Enterococcus faecalis Adapts to Antimicrobial Conjugated Oligoelectrolytes by Lipid Rearrangement and Differential Expression of Membrane Stress Response Genes.

Gayatri Shankar Chilambi,Scott A Rice,Guillermo C Bazan,Mary B Chan-Park,Xinyi Zhu,Kimberly A Kline,Jamie Hinks,Xianghui Liu,Pei Yi Choo,Artur Matysik

doi:10.3389/fmicb.2020.00155

Abstract

Conjugated oligoelectrolytes (COEs) are emerging antimicrobials with broad spectrum activity against Gram positive and Gram negative bacteria as well as fungi. Our previous in vitro evolution studies using Enterococcus faecalis grown in the presence of two related COEs (COE1-3C and COE1-3Py) led to the emergence of mutants (changes in liaF and liaR) with a moderate 4- to16-fold increased resistance to COEs. The contribution of liaF and liaR mutations to COE resistance was confirmed by complementation of the mutants, which restored sensitivity to COEs. To better understand the cellular target of COEs, and the mechanism of resistance to COEs, transcriptional changes associated with resistance in the evolved mutants were investigated in this study. The differentially transcribed genes encoded membrane transporters, in addition to proteins associated with cell envelope synthesis and stress responses. Genes encoding membrane transport proteins from the ATP binding cassette superfamily were the most significantly induced or repressed in COE tolerant mutants compared to the wild type when exposed to COEs. Additionally, differences in the membrane localization of a lipophilic dye in E. faecalis exposed to COEs suggested that resistance was associated with lipid rearrangement in the cell membrane. The membrane adaptation to COEs in EFC3C and EFC3Py resulted in an improved tolerance to bile salt and sodium chloride stress. Overall, this study showed that bacterial cell membranes are the primary target of COEs and that E. faecalis adapts to membrane interacting COE molecules by both lipid rearrangement and changes in membrane transporter activity. The level of resistance to COEs suggests that E. faecalis does not have a specific response pathway to elicit resistance against these molecules and this is supported by the rather broad and diverse suite of genes that are induced upon COE exposure as well as cross-resistance to membrane perturbing stressors.

Highlights

Enterococci are commensal bacteria commonly found in the gut microflora of humans and animals (Van Tyne and Gilmore, 2014)
The serial passaging of E. faecalis in increasing concentration of COE1-3C and COE1-3Py led to the emergence of low-level resistance to COEs in EFC3C and EFC3Py via adaptive mutations in the liaFSR regulon
The results demonstrate that COE1-3C and COE1-3Py induce a subtly different but general membrane stress response, whereby they modulate the cell envelope–COE interactions as well as defense strategies such as ABC transporter induction

Summary

Introduction

Enterococci are commensal bacteria commonly found in the gut microflora of humans and animals (Van Tyne and Gilmore, 2014). A number of emerging antimicrobial agents in various stages of development are active against Gram positive pathogens including Enterococci. Many of these emerging antimicrobial drugs target cell envelope constituents including lipid II, sortases, lipoteichoic acid, and teichoic acid, among others (van Harten et al, 2017). While many of those compounds are promising as new therapeutics, the development of resistance remains as an issue for both the application of the compounds and for the companies who invest in their development. There is an urgent need to continue to develop new drugs that are active against multidrug resistant (MDR) pathogens but which elicit minimal resistance in target pathogens

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