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

Lookup NU author(s): Dr Paul Dean

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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 theEscherichia 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.

Publication metadata

Author(s): Gawthorne JA, Audry L, McQuitty C, Dean P, Christie JM, Enninga J, Roe AJ

Publication type: Article

Publication status: Published

Journal: Applied and Environmental Microbiology

Year: 2016

Volume: 82

Issue: 9

Pages: 2700-2708

Print publication date: 01/05/2016

Online publication date: 26/02/2016

Acceptance date: 26/02/2016

ISSN (print): 0099-2240

ISSN (electronic): 1098-5336

Publisher: American Society for Microbiology

URL: http://dx.doi.org/10.1128/AEM.03418-15

DOI: 10.1128/AEM.03418-15

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