Abstract Type III secretion systems (T3SS) are intricate molecular machines that allow Gram-negative pathogens to directly inject effector proteins into host cells. While the arsenal of injected effector proteins varies among pathogens and supports a wide variety of pathogenic lifestyles, the core apparatus is conserved. A key feature of T3SSs is that effector translocation is triggered by host-cell contact. In order to inject proteins, the bacterium attaches to the host cell and uses the T3SS to insert pore-forming translocator proteins into the host cell plasma membrane. The tip of the needle is docked to this pore, forming the translocon. Host cell contact is sensed by a conformational change in the pore that is propagated to the base of the apparatus. While there is ample evidence that translocon assembly and function are modulated by the host cell, which cellular functions are involved, and what step in the translocation process they influence is poorly understood. We are proposing to perform a genome wide saturation mutagenesis selection in a haploid human cell line to identify cellular pathways that impact effector translocation by the Pseudomonas aeruginosa type III secretion system. We will employ a transposon that also harbors a constitutive promoter and splice donor site, which will allow us to not only identify loss of function mutations, but also gain of function mutations resulting from artificial production of a downstream gene. Moreover, we will take advantage of the P. aeruginosa system and perform the selection with either wild type bacteria or a mutant strain that does not require a specific host cell trigger to commence effector secretion. This will allow us to isolate the host cell process that triggers effector secretion. To achieve these goals, the application is divided into two specific aims. Aim 1 encompasses the selection for transposon insertion mutations that confer resistance to the P. aeruginosa T3SS, as well as the high throughput sequencing and analysis needed to identify candidate hits. In Aim 2 we will validate mutations we identify. Taken together this work will shed light on an understudied aspect of the type III secretion process: the contribution of the host cell to translocon function. By identifying both loss- and gain of function mutants, and by performing the selection in a haploid cell line, this project will be more comprehensive than any study performed to date. The design of the experiments also allows us to specifically identify factors contributing to triggering of effector secretion on host cell contact, a hallmark feature of type III secretion systems that remains a mystery to this day.