Biology subjects

Fumeaux, C.

Publications and source records attributed to Fumeaux, C..

3 recordsLinked to original sources

bioRxiv2025

Disruption of undecaprenyl phosphate recycling suppresses ampC beta-lactamase induction in Pseudomonas aeruginosa

Beta-lactam antibiotics are widely used to treat bacterial infections, but their efficacy is compromised by resistance mechanisms such as the production of beta-lactamases. In Pseudomonas aeruginosa, the chromosomally encoded beta-lactamase AmpC is the primary mediator of beta-lactam resistance. ampC expression is regulated by the transcription factor AmpR, which responds to intracellular peptidoglycan (PG) fragments. Under normal conditions, AmpR binds the PG precursor (UDP-MurNAc-pentapeptide, UDP-M5) and represses ampC expression. However, during beta-lactam treatment or in PG recycling-deficient mutants such as ampD mutants, PG degradation products (anhydromuropeptides, AMP) accumulate and activate AmpR, resulting in elevated ampC expression and beta-lactam resistance. We hypothesized that shifting the balance of PG precursors could modulate AmpR activity and suppress beta-lactamase expression, even in derepressed strains. Undecaprenyl phosphate (UndP) is a lipid carrier essential for translocating PG precursors across the bacterial inner membrane. Recent work has identified members of the DedA superfamily as UndP flippases responsible for recycling this lipid carrier. Disruption of UndP recycling leads to cytoplasmic accumulation of UDP-M5, the known AmpR repressor. Here, we show that deletion of dedA4, which encodes a predicted UndP flippase in P. aeruginosa, causes UDP-M5 accumulation and significantly reduces AmpC production and beta-lactam resistance in an ampD mutant. These findings highlight the influence of PG precursor dynamics on beta-lactamase regulation and identify DedA4 as a promising therapeutic target. Inhibiting UndP recycling offers a novel strategy to counteract beta-lactam resistance in P. aeruginosa and potentially other AmpC-producing pathogens. IMPORTANCEBeta-lactam resistance remains a major clinical challenge, often driven by the overexpression of chromosomally encoded beta-lactamases such as AmpC in Pseudomonas aeruginosa. Strategies to limit ampC activation are urgently needed to preserve the efficacy of these frontline antibiotics. Our study identifies DedA4, a membrane protein involved in undecaprenyl phosphate (UndP) recycling, as a potential target for such intervention. Inactivation of DedA4 disrupts UndP transport, leading to the accumulation of soluble, membrane-unbound PG precursors which act as corepressors of AmpC expression. This disruption in the balance between cell wall synthesis and turnover restores susceptibility to {beta}-lactams, even in strains harboring clinically significant resistance mutations such as those in ampD or dacB. Targeting UndP flippases like DedA4 could therefore represent a novel adjuvant strategy to combat AmpC-mediated beta-lactam resistance.

microbiology↗

bioRxiv2023

A potential space making role for a lytic transglycosylase in cell wall biogenesis revealed by a beta-lactamase induction phenotype in Pseudomonas aeruginosa

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by fragments of the peptidoglycan (PG) cell wall released upon beta-lactam treatment. These drugs target transpeptidase enzymes that form cell wall crosslinks. However, they do not block the activity of the transglycosylases that polymerize the glycan chains. Thus, drug-treated cells produce uncrosslinked PG polymers that have been shown to be rapidly degraded in the related gram-negative bacterium Escherichia coli. This degradation is performed by enzymes called lytic transglycosylases (LTs), which generate the anhydro-muropeptide (AMP) products sensed by the AmpR regulator that activates ampC expression. To identify factors required for proper PG biogenesis in P. aeruginosa, we used a reporter gene fusion to the ampC promoter to screen for mutants induced for ampC expression in the absence of drug. To our surprise, we found that inactivation of SltB1, an LT enzyme expected to produce the AMP products required for ampC induction, counterintuitively led to elevated ampC expression. This induction required another LT enzyme called MltG, suggesting that inactivation of SltB1 reduces the efficiency of PG crosslinking, causing the degradation of a subset of nascent PG strands by MltG to generate the inducing signal. Our results therefore support a model in which SltB1 uses its LT activity to open space in the PG matrix for the efficient insertion of new material, a function commonly thought to be restricted to endopeptidases that cut cell wall crosslinks. IMPORTANCEInducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, an ampC induction phenotype for a mutant lacking an enzyme that cleaves cell wall glycans was used to uncover a potential role for glycan cleavage in making space in the wall matrix for the insertion of new material during cell growth.

microbiology↗

bioRxiv2016

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in Alpha-Proteobacteria

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of -proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5-GANTC-3 sequence motifs that are methylated by he DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different -proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.\n\nAuthor SummaryDNA methylation is the post-replicative addition of a methyl group to a base by a methyltransferase that recognise a specific sequence, and represents an epigenetic regulatory mechanism in both eukaryotes and prokaryotes. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. CcrM is a conserved, cell cycle regulated adenine methyltransferase that methylates GANTC sites in -proteobacteria. N6-methyl-adenine (m6A) patterns generated by CcrM can change the affinity of a given DNA-binding protein for its target sequence, and therefore affect gene expression. Here, we combine restriction enzyme cleavage-deep sequencing (REC-Seq) with SMRT sequencing to identify hypomethylated 5-GANTC-3 (GANTCs) in -proteobacterial genomes instated by conserved cell cycle factors. By comparing SMRT and REC-Seq data with chromatin immunoprecipitation-deep sequencing data (ChIP-Seq) we show that a conserved transcriptional regulator, MucR, induces local hypomethylation patterns by occluding GANTCs to the CcrM methylase and we provide evidence that this competition occurs during S-phase, but not in G1-phase cells. Furthermore, we find that environmental signals (such as phosphate depletion) are superimposed to the cell cycle control mechanism and can override the specific hypomethylation pattern imposed by the cell cycle transcriptional circuitry.

microbiology↗