Defining the spatiotemporal dynamics of Salmonella type-III secretion system effectors

The Type-III Secretion System (T3SS) is a sophisticated macromolecular machine enabling pathogenic bacteria to inject proteins into eukaryotic host cells. Salmonella enterica species encode two such T3SSs and translocate over 60 individual proteins into host cells. Upon protein entry, T3SS effectors work cooperatively to hijack cellular signaling pathways and reprogram the host cell to enable bacterial survival. Unfortunately, our understanding of the "when and where" of T3SS mediated protein injection has been hindered by a lack of live-cell methods to study the dynamics of this host-pathogen interaction. This is in part due to the requirement of unfolded T3SS substrates prior to injection, precluding tagging with Green Fluorescent Protein (GFP). We have described the use of a tetracysteine peptide tag in conjunction with the biarsenical small molecule FlAsH to fluorescently label T3SS proteins expressed in Salmonella. We have demonstrated that this methodology is minimally perturbing to T3SS function and allows for quantification of the rates of protein secretion into host cells using live-cell fluorescence microscopy. While useful for measuring translocation rates (e.g. "when" substrates are secreted), this method is less adequate for monitoring bacterial protein localization and trafficking within the host cell. To address the "where" of bacterial protein localization post-injection, we have adapted a split GFP system which allows for live-cell monitoring of effector protein dynamics. Together these complementary tools have enabled quantitative measurements of the "when and where" of Salmonella host-pathogen interactions.