Host and parasite-derived IKK activities direct distinct temporal phases of NF-κB activation and target gene expression followingToxoplasma gondiiinfection

Abstract

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.

Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

“…Activation of NF-κB is partially achieved by phosphorylation of a subset of host IκBα [21]. However, it soon became clear that manipulation of the NF-κB pathway is more complex [22]. To address this issue, we developed a stable NF-κB-GFP reporter cell line (PSH2) for screening of mutants of the T. gondii that fail to activate NF-κB.…”

“…We have previously shown that activation of NF-κB is partially achieved by phosphorylation of a subset of host IκBα [21]. However, it soon became clear that manipulation of this pathway is a complex process involving multiple elements [22], which lead us to develop the reporter cell line system to screen for NF-κB defective mutants of the parasite.…”

Host cell manipulation by the human pathogen Toxoplasma gondii

“…Cells depleted of IKKα/β are incapable of activating NFκB. Although the IKKβ subunit is the main player in the phosphorylation of IκBα, the IKKα subunit is also required to induce the expression of genes targeted by NF-κB [108]. Notably, T. gondii infected cells present a striking increase in phosphorylated IκBα (PIκB) concentrated at the PVM [99].…”

“…The contribution of parasite TgIKK in the activation of the NF-κB pathway by infection was investigated recently by Molestina et al [108]. They showed that while the host IKK signalosome is essential for translocating NF-κB to the nucleus and for the observed upregulation of NF-κB target genes early after T. gondii infection, maintenance of the response appears to require the contribution of parasite TgIKK during the intermediate phase of the infection at a stage where host IKK dampening is normally seen.…”

Quantification of the gene expression of bell peppers (Capsicum annuum) ripening gene(s) using real -time PCR

“…The target amount was normalized to an endogenous reference and relative to a calibrator (fold differences), is given by 2-CT. Using the expression level of target genes in the control tissue as a base line, the expression folds have been detected in the treated ones assuming that both standard and target have same efficiencies (Molestina and Sinai, 2005). Threshold cycle values (Ct) were used, as Ct indicates the PCR cycle number at which the amount of amplified target reaches a fixed threshold in order to convert threshold cycles in copy numbers.…”