Long-term live-cell imaging reveals new roles forSalmonellaeffector proteins SseG and SteA

Abstract

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models.

Identification of Parameters of Host Cell Vulnerability during Salmonella Infection by Quantitative Image Analysis and Modeling

“…5A). We extracted all these elements using Icy, an image analysis software program (18) recently used for Salmonella infection studies in situ (19) (Fig. S5A gives an illustration of Icy cell segmentation).…”

Optimized Fluorescence Complementation Platform for Visualizing Salmonella Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types

“…We previously found that SteA and PipB2 accumulated on the SCV and membrane tubules in both HeLa and RAW cells 14 . However, primary BMDMs often lack a compact SCV, and instead internalized bacteria are more commonly enclosed within a membrane-bound compartment but spread throughout the cell 7 (Fig. S7), as was observed in infection of human monocyte-derived macrophages 41 .…”

“…The importance of live cell imaging for defining the interface between pathogen and host derives from the observation that isolated snap shots often fail to capture complex dynamic phenotypes such as dispersion and coalescence of the SCV 7 , and that cell fixation can alter infection phenotypes, such as the integrity of membrane tubules that emanate from the SCV 1,8 . The need for single cell resolution was motivated by widely observed heterogeneity in infection phenotypes from cell to cell.…”

“…The need for single cell resolution was motivated by widely observed heterogeneity in infection phenotypes from cell to cell. For example, Salmonella can use different mechanisms to invade individual epithelial cells 2,3,8–10 , can replicate inside the SCV or escape and hyper-replicate in the cytosol of epithelial cells 4,11 , and experience different fates in macrophages 5–7,12 . Collectively, these cell-to-cell variations in infection phenotypes are likely due, at least in part, to the differential presence and function of effector proteins 3,7 , demonstrating the need for techniques that capture effector protein localization and infection phenotypes, while preserving single cell heterogeneity.…”

“…For example, Salmonella can use different mechanisms to invade individual epithelial cells 2,3,8–10 , can replicate inside the SCV or escape and hyper-replicate in the cytosol of epithelial cells 4,11 , and experience different fates in macrophages 5–7,12 . Collectively, these cell-to-cell variations in infection phenotypes are likely due, at least in part, to the differential presence and function of effector proteins 3,7 , demonstrating the need for techniques that capture effector protein localization and infection phenotypes, while preserving single cell heterogeneity. Finally, transient transfection of tagged effector proteins often gives rise to different localization compared to translocated effector proteins 13,14 , and effector proteins work cooperatively to define the host-pathogen interface 2 , underscoring the importance of visualizing translocated effector proteins in the presence of the entire effector cohort.…”