Western Michigan University, Kalamazoo, MI 49008
The bacterium, genus Yersinia, most commonly known for its involvement in bubonic epidemics, has three human pathogenic strains that utilize a Type III Secretion System to harbor an attack on its host. Upon contact with the host’s membrane a needle like appendage is activated to release an invasion of six effector proteins into the host’s cytosol. These proteins called Yops (Yersinia Outer Proteins) perform an array of tasks all geared towards disarming and destroying the cell. Yop T has recently been shown to disorganize the cell’s cytoskeleton by disrupting actin filament formation. At the molecular level, YopT functions as a cysteine protease cleaving Rho family GTPases near the N terminus, thus releasing the RhoGTPase from the membrane inactivating this protein. RhoGTPases are essential signaling mechanism due to their control over cell growth, motility, cytokinesis, and organization of the cytoskeleton. To further investigate the role of YopT, we used the yeast Saccharomyces cerevisiae as our model system. The primary goal of this exploration is to identify suppressors of the lethal YopT phenotype as a step towards understanding the role YopT has within the cell. We used three major processes to create and isolate the spontaneous suppressors, beginning with strain construction, suppressor isolation, and suppressor specification. During strain construction, two separate plasmids were transformed into the diploid yeast JGY 709(Wt). Our plasmids contain a GAL 1 promoter allowing inducible expression of YopT, in which full induction of YopT results in a cytotoxic effect on the yeast. Independent colonies of the transformed strains were picked and grown under permissive conditions followed by plating at the restrictive condition (galactose). Each suppressor strain identified was characterized by a titering assay, tested for temperature sensitivity and examined whether they could suppress other lethal Yops. We have identified 16 suppressor strains of which all but two are YopT specific and five have some degree of temperature sensitivity. Titering assays illustrated that WT or wild type yeast expressing YopT die within two hours after galactose induction. This result is quite contradictory to the suppressor strains, which continuously grew within the five hour testing period. Six of the suppressors show an intermediate growth rate where death occurred within a few hours. Intermediate growth rates may identify these suppressor genes as partially functional. We have successfully isolated haploid strains from each suppressor and have begun examining linkage between each suppressor gene in an effort to identify the gene that has been mutated.
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