VERMONT COBRE: PROJECT 4: SUBVERSION OF HOST CELL SIGNALING BY TOXOPLASMA GOUDII

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. The objective of this proposal is to understand how T. gondii interferes with host cell signaling and determine the functional consequences for parasite survival. Invasion of a host by a pathogen usually activates the NF-kB family of transcription factors which play an important role in the regulation of the immune system and are frequently required for resistance to infection. Recent studies have shown that invasion of cells by T. gondii not only fails to activate NF-kB, but this parasite actively inhibits this signaling pathway, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. However, it remains unclear how T. gondii inhibits NF-kB and what is the important cellular target. We propose that when T. gondii infect macrophages the presence of a specific T. gondii factor associates with a p65 NF-kB protein in the cytoplasm masking its nuclear localization signals and preventing its translocation into the nucleus. As a result, expression of NF-kB-dependent cytokines in response to T. gondii in infected macrophages is impaired and, as a consequence, the adaptive immune response is also impaired in vivo. In Specific Aim_ 1 we will identify and characterize parasite factors that are responsible for the inhibition of the NF-kB signaling pathway. We will focus on an ankyrin-containing TgEST 1207539 protein, other parasite proteins that may interact with p65 NF-kB, and on generating parasite mutants that fail to inhibit the activation of NF-kB. In Specific Aim 2 we will examine if failure of T. gondii to inhibit NF-kB restores the ability of macrophages to produce cytokines in vitro. We will study cytokine production of macrophages overexpressing the TgEST 1207539 protein or infected with parasite mutants. In Specific Aim 3 we will evaluate if T. gondii mutants that fail to inhibit NF-kB are less virulent in an in vivo infection. The outcome of these studies will be useful to determine the functional significance of the NF-kB signaling pathway for the immune response to T. gondii. This research will yield new insights into the molecular mechanisms that this parasite uses to manipulate the host cell signaling.