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Volume 8
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10.3390/microorganisms8111786
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Article

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

by
Luit Barkalita
,
Athina G. Portaliou
,
Maria S. Loos
,
Biao Yuan
,
Spyridoula Karamanou
and
Anastassios Economou
*
Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Department of Microbiology, Immunology and Transplantation, KU Leuven, B-3000 Leuven, Belgium
*
Author to whom correspondence should be addressed.
Microorganisms 2020, 8(11), 1786; https://doi.org/10.3390/microorganisms8111786
Submission received: 9 October 2020 / Revised: 6 November 2020 / Accepted: 9 November 2020 / Published: 14 November 2020
(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)
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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.
Keywords: EPEC; Type 3 secretion; quantitative in vivo secretion assay; alkaline phosphatase activity; SctA-PhoA EPEC; Type 3 secretion; quantitative in vivo secretion assay; alkaline phosphatase activity; SctA-PhoA

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MDPI and ACS Style

Barkalita, L.; Portaliou, A.G.; Loos, M.S.; Yuan, B.; Karamanou, S.; Economou, A. A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli. Microorganisms 2020, 8, 1786. https://doi.org/10.3390/microorganisms8111786

AMA Style

Barkalita L, Portaliou AG, Loos MS, Yuan B, Karamanou S, Economou A. A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli. Microorganisms. 2020; 8(11):1786. https://doi.org/10.3390/microorganisms8111786

Chicago/Turabian Style

Barkalita, Luit, Athina G. Portaliou, Maria S. Loos, Biao Yuan, Spyridoula Karamanou, and Anastassios Economou. 2020. "A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli" Microorganisms 8, no. 11: 1786. https://doi.org/10.3390/microorganisms8111786

APA Style

Barkalita, L., Portaliou, A. G., Loos, M. S., Yuan, B., Karamanou, S., & Economou, A. (2020). A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli. Microorganisms, 8(11), 1786. https://doi.org/10.3390/microorganisms8111786

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