Blockade of Host Apoptosis by Toxoplasma Gondii - Scope

Toxoplasma gondii infected cells exhibit a profound blockade of apoptosis that manifests at multiple points in the apoptotic cascade. Our studies have shown an essential role for the hose transcription factor NFKB in the establishment of the parasite-directed anti-apoptotic state. Activation of NFKB by diverse cellular pathways occurs via the phosphorylation of specific Ser residues on its inhibitor IKB. This phosphorylation event is catalyzed by a unique cellular kinase complex defining the IKB kinase signalosome (IKK). In T. gondii infected cells Phospho-IKB localizes at the parasitophorous vacuole membrane (PVM), the oraganelle defining the intracellular replication-permissive niche. The detection of P-IKB at the PVM in cells devoid of all IKK activity suggested the presence of a parasite-encoded kinase (TgIKK) is responsible. We find just such an activity in parasite extracts and PVM-enriched fractions. We focus here on identifying the gene(s) encoding the TglKK activity and examining its role both in NFKB activation and the blockade of apoptosis. Our data indicate that TglKK activity alone is not sufficient to drive NFKB gene expression in cells with defects in IKK. Initial studies examining the temporal nature of NFKB activation reveal a biphasic pattern of NFKB expression suggesting independent, but temporally linked, contributions of the host and parasite IKK activities. We propose to use host cell lines with specific lesions in patheways upstream of IKK, as well as cell lines "locked" into a defined expression profile, to better characterize the pertinent cellular pathways subverted by T. gondii infection. Finally, we have developed a genetic screen to identify parasite genes involved in NFKB activation. Initial results bear out the evidence that the mechanism to subvert NFKB is multifactorial. Identification of these parasite genes and the elucidation of their roles in NFKB activation in the context of the studies on the cellular components will help define the signaling networks subverted by the parasite. Dissection of these pathways is particularly important given the targets of NFKB many of the cytokines implicated both in the pathogenesis of acute toxoplasmosis and the development of immunity to the parasite.