Published: Vol 4, Iss 20, Oct 20, 2014 DOI: 10.21769/BioProtoc.1270 Views: 11037
Reviewed by: Kanika GeraAnonymous reviewer(s)
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Abstract
Many gram-negative bacterial pathogens, including Shigella flexneri, are able to translocate bacterial proteins, dubbed effectors, across the host cell plasma membrane into the host cell cytosol using a syringe-like structure, the type three secretion apparatus (T3SA). While some bacteria use their T3SA to modulate their phagosomal environment (Salmonella spp.), establish pedestal structure to form microcolonies on the plasma membrane (Enteropathogenic Escherichi coli) or lyse their entry vacuole (Shigella spp.), they all have in common a tightly regulated activity of their T3SA. However, the tracking of the activity of the T3SA in infected cells and tissue has been difficult to perform. Using the property of MxiE-dependent promoters that are upregulated when the T3SA is active, we have recently designed a transcription-based secretion activity reporter (TSAR) that allows the following of the activity of the S. flexneri T3SA in real-time in tissue culture cells and in vivo using fast maturing GFP intrinsic fluorescence. Herein we describe the design of the TSAR and its application to fixed and live samples for microscopy and flow cytometry in a colonic epithelial cell model using TC7 tissue culture cells.
Keywords: ShigellaMaterials and Reagents
Equipment
Procedure
Notes
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Acknowledgments
We are grateful to Claude Parsot for his comments on the manuscript. FXCV was a CIHR, EMBO, Marie-Curie-IRG and FRM fellow. PS was an EMBO and Marie Curie-IEF fellow while PJS is a HHMI senior scholar. This work was supported by the ERC (PJS Advanced Grant, no. 232798).
References
Article Information
Copyright
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Campbell-Valois, F. X., Schnupf, P. and Sansonetti, P. J. (2014). Design of a Transcription-based Secretion Activity Reporter (TSAR) for the Type III Secretion Apparatus of Shigella flexneri and Uses Thereof. Bio-protocol 4(20): e1270. DOI: 10.21769/BioProtoc.1270.
Category
Microbiology > Microbial biochemistry > Protein
Molecular Biology > Protein > Expression
Cell Biology > Cell imaging > Fixed-cell imaging
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