The Role of GRA7, a Component in the ROP18 Protein Complex, in Toxoplasma gondii

<p>The obligate intracellular pathogen, Toxoplasma gondii, is adept at creating a safe niche within the rough environment of the host cytosol. By enclosing itself within a non-fusogenic parasitophorous vacuolar membrane (PVM), it cleverly resists lysosomal fusion. Despite this leverage, avirulent strains of Toxoplasma are cleared effectively from their host cells by the host's intrinsic cellular defenses. Forward genetic analysis has identified a virulence factor, the serine/threonine kinase ROP18, which plays an important role in the survival of virulent strains of Toxoplasma. In an infected cell ROP18 localizes to the PVM along with many members of the rhoptry (ROP) and dense granule (GRA) family of proteins. On the PVM, ROP18 kinase targets the host's dynamin-related immunity related GTPases (IRG) for phosphorylation on critical residues, leading to their inactivation. Avirulent strains that lack ROP18 are successfully targeted by the IRG proteins that are recruited to the PVM, resulting in ruffling and disruption of the membrane and hence death of the parasite within it. Although ROP18 is necessary and sufficient to subvert the IRG pathway, we were interested in whether it associated with other parasite proteins to carry out its virulence function in vivo. Using immunoprecipitation and mass spectrometric analysis we found that ROP18 partners with two parasite proteins, the rhoptry pseudokinase ROP2, and the dense granule protein GRA7. While GRA7 arises from a distinct organelle intracellularly, upon infection, we were able to localize GRA7 to the cytosolic face of the PVM similar to ROP18, using selectively permeable detergents and immunoflorescence microscopy. To resolve the biological significance of this complex, we constructed single as well as double deletion strains, using &#916;ku80, a genetically malleable parent parasite line. Although the single deletion strains did not affect virulence, the double deletion strain &#916;gra7&#916;rop18 displayed considerable attenuation. By performing cellular assays, we ascertained that the &#916;gra7&#916;rop18 parasites defective as they were significantly more vulnerable to the IRG pathway. Although GRA7 has been suspected to play a role in nutrient acquirement, we were unable to find a defect in the &#916;gra7&#61472;parasites growth in vitro, or in virulence in vivo. To establish a mechanism for GRA7 in defense against the IRGs, we performed in vitro biochemical assays testing the effect on GRA7 on the activity of Irga6. We found that sub molar amounts of GRA7 were capable of stimulating rapid oligomerzation of recombinant GTP-activated Irga6 to a much higher extent than without GRA7. In addition, GRA7-driven oligomers of Irga6 also resolved back into monomers much faster than oligomers without GRA7. Collectively, we propose that GRA7 is involved in mediating IRG resistance by stimulating the turnover of Irga6 near the PVM, and in effect, preventing them from accumulating and destroying the PVM, and/or providing its partner kinase, ROP18, with substrate. This finding adds to the growing list of parasite factors that subvert the IRG pathway, and suggests a novel mechanism for its action.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Year dc:date.available

2014

Author dc:creator

• Alaganan, Aditi

Contributors dc:contributor

• David L Sibley

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7TQ5ZK1

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-2375