T. gondii rhoptry protein ROP18 induces apoptosis of neural cells via endoplasmic reticulum stress pathway
- Lijuan Wan†1,
- Lingli Gong†1,
- Wei Wang2,
- Ran An1,
- Meijuan Zheng3,
- Zongru Jiang1,
- Yuewen Tang1,
- Yihua Zhang2, 4,
- He Chen3, 4,
- Li Yu4, 5,
- Jilong Shen2, 4 and
- Jian Du1, 4Email author
© Wan et al. 2015
Received: 10 September 2015
Accepted: 18 September 2015
Published: 21 October 2015
The neurotropic parasite T. gondii is widespread among mammalian hosts including humans. During the course of T. gondii infection, the central nervous system is the most commonly damaged of all invasive organs. The polymorphic rhoptry protein ROP18 has been identified as a key factor in the pathogenesis of T. gondii; however, the molecular mechanism by which this protein exerts neuropathogenesis remains elusive.
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.
Immunofluorescence staining reveals induction of the propidium iodide (PI) - positive neural cells in mouse cerebral cortex and hippocampus infected with ROP18 over-expressing transgenic tachyzoites. Western blotting analyses reveal that ROP18 increases the expressions of cleaved caspase-12, CHOP and cleaved caspase-3 when compared to the control groups. After the pretreatment of Z-ATAD-FMK (a specific caspase-12 inhibitor), the apoptotic level of neural cells had an apparent decline, and correspondingly, the expressions of those related proteins were notably decreased.
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.
T. gondii is a ubiquitous obligate intracellular parasite infecting a wide range of mammalian hosts including humans [1–3]. Infections with T. gondii are generally subclinical in healthy individuals, but toxoplasmosis remains a major problem in immunosuppressed adults or developing fetuses . In patients with severe immune dysfunction, a reactivation of the infection produces the neurological manifestations or even fatal Toxoplasmic encephalitis (TE) due to reactivation of Toxoplasma cysts in the brain . For those patients, dormant encysted bradyzoites can reactivate into fast replicating tachyzoites and cause severe damage to the brain . TE is the most vital outcome of toxoplasmosis in immunosuppressed individuals and the main symptoms including focal seizures, cranial nerve disturbances, altered mental state, ataxia, sensory abnormalities, hemiparesis, meningismus and neuropsychiatric disorders as well .
Although toxoplasmosis can be associated with multi-organ involvement, the central nervous system (CNS) is the most commonly affected of all invasive organs [7, 8]. As a neurotropic parasite, little is known about which effectors of the type I RH strain elicit neuropathogenesis during infection [7, 9]. Previous work revealed a polymorphic, parasite rhoptry protein, known as ROP18, is a key virulence determinant among different T. gondii clonal lineages [10–13]. The recent results have shown that ROP18 has several targets in the host cell, including IRGs (immunity-related GTPases) [14, 15] and NF-κB p65 . IRGs are strongly induced by interferon-γ (IFN-γ) and are important in the innate immune response against T. gondii . ROP18 contributes to avoidance of IRG recruitment to the parasitophorous vacuole membrane (PVM), thus protecting the parasite from clearance in interferon-activated macrophages. The nuclear factor NF-κB transcription factor has essential roles in immune and inflammatory responses. Its p65 subunit is regulated by several post-translational modifications, including phosphorylation, acetylation and ubiquitination. Our recent results show that ROP18 phosphorylates the host p65 and targets this protein to the ubiquitin-dependent degradation, thus inhibiting the NF-κB pathway in infected macrophages .
Our previous study also revealed that both the canonical type I RH strain and TgCtwh3, a representative strain prevalent in China, can induce the apoptosis of neural stem cells through ER stress signaling pathways [17, 18]. In this study, we investigated the effect of rhoptry protein ROP18 on the apoptosis of neural cells. Our results presented here showed that ROP18 can stimulate neural cell death through inducing ER stress-mediated apoptosis pathway.
The study protocol was approved from the Institutional Review Board of the Institute of Biomedicine at Anhui Medical University (Permit Number AMU 26–093628), which records and regulates all research activities in the school. All surgeries were performed under anesthesia, and all efforts were made to minimize animal suffering.
Parasite and cell lines
ROP18 over-expressing transgenic RH strain (ROP18-RH) was constructed as previously described. Briefly, the 5′-UTR-TUB promoter-ROP18 -Ty1-HXGPRT-3′ -UTR fragment was transfected into the Δku80Δhxgprt RH strain parasites (kindly provided by Professor John C. Boothroyd, Stanford University, USA) by electroporation. Stable integrants were selected in media with 50 μg/ml mycophenolic acid and 50 μg/ml xanthine and cloned by limiting dilution. Construction of ROP18-RH strain was confirmed by PCR, IF and Western-blotting . Then the parasites of ROP18-RH strain were harvested from the mouse peritoneal exudates by injection on the third day after infection, and were isolated by centrifugation to discard the contaminating host cells. Then the parasites were maintained by serial passage in the mouse N2A cells (ATCC, Neuro2a) for further experiments. The mouse neuroblastoma Neuro2a (N2a) cells were cultured in DMEM supplemented with 20 % FBS and 1 % penicillin/streptomycin in a humidified 5 % CO2 atmosphere. All parasite strains and cell lines were routinely assessed for mycoplasma contamination, and no contamination was detected.
Plasmid and reagents
Amplification of the open reading frame encoding T. gondii ROP18 (GenBank ID: AM075204.1) was achieved through RT-PCR of the RH tachyzoite RNA. Caspase-12, CHOP and caspase-3 antibodies were purchased from Cell Signaling Technology (USA); GAPDH antibody was purchased from Santa Cruz (USA).
Brain sections were hydrated and rinsed in PBS. Then they were placed in ACSF (artificial cerebrospinal fluid) containing 5 μg/ml PI for 30 min. After antigen retrieval, the brain sections were premeabilized, then incubated with primary antibody at 4 °C overnight. FITC-conjugated goat anti-mouse IgG, rhodamine-conjugated goat anti-rabbit IgG and DAPI dye were used for antigen and DNA visualization. The images were captured using a fluorescent microscopy (Olympus BX60, Tokyo, Japan).
Detection of apoptosis
The apoptosis of N2a cells were determined following the instruction of Annexin V-PE/7-AAD kit (BD, USA). Briefly, the N2a cells were harvested, then washed twice with cold PBS, and incubated in 1 × binding buffer (10 mM HEPES, 0.14 M NaCl, and 0.25 mM CaCl2) containing PE Annexin V and 7-AAD for 15 min. Then stained cells were analyzed using a Faces Calibur flow cytometer (BD Biosciences, USA) within 1 h, and the data were analyzed using FCS Express 4.0 software. Annexin V-PE+/7-AAD − cells represent the early apoptotic cells, and annexinV-PE+/7-AAD+ cells represent the late apoptotic cells.
Western blotting analysis
To further identify the apoptosis of N2a cells, the expressions of caspase-12, CHOP and caspase-3 were determined using Western blotting analysis. Western blotting was conducted as described previously . Briefly, after the N2a cells were infected with ROP18-RH or RH tachyzoites at an m.o.i of ~3 for 24 h, they were harvested, washed with PBS, and lysed in a lysis buffer (50 mM HEPES, pH7.4, 150 mM NaCl, 2 mM EGTA, 1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml pepstatin A) containing a protease inhibitor mixture (Sigma, USA). The cell lysates were separated through SDS-PAGE, transferred to nitrocellulose membranes, probed with the corresponding antibodies, and developed using an ECL kit.
All quantitative results were expressed as mean ± SD. Statistical analysis of variance used was a two-way ANOVA followed by the Scheffe’s test. P < 0.05 was considered statistically significant.
ROP18 induced the apoptosis of neural cells in vivo
ROP18 induced the apoptosis of neural cells in vitro
ROP18 induced the apoptosis of neural cells via ER stress pathway
Life-threatening toxoplasmic encephalitis is a common opportunistic infection of the CNS that usually results from reactivation of latent cysts into tachyzoites in the brain tissue of immunosuppressed individuals [19, 20]. After reaching the CNS, the parasites can invade all nucleated cells and initiate activation of resident microglia and astrocytes [21–23]. In the brain tissues, microglial cells, astrocytes and neurons are all susceptible to T. gondii infection [2, 24]. Microglia cells are implicated as essential mediators of the brain’s immune response to injury, inflammation or the presence of pathogens [25, 26]. During the course of T. gondii infection, microglia cells can secrete pro-inflammatory cytokines and lead to the neuronal cell damage of inflammatory surroundings . However, neurons are fundamental elements of the CNS, we hypothesized that there may be the possible direct mechanisms of T. gondii on neurons to disrupt their survival and function . Accumulated studies conducted in different animal experimental models have found that behavioural changes occur upon acute and chronic T. gondii infection . One of the most ‘convenient’ explanations for the neuropsychological and behavioural deficits could be that the parasites directly inject effective molecules into the neurons interfering with their activity and function .
As noted, ROP18 is a Ser/Thr kinase related to the ROP2 family, secreted from rhoptry organelles and relocalized to the PVM during invasion [14, 16, 29, 30]. During host cell invasion, ROP18 is secreted to the PVM where it is tethered to the cytosolic face of this host-pathogen interface. They are thought to modify the host cell environment, thus favoring intracellular parasitism . Although the functional role of rhoptry proteins in the biology of T. gondii has not been clearly elucidated, numerous studies have highlighted the role of ROP18 as the key virulence factor in the pathogenesis of T. gondii infection [10, 11, 31]. Indeed, we have previously demonstrated that ROP18 kinase activity is responsible for the phosphorylation and degradation of p65, thereby down-regulating Th1 responses and causing M2 phenotypes in macrophage . Here, we report that ROP18 is involved in the apoptosis of neural cells.
ER stress conditions have been observed in numerous diseases including ischemia/reperfusion injury, neurodegeneration, as well as infectious diseases [32–35]. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER trans-membrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. In eukaryotic cells, three ER trans-membrane proteins mediate the canonical UPR: the two kinases, IRE1 (inositol requiring enzyme 1) and PERK (PKR-like eukaryotic initiation factor 2a kinase), and the transcription factor precursor ATF6 (activating transcription factor 6). If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues through activation of caspase-12, CHOP and/or JNK (c-JUN NH2-terminal kinase) [36–38]. Many of these changes are induced by intracellular pathogens, so it is not surprising that ER stress is induced by cellular pathogens and viruses alike [32, 33]. Meanwhile, it has reported that T. gondii ROP18 is evolved mechanisms to mediate degradation of the host endoplasmic reticulum-localizing transcription factor, ATF6β, to down-regulate T cell-mediated type I adaptive immune responses .
In the present study, we detected the expressions of related proteins in the ER stress-mediated apoptosis pathway by Immunoblotting analysis, and the results showed that ROP18 enhanced the expressions of cleaved caspase-12, CHOP and cleaved caspase-3 in the neural cells (Fig. 4), and subsequently increased the apoptosis of the neural cells infected by ROP18 over-expressing parasite (Fig. 2). Moreover, with pretreatment of Z-ATAD-FMK, the protein level of cleaved caspase-12 and caspase-3 were significantly reduced and the apoptotic level of neural cells was down-regulated accordingly (Figs. 3 and 4). Taken together, our data indicate that the virulence factor ROP18 in T. gondii is involved in the apoptosis of neural cells through the ER stress pathway. Further studies focusing on ROP18-interacting host factors in ER stress pathway will help us to gain new insights into this pathological process.
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 contribute to better understanding the possible mechanism of brain pathology during T. gondii infection.
This work was supported by the National Natural Science Foundation of China (81271864, 30801329), Fok Ying Tung College Young Teachers Fund (131033) of the Chinese Ministry of Education, the Distinguished Young Scholar of Anhui Province (10040606Y19, 1308085JGD11) to Jian Du; the National Natural Science Foundation of China (81471983), the National Basic Research Program of China (973 program; Grant No. 2010CB530001) to Jilong Shen; the Natural Science Foundation of Anhui Educational Committee (No. KJ2015A330) to Wei Wang.
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