Anthony P. Sinai

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30–amino acid (aa) NH2-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH2-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities

The regulation of mitochondrial permeability, a key event in the initiation of apoptosis is governed by the opposing actions of the pro- and anti-apoptotic members of the BCL2-family of proteins. The BCL2-family can be classified further based on the number of BCL-homology (BH) domains they encode. Pathogen mediated modulation of BCL2-family members play a significant role in their ability to affect the apoptotic pathways in the infected host cell. The protozoan parasite Toxoplasma gondii establishes a profound blockade of apoptosis noted by a requirement for host NFκB activity and correlating with the selective degradation of pro-apoptotic BCL2-family members. In this study, we explore the potential activities associated with the inherent stability of the anti-apoptotic BCL2 as well as the selective degradation of the pro-apoptotic proteins BAX, BAD, and BID. We find that multiple activities govern the relative stability of BCL2-family members suggesting a complex and balanced network of stability-enhancing and–destabilizing activities are perturbed by parasite infection. The data leave open the possibility for both parasite induced host activities as well as the direct consequence of parasite effectors in governing the relative levels of BCL2-proteins in the course of infection.

Activation of NF-κB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IκB to the parasitophorous vacuole membrane

Mammalian cells infected with Toxoplasma gondii are resistant to apoptosis induced by a variety of stimuli. We have demonstrated that the host transcription factor NF-κB plays a pivotal role in the T.-gondii-mediated blockade of apoptosis because inhibition is lost in cells lacking the p65 (RelA) subunit of NF-κB (p65–/–). In the present study, we examined the effects of T. gondii infection on NF-κB activation and the expression of genes involved in the apoptotic cascade. Infection of wild-type mouse embryonic fibroblasts (MEFs) with T.-gondii-induced nuclear translocation of the p50 and p65 subunits of NF-κB as examined by immunoblotting of nuclear extracts, immunofluorescence and electrophoretic mobility shift assays. A comparison of apoptotic gene expression profiles from wild-type and p65–/– MEFs revealed distinct patterns of induction in response to T. gondii infection. In particular, the differences seen in the Bcl-2 and IAP families are consistent with the antiapoptotic responses observed in the resistant wild-type cells compared with the sensitive p65–/– fibroblasts. Consistent with NF-κB activation, T. gondii infection promoted phosphorylation of the inhibitor IκB. Interestingly, phosphorylated IκB was concentrated on the parasitophorous vacuole membrane (PVM), suggesting a parasite-directed event. Results from this study suggest that activation of NF-κB plays an important role in stimulation of antiapoptotic gene expression by T. gondii. Furthermore, recruitment of phosphorylated IκB to the PVM implies the presence of intrinsic factor(s) in T. gondii that might be used to manipulate the NF-κB signaling pathway in the host to elicit a survival response during infection.

Inhibition of caspase activation and a requirement for NF-κB function in the Toxoplasma gondii-mediated blockade of host apoptosis

Mammalian cells infected with the protozoan parasite Toxoplasma gondii are resistant to many apoptotic stimuli transmitted along both the mitochondrial and death receptor pathways. Apoptosis, and its inhibition in infected cells, was examined using multiple morphological, molecular and biochemical approaches. The data strongly indicate manipulation of the host apoptotic machinery at multiple levels, focusing on the inhibition of host caspases. Activation of the pro-apoptotic caspase family of proteases is a biochemical hallmark of apoptosis. Caspase activation occurs in a highly ordered cascade triggered by the initiator caspases 8 and 9, which activate the executioner caspase, caspase 3. Our findings indicate a profound blockade of caspase activation and activity as the molecular basis for the inhibition of apoptosis in T.-gondii-infected cells. Caspase inhibition was demonstrated using multiple intrinsic and synthetic substrates. Although the specific inhibitory molecule remains to be identified, data indicate an absolute requirement for the host transcription factor NF-κB and, by extension, genes regulated by it. We propose that T. gondii activates the host survival response, thereby increasing the overall resistance of infected cells to apoptotic stimuli.

Modulation of the Host Cell Proteome by the Intracellular Apicomplexan Parasite Toxoplasma gondii

ABSTRACT To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha.

Toxoplasma gondii activates the NF‐κB pathway in the infected host cell resulting in upregulation of pro‐survival genes and prevention of apoptosis. Manipulation of the NF‐κB cascade by T. gondii correlates with the localization of phosphorylated IκB at the parasitophorous vacuole membrane (PVM). This suggests a parasite‐mediated event, involving the recruitment and activation of the host IκB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKα, IKKβ, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IκBα at Ser 32/36 was observed at the PVM of T. gondii‐infected IKKα–/–, IKKβ–/– and IKKα/β double‐knockout (IKKα/β–/–) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IκB kinase was examined by in vitro kinase assays using GST–IκBα constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IκBα at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IκBα and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF‐κB pathway.

Suicide prevention: disruption of apoptotic pathways by protozoan parasites

The modulation of apoptosis has emerged as an important weapon in the pathogenic arsenal of multiple intracellular protozoan parasites. Cryptosporidium parvum, Leishmania spp., Trypanosoma cruzi, Theileria spp., Toxoplasma gondii and Plasmodium spp. have all been shown to inhibit the apoptotic response of their host cell. While the pathogen mediators responsible for this modulation are unknown, the parasites are interacting with multiple apoptotic regulatory systems to render their host cell refractory to apoptosis during critical phases of intracellular infection, including parasite invasion, establishment and replication. Additionally, emerging evidence suggests that the parasite life cycle stage impacts the modulation of apoptosis and possibly parasite differentiation. Dissection of the host–pathogen interactions involved in modulating apoptosis reveals a dynamic and complex interaction that recent studies are beginning to unravel.

Autophagy is a cell death mechanism in Toxoplasma gondii

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α‐P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3‐methyl adenine (3‐MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.

Toxoplasma gondii inhibits ultraviolet light-induced apoptosis through multiple interactions with the mitochondrion-dependent programmed cell death pathway.

Cells infected with the protozoan parasite Toxoplasma gondii are resistant to diverse apoptotic stimuli. In this study, we perform a detailed analysis of the manipulation of the mitochondrial arm of the apoptotic cascade by the parasite. Apoptosis was induced using irradiation with ultraviolet light (UV), and the kinetics of caspase activation, cytochrome c release and activation of the upstream signalling pathways were examined. The evidence clearly points to T. gondii targeting multiple steps in the transmission [inhibition of c‐Jun N‐terminal kinase (JNK) activation in response to UV], triggering (inhibition of cytochrome c release by affecting the balance of pro‐ and anti‐apoptotic BCL‐2 family members) and execution (inhibition of caspase 9 and caspase 3) phases of the apoptotic cascade. Interestingly, the multilevel pattern of inhibition that emerges suggests that the global inhibition of the mitochondrial arm of apoptosis is not likely to be contributed to by the small subset of mitochondria recruited to the T. gondii parasitophorous vacuole membrane.

Host and parasite-derived IKK activities direct distinct temporal phases of NF-κB activation and target gene expression following Toxoplasma gondii infection

Activation of NF-κB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IκBα to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IκB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-κB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-κB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-κB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-κB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-κB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.