1. Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System
- Author
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Paul Dean, Jayde A. Gawthorne, John M. Christie, Laurent Audry, Claire McQuitty, Jost Enninga, Andrew J. Roe, University of Glasgow, Institut Pasteur [Paris], Newcastle University [Newcastle], This study was supported by a grant from the BBSRC Tools and Development Fund (BB/H023518=/1) to A.J.R. and J.M.C. C.M. was supported under a BBSRC DTP studentship., and Institut Pasteur [Paris] (IP)
- Subjects
0301 basic medicine ,MESH: Shigella flexneri ,030106 microbiology ,Virulence ,Chromosomal translocation ,MESH: Escherichia coli Proteins ,Biology ,medicine.disease_cause ,Escherichia coli O157 ,Applied Microbiology and Biotechnology ,Type three secretion system ,Shigella flexneri ,03 medical and health sciences ,MESH: Type III Secretion Systems ,Bacterial Proteins ,Protein Domains ,Genes, Reporter ,[SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB] ,medicine ,Type III Secretion Systems ,Methods ,Humans ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Escherichia coli ,MESH: Bacterial Proteins ,Genetics ,Host cell membrane ,MESH: Optical Imaging ,MESH: Humans ,Ecology ,Effector ,C-terminus ,Escherichia coli Proteins ,Optical Imaging ,MESH: Genes, Reporter ,MESH: Host-Pathogen Interactions ,biology.organism_classification ,Cell biology ,030104 developmental biology ,MESH: Escherichia coli O157 ,Host-Pathogen Interactions ,MESH: HeLa Cells ,MESH: Protein Domains ,MESH: Genetic Engineering ,Genetic Engineering ,Food Science ,Biotechnology ,HeLa Cells - 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.
- Published
- 2016