uninfected cells, but it does accumulate on the PVM after Toxoplasma infection and changes to the GTP-bound form. Accumulation of Irga6on the T. gondii PVM is associated with vesiculation and ultimately disruption of the vacuolar membrane in a process that requires an intact GTP-binding domain. ARF6is recruited to the PVM of T. gondii RH strain and plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP2and PIP3 to the PV of T. gondii. The significance of some GTPases in the Toxoplasma invasion process has prompted us to further investigate whether other members of the small GTPases are also involved in host cell invasion.Objectives:1. To testify if RhoA/Rac1GTPases of host cell also accumulate on the PVM of T.gondii when the parasite invading the host cell.2. To learn about if dominant negative RhoA/Rac1GTPases of host cell also accumulate on the PVM when the T. gondii tachyzoites invading the host cell.3. To determine the activation states of the RhoA/Rac1GTPases in the host cell after nvasion of the T. gondii.4. To explore what motifs are essential for the recruitment of Rho GTPases to the PVM.5.To analyze the activity status of the Rho/Rac GTPases accumulated on the PVM.6. To study if the deploite of host cell RhoA and Rac1GTPases during the tachyzoites invasion.Methods:1. pECFP-RhoA-WT and pECFP-Rac1-WT plasmids were Transfected into in COS-7cells.48hours post transfection,the cells were infected with different virulent strains of RH and Pru tachyzoites for2hours, respectively. Then the cells were fixed wtih4%polyformaldehyde for30min. Fixed cells were stained with DAPI for10min at room temperature. The accumulation of the CFP tagged RhoA and Racl was visualized under the fluorescence microscope.2. The tachyzoites of T. gondii RH strain were used to infect human16HBE cells.The endogenous RhoA and Racl of the host cell were localized by indirect immunofluorescence assay.3. COS-7cells transfected with pECFP-RhoA-WT were infected with T. gondii RH tachyzoites. The real-time photographs were taken at5min post-infection and every10minutes thereafter using a confocal fluorescence microscopy.4. Dominant negative mutants of Rho and Racl(RhoA-N19and Racl-N17) were transfected into COS-7cells.48hours post infection, the cells were transfected with T. gondii RH tachyzoitesfor2hours. The cells were fixed with4%poly formaldehyde for30min, cells were stained with DAPI for10min at room temperature. The cell localization of these truncated RhoA was visualized under the fluorescence microscope.5. The activation of the host cell RhoA/Racl was detected with active RhoA or Racl pull down assay.6. The10aa sequentially truncated RhoA mutants were constructed by site directed mutation with pECFP-RhoA-WT as the parental plasmid, namely M1-M19. These mutants were transfected into COS-7cells,48hours post transfection, the cells were infected with T. gondii RH tachyzoites for2hours. The cells were fixed wtih4%polyformaldehyde for30min, then cells were stained with DAPI for10min at room temperature. The distribution of the CFP tagged truncated RhoA mutants was visualized under the fluorescence microscope.7. COS-7cells was transfected with pECFP-RhoA-WT48hours post transfection, the cells were infected with T. gondii RH tachyzoites for2hours.100nMEGF was added to one corner of the coverslips to point activate the cells for5min. After the cells were fixed with polyformaldehyde, cells were stained with DAPI for10min at room temperature. The translocation of the RhoA was observed under a fluorescence microscope.8. The detection of the activity and existence of RhoA and Racl in host cells related to the infection rate of T. gondii:a. The dominant negative mutant plasmids of RhoA and Racl(pECFP-RhoA-N19 and pECFP-Rac1-N17) were transfected into COS-7cells,48hours post transfection, the cells were infected with T. gondii RH tachyzoites for2hours. The cells were fixed with poly formaldehyde, cells were stained with Giemsa. The infection rates of different groups of cells were calculated, and the statistical difference was analyzed with SPSS13.0software,b. Human16HBE cells were transfected with RhoA-siRNA and Racl-siRNA to silent the endogenous expression of RhoA and Racl.48hours post transfection, the cells were infection with T. gondii RH tachyzoites for2hours. The cells were fixed with polyformaldehyde, cells were stained with Giemsa. The infection rates of different groups of cells were calculated, and the statistical difference was analyzed with SPSS13.0software.Results:1. The small GTPases-RhoA and Racl accumulated on the PVM of T. gondii in the whole processes of infection.2. The recruitment of RhoA and Racl GTPases into PVM is dependent on the GTPase activity.3. The Rho A and Racl GTPases were activated upon T. gondii tachyzoite invasion4. The recruitment of RhoA to T. gondii PVM is dependent on different RhoA domains.5. The PVM-localized Rho and Rac GTPases do not respond to epithelial growth factor (EGF) activation, implying that the RhoA and Racl GTPases on the PVM are in GTP-bound active form.6. Interference with host cell RhoA and Racl endogenous activity, the T. gondii tachyzoites cell invasion efficiency was inhibited significantly.Conclusion:1. RhoA and Racl GTPases from the host cell were found accumulating on the PVM after T. gondii invasion regardless of the virulence of the parasitic strain,and this accumulation depended on their GTPase activity.2. RhoA GTPase was recruited to the PVM as soon as the T. gondii tachyzoite invading the host cell through host cell membrane or from the cytosol. Host cell RhoA and Racl were activated after T. gondii invasion.3. The decisive domains for the RhoA accumulation on the PVM were identified as the GTP/Mg2+binding site, media effector interaction site, and G1box/G2box/G5box, respectively.4. The recruited RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, which indicated that the recruited RhoA or Racl on the PVM might be GTP bound active form.5. Wild type RhoA or Racl overexpressed cells and RhoA or Racl siRNA-treated cells showed that the normal activity of RhoA and Racl GTPases are indispensable to the internalization of the tachyzoite. The accumulation of the RhoA and Racl on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.