Date of Award
Doctor of Philosophy
Dr. John Geiser
Dr. Bruce Bejcek
Dr. Silvia Rossbach
Dr. Richard VanEnk
Yersinia species use a type three secretion system (TTSS) to inject Yersinia outer proteins (Yops) into the target cell. The effector YopO is a serine/threonine kinase which upon association with actin becomes active. However, activation of the kinase is not required for actin disruption. YopO also contains a Rho/Rac binding domain, but it is unclear how it affects these proteins.
We have established an expression system in Saccharomyces cerevisiae to study YopO. The expression vector contains a GAL1 promoter for inducible expression of YopO as well as a V5 epitope used for visualization of each Yop. We have shown that YopO is lethal in yeast. YopO containing strains were visualized using immunofluorescent microscopy which showed YopO localization near the cell membrane. YopO exerts an effect over the actin cytoskeleton, causing a loss of actin cables and a decrease in the amount of cortical patches by hour three post induction.
Co-immunoprecipitation assays were used to elucidate the cellular targets of YopO. Utilizing the V5 epitope present on the YopO expression vector, and an HA epitope present on the Rho construct expression vector, we were able to precipitate the protein complex. The co-immunoprecipitation assay showed that Rho1, Rho2, Rho3, Rho5 and Cdc42 all bind physically to YopO.
Nejedlik, Laura, "Construction of a New Model System in Saccharomyces Cerevisa to Study YopO and Identify Its Targets" (2008). Dissertations. 800.