This item is under embargo and not available online per the author's request. For access information, please visit http://libanswers.wustl.edu/faq/5640.

Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Toxoplasma gondii is an obligate-intracellular parasite, which causes disease in immunocompromised individuals and the developing fetus. Dissemination of the parasite requires efficient replication within its host cell, lysis and reinfection of neighboring cells. One mechanism of immune control of T. gondii is to target intracellular parasites through the IFNγ-activated GTPases including the immunity-related GTPases (IRGs), which accumulate on the parasitophorous vacuole membrane (PVM) surrounding the parasite leading to its rupture and exposure of the parasite to the host cell cytosol. The parasite is able to protect its PVM through the function of the active serine/threonine kinase ROP18, which phosphorylates the IRGs and presumably disrupts their function. The guanylate-binding proteins (GBPs) are related to the IRGs and are maintained in the human genome. To determine if the GBPs play a role in cell-autonomous immunity to T. gondii in the mouse, localization of Gbp1 to canonical strains of T. gondii and their localization in the absence of ROP18 was examined. Gbp1-/- bone marrow derived macrophages and mice were used to assess the role of Gbp1 in clearance of T. gondii and its role in vivo during T. gondii infection. Gbp1 plays a role in the cell-autonomous immunity to T. gondii in cells and in vivo, and its recruitment is dependent on ROP18. In vitro kinase assays determined ROP18 was able to phosphorylate GBPs, including Gbp1, Gbp2, human GBP1 and GBP2. These proteins were phosphorylated in their GTPase domain. hGBP1 was not recruited to high levels to T. gondii in IFNγ-activated human cells, but multiple autophagy factors were recruited to the PVM, including ubiquitin, p62, NDP52 and LC3. The recruitment of these factors was dependent on the strain of the parasite, with type 1 parasites resisting recruitment and type 2 and 3 parasites showing high levels of recruitment. The targeted T. gondii vacuoles were stunted and wrapped in multiple membranes, but did not fuse with LAMP-1 positive compartments. In the absence of ATG proteins, the stunting of vacuoles was relieved. Collectively, these data demonstrate that in IFNγ-activated murine cells, interferon-inducible GTPases including Gbp1 are required for cell-autonomous immunity to T. gondii. In human cells, autophagy-mediated stunting of T. gondii is an important strain-dependent IFNγ-induced restriction mechanism.

Language

English (en)

Chair and Committee

L. David Sibley

Committee Members

Mary C Dinauer, Brian T Edelson, Daniel E Goldberg, Christina L Stallings, Herbert W Virgin,

Comments

Permanent URL: https://doi.org/10.7936/K7125QTK

Available for download on Thursday, August 15, 2115

Included in

Biology Commons

Share

COinS