title: Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.
abstract: Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles.
authors.affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Jayde A
authors.initials: JA
authors.lastname: Gawthorne
authors.affiliation: Institut Pasteur, Paris, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Audry
authors.affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: McQuitty
authors.affiliation: School of Biomedical Sciences, Newcastle University, Newcastle, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Dean
authors.affiliation: Institute of Molecular, Cell, and Systems Biology, University of Glasgow, Glasgow, United Kingdom.
authors.forename: John M
authors.initials: JM
authors.lastname: Christie
authors.affiliation: Institut Pasteur, Paris, France.
authors.forename: Jost
authors.initials: J
authors.lastname: Enninga
authors.affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom andrew.roe@glasgow.ac.uk.
authors.forename: Andrew J
authors.identifier: http://orcid.org/0000-0003-3698-6134
authors.initials: AJ
authors.lastname: Roe
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D061848:Optical Imaging; D000072417:Protein Domains; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism; D000069296:Type III Secretion Systems / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000069296:Type III Secretion Systems; C087447:ipaB protein, Shigella