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 NFxB by diverse cellular pathways occurs via the phosphorylation of specific Ser residues on its inhibitor kB. This phosphorylation event is catalyzed by a unique cellular kinase complex defining the kB kinase signalosome (IKK). In T.gondii infected cells Phospho-kB localizes at the parasitophorous vacuole membrane (PVM), the oraganelle defining the intracellular replication-permissive niche. The detection of P-kBat the PVM in cells devoid of all IKK activity suggested the presence of a parasite-encoded kinase (TglKK) 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 NFxB 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 NFxB 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI049367-08
Application #
7586189
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Wali, Tonu M
Project Start
2001-03-01
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
8
Fiscal Year
2009
Total Cost
$313,985
Indirect Cost
Name
University of Kentucky
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Sinai, Anthony P; Watts, Elizabeth A; Dhara, Animesh et al. (2016) Reexamining Chronic Toxoplasma gondii Infection: Surprising Activity for a ""Dormant"" Parasite. Curr Clin Microbiol Rep 3:175-185
Gaviria, David; Paguio, Michelle F; Turnbull, Lindsey B et al. (2013) A process similar to autophagy is associated with cytocidal chloroquine resistance in Plasmodium falciparum. PLoS One 8:e79059
Sinai, Anthony P; Roepe, Paul D (2012) Autophagy in Apicomplexa: a life sustaining death mechanism? Trends Parasitol 28:358-64
Ghosh, Debasish; Walton, Julia L; Roepe, Paul D et al. (2012) Autophagy is a cell death mechanism in Toxoplasma gondii. Cell Microbiol 14:589-607
Carmen, John C; Southard, R Chase; Sinai, Anthony P (2008) The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation. Exp Cell Res 314:3724-36
El-Guendy, Nadia; Sinai, Anthony P (2008) Potential problems inherent in cell-based stable NF-kappaB-GFP reporter systems. Mol Cell Biochem 312:147-55
Molestina, Robert E; El-Guendy, Nadia; Sinai, Anthony P (2008) Infection with Toxoplasma gondii results in dysregulation of the host cell cycle. Cell Microbiol 10:1153-65
Carmen, John C; Sinai, Anthony P (2007) Suicide prevention: disruption of apoptotic pathways by protozoan parasites. Mol Microbiol 64:904-16
Herman, Rebecca K; Molestina, Robert E; Sinai, Anthony P et al. (2007) The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation. Infect Immun 75:4255-62
Petersen, Christine A; Krumholz, Katherine A; Carmen, John et al. (2006) Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells. Infect Immun 74:1580-7

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