Introduction: Salmonella enterica Typhimurium (STM) relies on the translocation of effector proteins by two type III secretion systems (T3SS). While T3SS-1 is required for invasion of non-phagocytic cells, T3SS-2 allows formation of a replication-permissive niche within host cells. Goals: This project aims to study the translocation, targeting, and interaction of T3SS-1 and T3SS-2 effectors with host structures and to assess the individual contribution of the latter to endosomal remodeling, resulting in the formation of an extended interconnected network of Salmonella-induced filaments (SIF). Materials & Methods: Our recent work demonstrated the applicability of genetically encoded self-labeling enzyme (SLE) tags to investigate the subcellular localization and dynamics of T3SS-2 effector proteins in living cells¹. Here, we further improve this approach by generating different HaloTag variants with improved detection sensitivity and less interference with translocation by T3SS. Additionally, we employ SPLIT-GFP-based contact site sensors for live-cell imaging and super-resolution microscopy to assess the contribution of T3SS-2 to membrane contact sites formed during SIF biogenesis. Results: Volumetric electron microscopy of STM-infected HeLa cells revealed that endosomal remodeling creates novel contact sites between mitochondria and the SIF network. SPLIT-GFP-based contact site sensors are used to assess the function of T3SS effectors in this process. To enable analyses of effector translocation with high spatial and temporal resolution, we identified HaloTag variants with improved T3SS translocation and high signal intensity after ligand binding. The fusion proteins were functional as effectors after translocation into host cells, allowing the analysis of T3SS effector translocation early during infections and monitoring of effector fates. Summary: Here, we present improved tools to study the function and translocation dynamics of STM effector proteins in vivo in real-time during infection.

¹Göser V et al., (2023). Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nat Commun 14(1):1240.