The Mechanism Of Neural Cells Apoptosis Induced By Toxoplasma Virulence Factor ROP18

Background:T. gondii is a ubiquitous obligate intracellular parasite, infecting a wide range of mammalian hosts. Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Infections with T. gondii are generally subclinical in healthy individuals, but infection in immunocompromise would lead to serious complications, and the main symptoms including the central nervous system, eyes, heart, lungs and liver disease. During the course of T. gondii infection, the central nervous system is the most commonly damaged of all invasive organs. It would cause the microcephaly, hydrocephalus, chorioretinitis and other serious symptoms. ROP18 is a protein kinases, which is secreted by the toxoplasma rhoptries and identified as a key virulence molecule conferring a high mortality phenotype characteristic of type I T.gondii strains. In this study, our results confirmed T. gondii rhoptry protein ROP18 promotes the neuronal apoptosis via endoplasmic reticulum stress pathway.Aim:To investigate the effect of rhoptry protein ROP18 on the apoptosis of neural cells, which will help us to gain new insights into the molecular mechanisms of neuropathogenesis during T. gondii infection.Methods:Primers were designed by OMIGA software, we constructed ROP18-pEGFP-C2 plasmid. Immunofluorescence staining was performed to detect neuropathogenesis of the mouse brain tissues. The apoptosis of neural cells and the expressions of related proteins in the endoplasmic reticulum stress (ER Stress)-mediated apoptosis pathway were detected by flow cytometry and Western blotting.Results:1. Constructed ROP18-pEGFP-C2 eukaryotic expression vector. The expression of ROP18 was confirmed in N2a nerve cells using western blotting.2. Immunofluorescence analysis of the brain from mice infected with over-expressing ROP18 transgenic parasites showed ROP18 induced the apoptosis of neural cells in vivo.We infected BALB/c mice intraperitoneally with 1000 tachyzoites from ROP18 over-expressing parasites (ROP18-RH) or RH tachyzoites, then used immunofluorescen-ce to analysis the brain from mice. Brain sections were stained with NeuN (green), PI (red), DAPI (blue). We found that in the un-infected group, the cerebral cortex was intact and there were many neurons there. However, the loss of neurons in the hippocampus and cerebral cortex of the mice infected with ROP18-RH or RH tachyzoites higher than those in the un-infected group. In addition, the numbers of dead neural cells in the cerebral cortex as well as in the hippocampus of the mice infected with ROP18-RH tachyzoites were significantly higher than RH tachyzoites infected group, accordingly. The results suggest ROP18 may induce the loss of neurons during infection in vivo.3. ROP18 induced the apoptosis of neural cells in vitro(1)The N2a cells were transfected with ROP18-GFP,14-3-3-GFP or control GFP vector for 24 h. Strikingly, the over-expression of ROP18, but not 14-3-3, in N2a cells led to high levels of apoptosis by using flow cytometry. (2) The N2a cells were infected with ROP18-RH, RH tachyzoites at an m.o.i of～3 for 24 h. The apoptotic rates of the cells infected with ROP18-RH were significantly higher than RH tachyzoites infected group.4. ROP18 induced the apoptosis of neural cells via ER stress pathwayImmunoblotting analysis further displays that the expression levels of cleaved Caspase-12, CHOP and cleaved Caspase-3 in the N2a cells increased significantly in both the ROP18-RH tachyzoites infected group and ROP18-GFP transfected group when compared to the control group. When the cells were pretreated with a Caspase-12 inhibitor, Z-ATAD-FMK, the expressions of cleaved Caspase-12 and Caspase-3 in the inhibitor-pretreated groups significantly decreased, whereas no significant differences were detected in the levels of CHOP when they were compared to the control groups. Flow cytometry analysis showed that the apoptotic rates of the cells transfected with ROP18-GFP or infected with ROP18-RH were lower than those in the Z-ATAD-FMK untreated ROP18-GFP or ROP18-RH group.Conclusions:Our findings here highlight that the virulence factor ROP18 in T. gondii may contribute to neuronal apoptosis through the ER stress-mediated apoptosis pathway, which may be a potential molecular mechanism responsible for neurological disorders of toxoplasmosis.