A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli

Microorganisms
Luit BarkalitaAnastassios Economou

Abstract

The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening.

References

Mar 1, 1991·Journal of Bacteriology·T Michiels, G R Cornelis
Feb 1, 1990·Journal of Bacteriology·C ManoilJ Beckwith
Oct 14, 1970·Journal of Molecular Biology·M Mandel, A Higa
Mar 7, 1997·The Journal of Biological Chemistry·M SoneK Ito
Jul 22, 1997·Proceedings of the National Academy of Sciences of the United States of America·D A Siegele, J C Hu
Feb 11, 1998·Clinical Microbiology Reviews·J P Nataro, J B Kaper
Mar 31, 1999·Proceedings of the National Academy of Sciences of the United States of America·C A RohlR L Baldwin
Apr 12, 2001·Cellular Microbiology·R K ShawS Knutton
Jul 12, 2003·Archives of Microbiology·Hao-Jie ChiuWan-Jr Syu
Feb 28, 2004·Proceedings of the National Academy of Sciences of the United States of America·Wanyin DengB Brett Finlay
Dec 28, 2004·Nature Structural & Molecular Biology·Calvin K YipNatalie C J Strynadka
Mar 21, 2008·Journal of Molecular Biology·Valerie F CrepinSarah J Daniell
Oct 16, 2008·Cell Host & Microbe·Heather B FeliseSamuel I Miller
Nov 19, 2008·Antimicrobial Agents and Chemotherapy·Ning J PanJon D Goguen
Apr 27, 2010·Methods in Molecular Biology·Giorgos GouridisAnastassios Economou
Mar 10, 2011·Science·Oliver Schraidt, Thomas C Marlovits
Aug 2, 2011·Molecular Microbiology·Julie E Button, Jorge E Galán
Dec 14, 2011·Molecular Cell·Li ChenCharalampos G Kalodimos
Jul 14, 2012·Molecular Microbiology·Andreas DiepoldGuy R Cornelis
Dec 10, 2013·Nature Structural & Molecular Biology·Julia RadicsThomas C Marlovits
Feb 4, 2014·FEMS Microbiology Reviews·Andreas Diepold, Samuel Wagner
Feb 12, 2014·Biochimica Et Biophysica Acta·Brianne J Burkinshaw, Natalie C J Strynadka
Sep 12, 2014·Molecular & Cellular Proteomics : MCP·Georgia Orfanoudaki, Anastassios Economou
Oct 13, 2015·Acta Crystallographica. Section F, Structural Biology Communications·Brianne J BurkinshawNatalie C J Strynadka
Nov 2, 2015·Trends in Biochemical Sciences·Athina G PortaliouAnastassios Economou
Nov 29, 2016·Nature Reviews. Microbiology·Alexandra TsirigotakiSpyridoula Karamanou
Dec 25, 2016·Microbial Cell Factories·Kevin James MetcalfDanielle Tullman-Ercek
Apr 11, 2017·Nature Reviews. Microbiology·Wanyin DengB Brett Finlay
Apr 14, 2017·The Journal of Cell Biology·Katerina E ChatziAnastassios Economou
Jun 20, 2017·Structure·Marios Frantzeskos SardisAnastassios Economou

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