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.
Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. While normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein Latent Membrane Protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.
The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG and SlrP, that have never before been visualized over time during infection of live cells. Using this platform, we developed a methodology for visualizing effector proteins in primary macrophage cells for the first time and reveal distinct differences in effector defined intracellular niche between primary macrophage and commonly used HeLa and RAW cell lines.
A human herpesvirus, Epstein Barr virus (EBV), establishes lifelong infection through memory B cells and often -leads to lymphoid malignancies and lymphoproliferative syndromes. Although the detailed mechanism of LMP 1 -activity is not clearly known, previous studies indicate that EBV uses the viral latent membrane protein 1 (LMP 1) for B lymphocyte immortalization. Our study demonstrates the fifth transmembrane helix (TM5) of -LMP 1 form homotrimeric complexes. The polar aspartic acid residue (D150) of TM5 embedded in the membrane -mediates the self association of TM5.In vivo and in vitro studies indicate that the trimerization of TM5 plays a key role in constitutive activation of signaling of LMP 1. In addition, we developed small molecule inhibitors -specifically disrupting the TM5 trimerization, suggesting a new strategy for drug development targeting transmembrane protein protein interactions.
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