Visualizing translocation and localization of bacterial type III effector proteins using a genetically encoded reporter system

Gawthorne, J. A., Audry, L., McQuitty, C., Dean, P., Christie, J. M. , Enninga, J. and Roe, A. J. (2016) Visualizing translocation and localization of bacterial type III effector proteins using a genetically encoded reporter system. Applied and Environmental Microbiology, 82(9), pp. 2700-2708. (doi: 10.1128/AEM.03418-15) (PMID:25576618) (PMCID:PMC4345375)

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Abstract

Bacterial Type Three 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gawthorne, Ms Jayde and Christie, Professor John and Roe, Professor Andrew
Authors: Gawthorne, J. A., Audry, L., McQuitty, C., Dean, P., Christie, J. M., Enninga, J., and Roe, A. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Applied and Environmental Microbiology
Publisher:American Society for Microbiology.
ISSN:0099-2240
ISSN (Online):1098-5336
Published Online:26 February 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Applied and Environmental Microbiology 82(9):2700-2708
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
535461Utility of LOV-based Fluorescent Reporters To Monitor the Real-Time Secretion and Localisation of Bacterial Effector ProteinsAndrew RoeBiotechnology and Biological Sciences Research Council (BBSRC)BB/H023518/1III - BACTERIOLOGY