Quorum Sensing

Chapter 17

AssessingPseudomonas aeruginosaAutoinducer Effects

on Mammalian Epithelial Cells

Jake Everett, Rebecca Gabrilska, Kendra P. Rumbaugh,

and Elena Vikstro ̈m

Abstract

The human mucosal environment in the gut is rich with interactions between microbiota and mammalian
epithelia. Microbes such as the Gram-negative bacteriumPseudomonas aeruginosamay use quorum sensing
to communicate with other microorganisms and mammalian cells to alter gene expression. Here, we present
methodologies to evaluate the effects ofP. aeruginosa N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-
C 12 -HSL) on Caco-2 cell monolayers. First, we describe a method for assessing barrier function and
permeability of epithelial cells when exposed to 3O-C 12 -HSL by measuring transepithelial electrical
resistance (TER) and paracellular flow using fluorescently labeled dextran. Secondly, we detail methods
to investigate the effect of 3O-C 12 -HSL on protein–protein interactions of epithelial junction proteins.
Lastly, we will detail imaging techniques to visualize Caco-2 barrier disruption following exposure to 3O-
C 12 -HSL through the use of confocal laser scanning microscopy (CLSM) and a super resolution technique,
stimulated emission depletion (STED) microscopy, to achieve nanoscale visualization of Caco-
2 monolayers.

Key wordsHost pathogen interaction,Pseudomonas aeruginosa, Quorum sensing, Acyl homoserine

lactone, Transepithelial electrical resistance, Paracellular permeability, Cell junction, IQGAP1,

Imaging

1 Introduction

Quorum sensing is a cell signaling system whereby microbes secrete

and detect small molecule signals to orchestrate gene expression

based on population density. This system allows Gram-negative

bacteria to use these signals, termed autoinducers, to modulate

growth, virulence, motility, and biofilm formation in a coordinated

fashion.Pseudomonas aeruginosais a model Gram-negative oppor-

tunistic pathogen that utilizes twoN-acyl homoserine lactones as

autoinducers,N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-

C 12 -HSL) and N-butyryl-L-homoserine lactone (C 4 -HSL), to

coordinate these responses. 3O-C 12 -HSL and C 4 -HSL bind to

Livia Leoni and Giordano Rampioni (eds.),Quorum Sensing: Methods and Protocols, Methods in Molecular Biology,
vol. 1673,https://doi.org/10.1007/978-1-4939-7309-5_17,©Springer Science+Business Media LLC 2018

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