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

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...

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Autores principales:	Vera Göser, Carina Kommnick, Viktoria Liss, Michael Hensel
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Salmonella enterica cell invasion facultative intracellular pathogens live-cell imaging super-resolution microscopy type III secretion system Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/e838e318789a44058cde6487b1f4be13
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spelling	oai:doaj.org-article:e838e318789a44058cde6487b1f4be132021-11-15T15:55:24ZSelf-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins10.1128/mBio.00769-192150-7511https://doaj.org/article/e838e318789a44058cde6487b1f4be132019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00769-19https://doaj.org/toc/2150-7511ABSTRACT 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.Vera GöserCarina KommnickViktoria LissMichael HenselAmerican Society for MicrobiologyarticleSalmonella entericacell invasionfacultative intracellular pathogenslive-cell imagingsuper-resolution microscopytype III secretion systemMicrobiologyQR1-502ENmBio, Vol 10, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Salmonella enterica cell invasion facultative intracellular pathogens live-cell imaging super-resolution microscopy type III secretion system Microbiology QR1-502
spellingShingle	Salmonella enterica cell invasion facultative intracellular pathogens live-cell imaging super-resolution microscopy type III secretion system Microbiology QR1-502 Vera Göser Carina Kommnick Viktoria Liss Michael Hensel Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
description	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.
format	article
author	Vera Göser Carina Kommnick Viktoria Liss Michael Hensel
author_facet	Vera Göser Carina Kommnick Viktoria Liss Michael Hensel
author_sort	Vera Göser
title	Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
title_short	Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
title_full	Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
title_fullStr	Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
title_full_unstemmed	Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins
title_sort	self-labeling enzyme tags for analyses of translocation of type iii secretion system effector proteins
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/e838e318789a44058cde6487b1f4be13
work_keys_str_mv	AT veragoser selflabelingenzymetagsforanalysesoftranslocationoftypeiiisecretionsystemeffectorproteins AT carinakommnick selflabelingenzymetagsforanalysesoftranslocationoftypeiiisecretionsystemeffectorproteins AT viktorialiss selflabelingenzymetagsforanalysesoftranslocationoftypeiiisecretionsystemeffectorproteins AT michaelhensel selflabelingenzymetagsforanalysesoftranslocationoftypeiiisecretionsystemeffectorproteins
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Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins

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