Research statement: Intracellular pathogens like the protozoan parasite Toxoplasma gondii subvert host cell functions to establish a successful infection. We are particularly interested in understanding how parasites manipulate the NFkappaB pathway as we have shown that activation of this cascade is required for the parasite induced blockade of host cell apoptosis. A central player in the activation of NFkappaB by the parasite is a parasite derived kinase activity termed TgIKK that is localized to the vacuolar membrane surrounding the intracellular parasite. TgIKK activity appears directed at the host inhibitor of NFkappaB protein (IkappaB) targeting residues critical for NFkappaB activation. The activation of NFkappaB by the parasite is multifactorial as mutants with diverse lesions (in both known and hypothetical loci) have been isolated using a flow cytometry based genetic screen. Investigations into the blockade of apoptosis and NFkappaB activation have lead to investigations in other cellular functions including the impact of infection on the host cell cycle and energetics. We are beginning to investigate whether these changes impact parasite physiology and may serve as critical cues in differentiation into the relatively dormant cyst forms.

As the interface between the parasite and the host cell, the parasitophorous vacuole membrane (PVM) serves as a platform for diverse activities in the pathogen-host cell interaction. Investigation into PVM biology has been limited due to its being present only in infected cells. In addition the intimate association of the PVM with host organelles that precludes its purification for biochemical analysis. We developed a pair of novel antibody reagents against the PVM-fraction and used them to identify novel PVM-localized activities. This study relied on the development of sample preparation techniques and state of the art proteomics (in collaboration with Dr. Bert Lynn, Department of Chemistry) and has resulted in the provisional identification of 70 novel proteins. Several of these targets have been confirmed and include a PVM –localized E2- Ubiquitin ligase (potentially affecting NFkappaB signaling) and a family of predicted proteophosphoglycans (PPG’s) that one may expect in a Toxoplasma cyst wall. The detection of several PPG’s at the PVM of RH strain parasites that typically do not form cysts was truly surprising. The PVM proteome presents an excellent opportunity to analyze diverse and interesting parasite proteins and reveal the complexity of the Toxoplasma-host cell interaction.

By combining state of the art cell biological, biochemical and increasingly molecular biological approaches to dissect the mechanisms underlying the disruption of cellular function by Toxoplasma we hope to gain new insights into parasite biology as well as broader questions on the microbial pathogenesis.