Supplementary MaterialsFigures. of tools to study these proteins in the native cellular environment. We report a method to label and track T3SS effectors during infection using a split-GFP system. The breadth of this technique is demonstrated by labeling three effectors from (PipB2, SteA, and SteC) and characterizing their localizations within host cells. PipB2 displays highly dynamic behavior on tubules emanating from the containing vacuole labeled with both endo- and exocytic markers. SteA is preferentially enriched on tubules localizing with Golgi markers. This segregation suggests effector targeting and localization may play a functional role during infection. Introduction causes acute gastroenteritis and systemic typhoid fever in humans. This gram-negative bacterial pathogen establishes a replicative niche within the host, enabled by type-III secretion systems (T3SS) encoded within the genome. The T3SS is a multi-subunit protein complex capable of injecting proteins, termed effectors, directly from bacterial cytosol into host cells 1. Upon translocation, effector proteins, which possess an array of biochemical functions, commandeer sponsor cell signaling to allow bacterial replication and admittance. effectors are classified in two methods, those translocated in to the sponsor cell from the T3SS, encoded on pathogenicity isle-1 (SPI-1) which allow bacterias to gain admittance into sponsor cells and assist in the biogenesis from the nascent Including Vacuole (SCV), and the ones secreted after internalization from the bacteria from the T3SS encoded within pathogenicity isle-2 (SPI-2) which arerequired for SCV maturation, trafficking, and intracellular replication, 2. More than 60 effectors have already been characterized as T3SS substrates and biochemical features have already been ascribed to a subset of the proteins. Emerging proof shows that T3SS effector activity can be controlled both spatially within sponsor cells and temporally by shot/degradation to organize the hijacking of sponsor cell signaling equipment Tenofovir Disoproxil Fumarate 3. Further diversification of effector focusing on can be attained by posttranslational changes of effectors inside the sponsor cell 4-6. This difficulty of effector rules underscores the need for having the ability to straight localize and monitor dynamics of specific TET2 effectors in living contaminated sponsor cells. Numerous research possess localized T3SS effectors in sponsor cells by immunofluorescence at an individual (or little subset) of time points. Unfortunately, immunofluorescence studies require fixation of infected cells which has been shown to perturb effector localization Tenofovir Disoproxil Fumarate 7,8, thus confounding some studies of effector function. A widely used technique for tracking the movement of proteins in living cells is to genetically fuse the protein of interest to a fluorescent protein, such as GFP. Tenofovir Disoproxil Fumarate However, fusion of effector proteins to GFP perturbs secretion, perhaps because GFP cannot be unfolded to fit through the T3SS needle complex 9. Effector proteins fused to GFP have been transiently transfected into host cells to attempt to overcome these limitations 7. However, host cell expressed effectors often display different Tenofovir Disoproxil Fumarate localizations compared with bacterially derived and T3SS delivered effectors 2, highlighting the importance of studying translocated effectors in the presence of the entire effector cohort at a given time post-infection. Recently, methods developed by our laboratory 10 and others 11,12 have enabled the measurement of SPI-1 type-III translocation by time-lapse microscopy. These approaches are not easily adaptable for SPI-2 effectors which are upregulated in the environment of the SCV and only secreted after bacterial internalization. Several techniques exist to measure accumulation of T3SS effectors within infected host cells at distinct time points 13,14. However, these methods necessitate host cell lysis and bulk measurement of effector populations which precludes their make use of in monitoring subcellular dynamics of effectors in specific cells. Currently, no technique exists to label and visualize the dynamics of T3SS directly.