Paradis-Bleau, C. | bioRxiv Science

bioRxiv2025

High-throughput LacZ/CPRG screen identifies novel potential antibiotics targeting Gram-negative bacterial envelopes to combat resistance

BackgroundBacterial resistance, exacerbated by multidrug-resistant Gram-negative (GN) pathogens, poses a public health threat due to their impermeable envelopes, which block many antibiotics. ObjectivesWe aimed to develop a high-throughput screening (HTS) method to identify small molecules targeting GN bacterial envelopes and assess their antibacterial potential. MethodsEnvelope disruption in Escherichia coli and Pseudomonas aeruginosa was assessed using a {beta}-galactosidase (LacZ)/CPRG reporter assay in LB at 37{degrees}C. The assay was validated through screening the LOPAC1280 and KD24761 compound libraries. Concentration-response relationships, permeabilisation constants (K50), co-permeabilisation assays, minimal inhibitory concentration (MIC) measurements, and bacterial microscopy post-MICs were performed. ResultsThe assay demonstrated robust performance, evidenced by high Z-factor and signal-to-noise (S/N ratios. Screening identified 57 active compounds (1.2% of the library), including {beta}-lactams and three non-antibiotic molecules--suloctidil, isorotenone, and alexidine--that exhibited concentration-dependent antibacterial activity. Alexidine showed the most potent activity, with the lowest K50 (2.7x10-3 mM) and MICs of 0.004 mM for E. coli and 0.015 mM for P. aeruginosa. Suloctidil and isorotenone induced spherical cell morphology, while alexidine induced a filamentous phenotype, indicative of envelope disruption. The assay also identified antibiotics for monotherapy and combination therapy, with ampicillin, alexidine, and suloctidil enhancing chloramphenicols efficacy against E. coli MG1655. ConclusionsThe LacZ/CPRG reporter assay effectively identified compounds targeting bacterial envelopes, including novel molecules with antibacterial activity against GN pathogens, making it a promising tool for antibiotic discovery or combination therapy.

microbiology↗

bioRxiv2024

Phenotypic and Transcriptomic Characterization of ElyC-Defective Escherichia coli Cells Reveal the Importance of ElyC in Cell Envelope Biology at Optimal and Sub-Optimal Temperatures

The bacterial cell envelope acts as the frontline defense against environmental and internal stress, maintaining cellular homeostasis. Understanding envelope biology is crucial for both fundamental research and practical applications. Peptidoglycan (PG) is a key structural element, protecting against mechanical and osmotic stress while maintaining cell shape and integrity. In a previous study, we discovered the importance of ElyC, a highly conserved Escherichia coli protein with an unknown function, in maintaining envelope integrity at low temperatures. ElyC is essential for PG assembly at 21{degrees}C and plays a role in lipid carrier metabolism, a crucial step in PG and other bacterial envelope polysaccharide biosynthesis. At 21{degrees}C, ElyC deficiency leads to complete PG assembly blockage and cell lysis. However, the significance of ElyC in cells grown at 37{degrees}C remained unexplored. In our recent study, we conducted phenotypic and transcriptomic profiling of ElyC-defective E. coli cells grown at 37{degrees}C and 21{degrees}C, compared to wild-type cells. While{Delta} elyC mutant cells grow similarly to wild-type cells at 37{degrees}C, microscopy revealed altered cell morphology due to ElyCs absence. PG quantification confirmed significantly inhibited PG biosynthesis at 37{degrees}C without ElyC, and these mutants showed increased sensitivity to PG-targeting {beta}-lactam antibiotics compared to wild-type cells at the same temperature. RNA-Seq analysis of{Delta} elyC mutant and WT strains at 21{degrees}C and 37{degrees}C revealed that ElyC deletion severely affects the cell envelope at 21{degrees}C and moderately at 37{degrees}C. Several pathways and genes, especially stress response pathways, impact cell envelope functions, including biogenesis, maintenance, repair, metabolism, respiratory chain, peptidoglycan, lipopolysaccharide, membrane, cell wall, oxidative stress, osmotic stress, trehalose, chaperone, oxidoreductase, amino sugar synthesis and metabolism, vancomycin and beta-lactam resistance pathways and are affected. Downregulated transcripts are associated with mobility, arginine metabolism, membrane transport, regulation, outer membrane, transferase, and unknown functions. Our data highlights ElyCs broad role in bacterial cell envelope and peptidoglycan biosynthesis at varying temperatures. IMPORTANCEThe molecular pathways governing bacterial envelope biosynthesis, assembly, regulation, and adaptation remain incompletely understood. Envelope biology is vital for both fundamental microbiological research and the development of novel therapeutic targets. We previously established ElyCs role in sub-optimal temperature envelope biology, showing its essentiality for PG assembly and bacterial survival at 21{degrees}C. In this study, we show that ElyC, a protein containing the highly conserved DUF218 domain of unknown function, is crucial for proper cell morphology, PG biosynthesis, antibiotic tolerance and envelope homeostasis at 37{degrees}C. Our findings emphasize the significance of DUF218-containing ElyC in envelope biology at physiological temperatures and uncover a novel cold-sensitive process in bacterial envelope biology.

microbiology↗