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

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

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

“…Fluorescence distribution of Ebp staining was quantified as described previously (Chilambi et al, 2020; Kandaswamy et al, 2013). Briefly, mid log phase cells (1.5 µm −2 µm in length) were first detected using a Matlab function, Projected System of Internal Coordinates from Interpolated Contours (PSICIC) (Guberman et al, 2008).…”

Membrane-intercalating conjugated oligoelectrolytes

“…74 Meanwhile, transcriptomic analysis also supported that membrane disruption is a key MoA because genes related to the membrane stress response were differentially expressed in cells that were exposed to MICOE over a long period of time. 75…”

Enterococcal biofilm—A nidus for antibiotic resistance transfer?

“…The search for regulatory systems central to enterococcal biofilm formation and the regulatory systems that control them has been an area of active research (Grand et al, 2019 , 2020 ; Manias & Dunny, 2018 ; Zheng et al, 2017 ). Chilambi et al ( 2020 ) have further extended our understanding of these complex and diverse regulatory systems by reporting on the evolution of Efs in immunocompromised patients finding that vancomycin‐resistant strains adapted during colonization and mutations accumulated that contributed to increased biofilm formation. There has been considerable effort put into defining Efs biofilm regulation in the hope of identifying a central control point that might become a novel antimicrobial drug target.…”