Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins.

Vera Göser,Viktoria Liss,Michael Hensel,Carina Kommnick,Reinhard Fischer

doi:10.1128/mbio.00769-19

Abstract

Type III secretion systems (T3SS) are molecular machines in Gram-negative 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 host-pathogen 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.

Highlights

Type III secretion systems (T3SS) are molecular machines in Gramnegative pathogens that translocate effector proteins with central roles in virulence
The Salmonella pathogenicity island 1 (SPI1)-encoded T3SS translocates a set of preformed effector proteins mediating host cell invasion, while the Salmonella
The strong labeling of effector-self-labeling enzymes (SLE) fusion proteins in bacterial cells observed in this study suggests that the SLE moiety is enzymatically active in self-labeling of preformed proteins in the bacterial cytosol

Summary

Introduction

Type III secretion systems (T3SS) are molecular machines in Gramnegative pathogens that translocate effector proteins with central roles in virulence. 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. 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. 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. To localize effector proteins in host cells, direct detection by immunolabeling of the effector protein or recombinantly introduced epitope tags has been frequently used This approach, is restricted to fixed, permeabilized cells and cannot address the kinetics and dynamics of effector translocation and distribution. Other tags have been used to follow effector protein translocation, such as the tetracysteine tag in complex with biarsenic dyes [4, 5]

Methods

Results

Conclusion