Testing the Association of SpcU and ExoU with the Type Three Secretion System Loading Platform of Pseudomonas aeruginosa via Two Hybrid Analysis

One mechanism used by Pseudomonas aeruginosa to promote infection of eukaryotic host organisms is the type three secretion system. The Type III secretion system is also used by other types of gram- negative pathogenic bacteria. The type three secretion system is made up of about 20 different protein subunits. Recently, three proteins, PscK, PscL, and PscQ, were proposed to form a loading platform. We are testing whether homologs of the three proposed loading platform proteins found in Pseudomonas aeruginosa form a complex and whether the chaperone protein SpcU and the effector ExoU interact with any of the proposed loading platform proteins. To test their interaction, we are using the yeast two hybrid system. For those experiments, we first cloned each of the genes for the three platform proteins into yeast plasmids. Preliminary results with the constructed plasmids have suggested that PscL and PscQ, and PscL and SpcU, may interact. However, we have encountered some technical problems with the assay that may be preventing the detection of interaction. These results would suggest that PscK is not part of the complex, however, that would need to be confirmed by other methods