Intracellular pHluorin as Sensor for Easy Assessment of Bacteriocin-Induced Membrane-Damage in Listeria monocytogenes.

Peter Crauwels,Dzung B Diep,Christian U Riedel,Dominik Weixler,Nadav S Bar,Gerd M Seibold,Leonie Schäfer

doi:10.3389/fmicb.2018.03038

Abstract

Bacteriocins are antimicrobial peptides naturally produced by many bacteria and were shown to be effective against various pathogens including Listeria monocytogenes. L. monocytogenes is a food-borne pathogen that frequently causes disease outbreaks around the world with fatal outcomes in at-risk individuals. Thus, bacteriocins are a promising solution to prevent contaminations with L. monocytogenes and other microorganisms during food production and preservation. In the present study, we constructed L. monocytogenes EGD-e/pNZ-Phelp-pHluorin, a strain that constitutively expresses the pH-sensitive fluorescent protein pHluorin, as a sensor strain to detect disruption of the pH gradient by the membrane-damaging activity of bacteriocins. The ratiometric fluorescence properties of pHluorin were validated both in crude extracts and permeabilized cells of this sensor strain. L. monocytogenes EGD-e/pNZ-Phelp-pHluorin was used to assess membrane damaging activity of the bacteriocins nisin A and pediocin PA-1 and to determine the minimal concentrations required for full disruption of the pH gradient across the membrane. Moreover, the sensor strain proved useful to analyze the presence of compounds affecting membrane integrity in supernatants of a nisin Z-producing Lactococcus lactis strain at different timepoints during growth. Supernatants of this strain that were active in disrupting the pH gradient across the membrane were also shown to inhibit growth of L. monocytogenes. In summary, the presented results suggest that the generated sensor strain is a convenient, fast and reliable tool to identify and characterize novel bacteriocins and other compounds that target membrane integrity.

Highlights

Listeria monocytogenes is a saprophytic soil organism found in a wide range of habitats as well as a food-spoiling bacterium with the potential to cause life-threatening disease in humans (Vivant et al, 2013; Ferreira et al, 2014)
In order to generate a sensor strain for the detection of membrane damage, a plasmid was constructed based on the broad host-range vector pNZ44 (McGrath et al, 2001) containing the gene for the pH-sensitive protein ratiometric pHluorin (Miesenböck et al, 1998) codon-optimized for L. monocytogenes under the control of the strong, constitutive, synthetic promoter Phelp (Riedel et al, 2007)
PHluorin has been successfully used in B. subtilis both on population and single cell level to study antibacterial effects of weak organic acids to assess their potential as food preservation strategies

Summary

Introduction

Listeria monocytogenes is a saprophytic soil organism found in a wide range of habitats as well as a food-spoiling bacterium with the potential to cause life-threatening disease in humans (Vivant et al, 2013; Ferreira et al, 2014). Despite an increased risk of bacterial contamination with human pathogens such as L. monocytogenes and the associated, far reaching consequences for safety, health, environment and profitability of consumers and producers, the interest in minimally processed foods is steadily increasing. Current approaches to identify, purify and characterize bacteriocins are limited to time- and labor-intensive genome mining and growthdependent assays (Sandiford, 2017; Zou et al, 2018) These approaches do not provide mechanistic information as to whether a bacteriocin is biostatic, biocidal, or bacteriolytic and whether it is able to cause membrane damage or pore formation

Methods

Results

Conclusion