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Integrated omics-based mechanistical insights into a membrane-targeting antibacterial

ABSTRACT: The continuous risk of antibiotic resistance development underscores the demand for new agents with mechanisms distinct from existing antibacterial drugs. Here, we investigated HSI#6, a small-molecule antibacterial previously identified as a SecA activator, using integrated omics and functional assays. HSI#6 exhibits broad-spectrum bacteriostatic activity and acts through a dual-phase mechanism: transient activation of SecA-dependent secretion followed by membrane perturbation and global stress reprogramming. Time-resolved transcriptomics and proteomics revealed early activation of envelope stress regulons (Cpx, Rcs, Pho), efflux systems, and oxidative stress pathways, followed by suppression of ribosome biogenesis and central metabolism. Comparative analysis and biomarker-based principle component analysis (PCA) positioned HSI#6 within the envelope stress mechanistic space, closely aligned with membrane-active antibiotics yet displaying a distinct signature. Adaptive laboratory evolution (ALE) combined with whole-genome sequencing (WGS) revealed compensatory mutations in topoisomerase 1A gene (topA) and transcriptional regulators, without adaptive resistance emerged even under prolonged selection pressure. These findings establish HSI#6 as a mechanistically unique antibacterial targeting membrane homeostasis with low resistance potential.

ORGANISM(S): Escherichia coli K-12 Escherichia coli O127:H6

PROVIDER: GSE325691 | GEO | 2026/05/22

REPOSITORIES: GEO

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Integrated omics-based mechanistical insights into a membrane-targeting antibacterial

Project description:Here, we investigated HSI#6, a small-molecule antibacterial previously identified as a SecA activator, using integrated omics and functional assays. HSI#6 exhibits broad-spectrum bacteriostatic activity and acts through a dual-phase mechanism: transient activation of SecA-dependent secretion followed by membrane perturbation and global stress reprogramming. Time-resolved transcriptomics and proteomics revealed early activation of envelope stress regulons (Cpx, Rcs, Pho), efflux systems, and oxidative stress pathways, followed by suppression of ribosome biogenesis and central metabolism. Comparative analysis and biomarker-based principle component analysis (PCA) positioned HSI#6 within the envelope stress mechanistic space, closely aligned with membrane-active antibiotics yet displaying a distinct signature. Adaptive laboratory evolution (ALE) combined with whole-genome sequencing (WGS) revealed compensatory mutations in topoisomerase 1A gene (topA) and transcriptional regulators, without adaptive resistance emerged even under prolonged selection pressure. These findings establish HSI#6 as a mechanistically unique antibacterial targeting membrane homeostasis with low resistance potential.

2026-05-22 | PXD075099 | Pride

Project description:Cpx envelope stress sensing in Pseudomonas aeruginosa

| PRJNA1238893 | ENA

A dual-mechanism antimicrobial peptide with antimutagenic activity targets the replisome and induces cell envelope stress

Project description:To combat the growing threat of multidrug-resistant bacteria, we need to develop novel antibiotics with unique modes of action. This study investigates the antibacterial properties of BTP-001 towards Escherichia coli. BTP-001 is a β-clamp targeting antimicrobial peptide containing an AlkB homologu 2 PCNA-interacting motif (APIM) linked to a cell penetrating peptide composed of 11 arginine residues (R11). Our data indicates that R11 mediates energy-dependent transport of BTP-001 across the cell membrane, possibly via iron transport systems such as the TonB-system. The full-length BTP-001 peptide rapidly affects the bacterial membrane, inducing increased expression and activation of the Cpx, E and Rcs cell envelope stress responses, which trigger the production of reaction oxygen species (ROS). This contributes to a rapid bactericidal effect as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, we show that BTP-001 reduces the development of resistance to ciprofloxacin likely by binding to the β-clamp via APIM, thereby inhibiting translesion synthesis. In addition, our multi-omics data suggests that the -clamp is part of ribosomal complexes, and that regulation of translation is targeted by BTP-001. In conclusion, BTP-001 exhibits a multifaceted mode of action which strengthens its potential as a novel therapeutic drug against antibiotic resistant bacteria

2025-08-25 | GSE276254 | GEO

A dual-mechanism antimicrobial peptide with antimutagenic activity targets the replisome and induces cell envelope stress

Project description:To combat the growing threat of multidrug-resistant bacteria, we need to develop novel antibiotics with unique modes of action. This study investigates the antibacterial properties of BTP-001 towards Escherichia coli. BTP-001 is a β-clamp targeting antimicrobial peptide containing an AlkB homologu 2 PCNA-interacting motif (APIM) linked to a cell penetrating peptide composed of 11 arginine residues (R11). Our data indicates that R11 mediates energy-dependent transport of BTP-001 across the cell membrane, possibly via iron transport systems such as the TonB-system. The full-length BTP-001 peptide rapidly affects the bacterial membrane, inducing increased expression and activation of the Cpx, E and Rcs cell envelope stress responses, which trigger the production of reaction oxygen species (ROS). This contributes to a rapid bactericidal effect as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, we show that BTP-001 reduces the development of resistance to ciprofloxacin likely by binding to the β-clamp via APIM, thereby inhibiting translesion synthesis. In addition, our multi-omics data suggests that the -clamp is part of ribosomal complexes, and that regulation of translation is targeted by BTP-001. In conclusion, BTP-001 exhibits a multifaceted mode of action which strengthens its potential as a novel therapeutic drug against antibiotic resistant bacteria.

2025-08-24 | PXD055475 | Pride

Modulation of global transcriptional regulatory networks as a strategy for increasing kanamycin resistance of EF-G mutants

Project description:Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics by the gain of genomic mutations. In our previous work, we used short term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin. We had reported the gain of resistance to kanamycin via multiple different point mutations in the translation elongation factor G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD / cpxA / topA / cyaA. In this work we expand on our understanding of these second site mutations. Using genetic tools we asked how mutations in the cell envelope stress sensor kinase (CpxAF218Y) and adenylate cyclase (CyaAN600Y) could alter their activities to result in resistance. We found that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown non-catalytic role for CyaA in gene expression. From the transcriptomes of double and single mutants we describe the epistasis between EF-G mutant and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result in supercoiling changes in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the mis-regulated genes in the mutants.

2016-07-14 | GSE82343 | GEO

Antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Project description:Antimicrobial resistance (AMR) is a major challenge for global health. Multi-drug treatments can help to reduce AMR and be implemented through the sequential administration of antimicrobials (Roemhild and Schulenburg 2019). Such sequential treatments can achieve high efficacy through negative cellular hysteresis: the antibiotic-induced sensitization of bacterial cells towards a subsequently administered antibiotic (Roemhild et al. 2018). However, to date, the exact characteristics of antibiotic sensitization via negative hysteresis are unknown. Therefore, our study aimed at providing fundamental insights into the nature of hysteresis and its possible applicability to treatment, focusing on the high-risk human pathogen Pseudomonas aeruginosa. We found that negative hysteresis is robustly expressed across the species P. aeruginosa, particularly upon switches from β-lactam antibiotics to aminoglycosides, even if the β-lactam is administered at sub-MIC levels. We further characterized the molecular basis of hysteresis by a transcriptomic analysis of wildtype Pseudomonas PA-14 and a PA-14 CpxS T163P mutant that is described here. We demonstrate that the Cpx envelope stress response system is centrally involved in inducible cellular sensitization. Furthermore, negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions. Overall, our study reveals negative hysteresis to be associated with β-lactam-induced membrane stress, widely expressed across P. aeruginosa, thus identifying this phenomenon as a promising focus for effective antimicrobial therapy.

2026-02-13 | GSE290299 | GEO

Transcriptomes of FA1090,, FA1090cpxA::kan, FA1090cpxR::erm mutant strains of Neisseria gonorrhoeae in late log phase

Project description:To better understand the role of Neisseria gonorrhoeae CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. We identified approximately 139 genes differentially expressed between cpxA (Cpx system active) and cpxR (Cpx system inactive) mutants. A large number of the differentially expressed genes encode envelope-localized proteins.

2016-09-01 | GSE83341 | GEO

Transcription profiling of Escherichia coli over-expressing genes involved in signaling systems that detect and respond to alterations of the bacterial envelope

Project description:The Bae, Cpx, Psp, Rcs and ?E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by i) determining the specificities of each system with respect to signal inducing conditions, and ii) monitoring global transcriptional changes in response to transient over-expression of each of the effectors.

2009-05-31 | E-MEXP-2139 | biostudies-arrayexpress

Stress Responses in Bacteria Induced by a Giant Channel

Project description:Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated trans-envelope multi-protein machineries, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria: type 2 and 3 secretion systems, the type 4 pilus biogenesis system and the filamentous phage assembly-secretion system (FFSS), contain large homologous gated channels in the outer membrane, called secretins. These channels are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum, which blocks the channel in its resting state. By analyzing the transcriptome of Escherichia coli producing the FFSS secretin pIV and an isogenic leaky-gate variant, we have expanded the known secretin responses to include the Sox, Cpx, Rcs and RyhB regulons, in addition to the known secretin responder, the Psp regulon. Furthermore, we show that responses dependent on transcriptional regulators CpxR, SoxS and RcsB (in addition to PspF) are required for survival of E. coli producing the leaky-gate variant of secretin pIV, while CpxR, SoxS and PspF are required for survival of cells producing wild-type T3SS secretin InvG. Our analysis shows that secretins induce three out of six envelope stress responses and responses to oxidative stress. Identification of the key stress response regulators required for survival of secretin stress suggests pathways that may be of interest in developing strategies for control of secretin-expressing pathogenic bacteria.

2021-01-31 | GSE85194 | GEO

Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus [RNA-seq]

Project description:The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (β-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR targets a common operator of divergent tipR and acrAB-nodT promoter in adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that either interfere with DNA-binding by TipR or induce its ClpXP-dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

2023-11-09 | GSE225488 | GEO

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