Thesis

Analysis of SNARE Protein Sec22b Interaction with Legionella pneumophila SdhB C-terminus

Legionella pneumophila is a gram-negative bacterium that causes a severe pneumonia known as Legionnaires' disease. L. pneumophila is engulfed by macrophages via phagocytosis, where it avoids being dismantled by the lysosomes through the establishment of, and proliferation inside a Legionella-containing phagosome (LCP). L. pneumophila secretes effector proteins into the cytosol of the host cell via the Dot/ICM type IV secretion system to allow its survival. SdhB is one of those effector proteins, and it has two paralogues, SidH and SdhA, which are also introduced into the cytoplasm of an infected cell. SdhA plays a role in inhibiting apoptosis. A previous yeast two-hybrid screen suggested that there was an interaction between SdhB and Sec22b, a Soluble NSF Attachment Protein REceptor (SNARE) on endoplasmic reticulum vesicles. Sec22b is involved in vesicular trafficking that takes place between the endoplasmic reticulum-Golgi intermediate compartment (ER-GIC) and the Golgi apparatus. Sec22b normally binds to three SNARES on the ER-Golgi intermediate compartment or on the Golgi apparatus. It was hypothesized that the N-terminus of SdhB interacts with the SNARE Sec22b; the regions of the N-terminus that are important for this interaction remain to be identified. To investigate potential protein-protein interactions between SdhB and Sec22b in vitro, the C-terminus portion of sdhB was cloned into a glutathione-S-transferase expression vector pGEX-5x-1 to create a protein fusion between glutatione S-transferase (GST) and SdhB. Expression of the GST-SdhB fusion protein in E. coli was optimized, and conditions for binding specific amounts of GST-SdhB from lysates cleared of cell debris onto glutathione-coated beads was determined. Beads previously coated with GST-SdhB were unable to bind His-tagged Sec22b. This was shown in both stained protein gels and by Western blotting. Sec22b was also unable to bind to GST-only controls. Similar results were obtained if Sec22b and the fusion protein underwent simultaneous incubation with the glutathione beads to control for the requirement of pre-association of proteins prior to glutathione binding. These results indicate the L. pneumophila effector protein SdhB C-terminus is not involved in a protein-protein interaction with Sec22b.

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