Transposon Screen for Yersinia pseudotuberculosis Genes Involved in Activating the HostTranscription Factor NFκB

Yersinia pseudotuberculosis is a bacterial pathogen that infects the human gut. Y. pseudotuberculosis uses a type III secretion system (T3SS) to inject six effector proteins into mammalian cells, where they act to disarm innate immune defenses. The T3SS of Y. pseudotuberculosis lacking the six effector proteins retains some activity against mammalian cells, as such strains trigger activation of the pro-inflammatory transcription factor NFκB, as well as other immune signaling pathways. We hypothesize that this residual activity stems from injection of unknown bacterial molecules inside host cells through the T3SS. In order to identify the Y. pseudotuberculosis genes involved in T3SS-dependent NFκB activation, we developed a library of 4400 Y. pseudotuberculosis transposon mutants lacking the six T3SS effector proteins. We have screened all 4400 mutants for the ability to express a functional T3SS on Petri plates through use of the T3SS indicator dye congo red. In addition, we have screened approximately 1,000 transposon mutants for the ability to trigger NFκB activation in HEK293T NFκB-luciferase reporter cells. We have isolated eight transposon mutants with defects in T3SS-dependent NFκB activation and have determined the location of the transposon insertion for each. One such mutant harbors a transposon insertion in the TatB gene, a component of the Yersinia twin arginine translocation (TAT) system. The Y. pseudotuberculosis TAT secretion system was previously shown to be important for virulence, yet has not been linked to the T3SS. We are currently investigating the nature of the connection between T3SS-dependent NFκB activation and the TAT secretion system.