: The long-term objective of this grant application is to understand the molecular pathogenesis of Toxoplasma gondii infection. This proposal focuses on macrophage intracellular signaling pathways during T. gondii infection. Macrophages serve as important host cells during infection, despite the fact that they are a potent source of inflammatory mediators when activated by microbial stimuli such as bacterial lipopolysaccharide (LPS). Recently, we found that parasite infection of macrophages results in suppression of the NFkappaB-dependent cytokines TNF-alpha and IL- 12. The mechanism by which this is achieved is due to a parasite-induced blockade in IkcappaB degradation, the latter of which is required to enable NFkappaB nuclear translocation. The objective of this proposal is to examine this phenomenon in detail, and to examine the effect of infection on other signaling pathways and host cells. There are four specific aims to be addressed. 1, Determine the mechanism by which infection interferes with the LPS-triggered NFkappaB activation pathway, and determine which components of this signaling cascade are triggered by the parasite. This will be accomplished employing immunochemical techniques to evaluate IkappaB ubiquitination, and transfection experiments using dominant negative mutants of signaling molecules lying upstream of IkappaB phosphorylation. 2, Determine the involvement of MAPK family members in tachyzoite-induced blockade of TNF-alpha, IL- 12, and NO production. This will be achieved by a combination of phospho-specific immunoblotting, and activity assays on each of the major MAPK. 3, Determine whether parasite-induced inhibition occurs through decreased gene transcription or increased mRNA stability, using nuclear run-on assays and RPA, respectively. Bio-array technology will also be employed to identify other macrophage genes up- or down-regulated by Toxoplasma infection. 4, Determine whether macrophages remain LPS non-responsive when intracellular parasites are killed, and when cells internalize parasites by phagocytosis. Also, determine whether Toxoplasma inhibits the ability of dendritic cells and intestinal epithelial cells to respond to LPS. These objectives will be accomplished using confocal fluorescence microscopy and electrophoretic mobility shift assay to detect NFkappaB nuclear translocation. The health relatedness of this proposal is that Toxoplasma is a major opportunistic infection in immunocompromised patient populations, and a serious congenital infection. Understanding the molecular pathology of infection will lead to better control strategies for this and related microbial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI050617-02
Application #
6657309
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Wali, Tonu M
Project Start
2002-09-15
Project End
2007-02-28
Budget Start
2003-03-01
Budget End
2004-02-28
Support Year
2
Fiscal Year
2003
Total Cost
$307,479
Indirect Cost
Name
Cornell University
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Schneider, Anne G; Abi Abdallah, Delbert S; Butcher, Barbara A et al. (2013) Toxoplasma gondii triggers phosphorylation and nuclear translocation of dendritic cell STAT1 while simultaneously blocking IFNýý-induced STAT1 transcriptional activity. PLoS One 8:e60215
Cohen, Sara B; Maurer, Kirk J; Egan, Charlotte E et al. (2013) CXCR3-dependent CD4? T cells are required to activate inflammatory monocytes for defense against intestinal infection. PLoS Pathog 9:e1003706
Smith, Norah L; Abi Abdallah, Delbert S; Butcher, Barbara A et al. (2013) Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase C?-mediated Ca(2+) mobilization. Front Microbiol 4:179
Mahamed, Deeqa A; Mills, Jeffrey H; Egan, Charlotte E et al. (2012) CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system. Proc Natl Acad Sci U S A 109:16312-7
Denkers, Eric Y; Bzik, David J; Fox, Barbara A et al. (2012) An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii. Infect Immun 80:476-82
Butcher, Barbara A; Fox, Barbara A; Rommereim, Leah M et al. (2011) Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS Pathog 7:e1002236
Denkers, Eric Y (2010) Toll-like receptor initiated host defense against Toxoplasma gondii. J Biomed Biotechnol 2010:737125
Leng, Jin; Butcher, Barbara A; Egan, Charlotte E et al. (2009) Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered macrophage activation. J Immunol 182:489-97
Egan, C E; Sukhumavasi, W; Butcher, B A et al. (2009) Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii. Clin Exp Immunol 156:17-24
Leng, Jin; Denkers, Eric Y (2009) Toxoplasma gondii inhibits covalent modification of histone H3 at the IL-10 promoter in infected macrophages. PLoS One 4:e7589

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