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

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.