Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

Jayde A Gawthorne,Jost Enninga,John M Christie,Laurent Audry,Claire Mcquitty,Paul Dean,Andrew J Roe

doi:10.1128/aem.03418-15

Jayde A Gawthorne, Jost Enninga + Show 5 more

Open Access

https://doi.org/10.1128/aem.03418-15

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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.

Highlights

The use of innovative imaging assays and probes to study the cellular microbiology of effector proteins is becoming commonplace as limitations in image capture, data processing, and suitable probes are overcome
The promoter region and coding sequence for the enterohemorrhagic E. coli (EHEC) effector proteins translocated intimin receptor (Tir) and mitochondrial associated protein (Map) were cloned from strain TUV93-0 into the pJAG03 backbone to create pJAG13 and pCMQ1, respectively, as described in Materials and Methods
To allow bacterial imaging without the need for antibody staining, the gene encoding a red fluorescent protein (RFP) optimized for bacterial expression was stably integrated into the EHEC genome in place of lacZ

Summary

Introduction

The use of innovative imaging assays and probes to study the cellular microbiology of effector proteins is becoming commonplace as limitations in image capture, data processing, and suitable probes are overcome. Translocation of an effector fused to TEM-1 induces catalytic cleavage of the CCF2 ␤-lactam ring, affecting the FRET This produces a detectable and measurable change in CCF2 fluorescence from green to blue emission. The use of a split-green fluorescent protein (GFP) system [6] overcomes the limitation of the type III secretion system (T3SS) to secrete partially folded polypeptides by fusing part of the GFP fluorophore to the effector and expressing the remaining component in the host cell. The two halves are united to form a functional molecule suitable for immunofluorescence, as demonstrated for Salmonella SPI2 effectors [6] The use of such a system may provide spatial information as the final localization can be determined, a marked advantage compared to the FRET-based system described above.

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