Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
|cell invasion; facultative intracellular pathogens; INTRACELLULAR SALMONELLA; live-cell imaging; LIVING CELLS; LOCALIZATION; Microbiology; PLASMID; Salmonella enterica; SALMONELLA PATHOGENICITY ISLAND-2; super-resolution microscopy; SUPERRESOLUTION MICROSCOPY; type III secretion system
|AMER SOC MICROBIOLOGY
Type III secretion systems (T3SS) are molecular machines in Gramnegative pathogens that translocate effector proteins with central roles in virulence. The analyses of the translocation, subcellular localization, and mode of action of T3SS effector proteins are of central importance for the understanding of hostpathogen interaction and pathogenesis of bacterial infections. The analysis of translocation requires dedicated techniques to address the temporal and spatial dynamics of translocation. Here we describe a novel approach to deploy self-labeling enzymes (SLE) as universal tags for localization and tracking of translocated effector proteins. Effector-SLE fusion proteins allow live-cell imaging of translocation by T3SS, superresolution microscopy, and single-molecule tracking of effector motility in living host cells. We describe the application of the approach to T3SS effector proteins for invasion and intracellular lifestyle of Salmonella enterica serovar Typhimurium and to a T3SS effector of Yersinia enterocolitica. The novel approach enables analyses of the role of T3SS in host-pathogen interaction at the highest temporal and spatial resolution, toward understanding the molecular mechanisms of their effector proteins. IMPORTANCE Type III secretion systems mediate translocation of effector proteins into mammalian cells. These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. The new approach of self-labeling enzyme tags fusions will enable analyses of type III secretion system effector proteins with new dimensions of temporal and spatial resolution.
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checked on Mar 2, 2024