Biology subjects

Berney, M.

Publications and source records attributed to Berney, M..

7 recordsLinked to original sources

bioRxiv2026

A new Persister Enrichment Model in Mycobacterium tuberculosis links multidrug persistence to drug sequencing and maltokinase-dependent carbon distribution.

Prolonged combination chemotherapy is required to cure Tuberculosis, in part because Mycobacterium tuberculosis (Mtb) forms antibiotic-tolerant persister cells that survive exposure to bactericidal drugs. Experimental systems to study multidrug persistence in Mtb remain limited. Here, we describe a rapid and tractable Persister Enrichment Model (PEM) in which removal of glycerol markedly increases the frequency of multidrug-tolerant Mtb cells that survive exposure to high concentrations of bactericidal antibiotics with distinct mechanisms of action. Persister enrichment in PEM depends on carbon source identity rather than carbon abundance and enables direct interrogation at the transition from active growth into multidrug tolerance. Using PEM, we show that the temporal order of isoniazid (INH) and rifampicin (RIF) exposure shapes the magnitude of multidrug persistence. A transposon sequencing screen under combination drug pressure identified maltokinase (mak) as a major determinant of multidrug persistence. Deletion of mak enhanced susceptibility to INH+RIF in PEM and impaired bacterial survival during the persistent phase of infection in mice. Metabolic analyses reveal that loss of mak destabilizes carbon distribution during co-catabolism of glycolytic carbon sources, leading to defective growth and antibiotic persistence. This defect is ameliorated by inactivating mutations in glucose-1-phosphate adenylyltransferase (glgC), which alleviate the buildup of ADP-glucose. Together, these findings establish PEM as a practical platform to study multidrug persistence, reveal that drug sequencing can influence entry into multidrug tolerance, and identify maltokinase-dependent carbon distribution as a metabolic vulnerability that constrains Mtb survival. ImportanceTuberculosis treatment requires prolonged multidrug chemotherapy in part because Mycobacterium tuberculosis forms antibiotic-tolerant persister cells that survive bactericidal drug exposure. However, experimental systems to study multidrug persistence remain limited, and the metabolic mechanisms underlying this phenotype are poorly understood. Here, we establish a rapid and experimentally tractable Persister Enrichment Model (PEM) that enriches multidrug-tolerant M. tuberculosis populations by removing glycerol from culture medium. Using this model, we demonstrate that the order of antibiotic exposure influences multidrug persistence both in vitro and in vivo and identify maltokinase-dependent carbon distribution as a critical determinant of persistence and long-term bacterial survival. These findings provide a new platform for studying multidrug tolerance and reveal carbon buffering pathways as potential therapeutic vulnerabilities that could help shorten tuberculosis treatment.

microbiology↗

bioRxiv2025

A functional amyloid matrix underpins the PDIM-architected corded superstructure of the Mycobacterium tuberculosis biofilm

Bacterial biofilms generate collective properties that cannot be explained by individual cells alone. Yet the structural principles that organize these communities and their relationship to drug tolerance remain poorly understood, including in major human pathogens such as Mycobacterium tuberculosis (Mtb). Here we show that submerged Mtb biofilms form organized multicellular architectures in which bacterial cords are integrated with a composite extracellular matrix. The virulence lipid phthiocerol dimycocerosate (PDIM) governs the higher-order organization of cords, proteinaceous matrix interactions support cohesion and attachment, an amyloid-like component contributes to biofilm integrity and establishment, and the ESX-1 secretion system increases matrix biochemical complexity. Biofilm growth conferred antibiotic tolerance across genetic backgrounds, while PDIM-dependent organization provided additional protection against first-line anti-tuberculosis drugs, most clearly isoniazid. Targeting amyloid assembly with epigallocatechin gallate impaired biofilm establishment without inhibiting planktonic growth. Together, these findings establish a cording-centred framework linking bacterial surface composition, cellular organization and extracellular matrix assembly to antibiotic tolerance, and show how resolving higher-order biofilm architecture can reveal processes amenable to chemical perturbation.

microbiology↗

bioRxiv2025

A bioluminescence-based chemical screen identifies a bactericidal naphthalene scaffold targeting MmpL3 in Mycobacterium abscessus

Mycobacterium abscessus pulmonary disease (Mabs-PD) presents a significant and growing global health threat, particularly in individuals with underlying lung conditions like cystic fibrosis and chronic obstructive pulmonary disease. A key challenge in treating Mabs-PD is the lack of bactericidal antibiotics effective at therapeutically relevant concentrations, underscoring an urgent need for drug discovery. Targeting cell-wall synthesis is a promising approach, as evidenced by the success of broad-spectrum {beta}-lactam antibiotics and the frontline antituberculosis drug isoniazid. However, these agents exhibit limited efficacy against Mabs, often requiring concentrations unachievable in lung tissues. Here, we used a bioluminescence-based whole-cell assay optimized to identify drugs targeting both cell-wall synthesis and the oxidative phosphorylation pathway. Screening a small drug library against Mabs revealed multiple hits, including {beta}-lactam antibiotics, validating the effectiveness of this approach to identify cell wall-targeting agents. Among these, we identified a chemically tractable naphthalene scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, disrupting cell wall integrity, inducing ATP leakage into the extracellular milieu, and uncoupling respiration, predominantly through the cytochrome bcc:aa3 branch. Further chemical optimization resulted in a new derivative exhibiting a nanomolar minimum inhibitory concentration, with potent activity against intracellular Mabs and in a zebrafish model of infection. This study offers a promising scaffold for future therapeutic development and highlights the utility of this approach as a rapid assay platform for identifying bactericidal compounds against Mabs.

microbiology↗

bioRxiv2025

The mycobacterial ATP burst is a lysis artifact and serves as an assay for drug-induced cell wall damage

Antibiotics targeting the mycobacterial cell wall are a cornerstone of tuberculosis treatment. Understanding how these drugs facilitate bacterial killing has important implications for identifying novel therapeutic targets and optimizing drug combinations. Previous studies on mycobacteria using the BacTiter-Glo luminescence assay have reported increased ATP levels after treatment with cell wall inhibitors such as isoniazid, a phenomenon referred to as an ATP burst. This phenomenon has been proposed to contribute to drug-induced killing. In this study, we demonstrate that the ATP burst is not a biological response but rather an experimental artifact resulting from enhanced cell lysis induced by cell wall-targeting drugs. The addition of a bead-beating lysis step prior to luminescence measurement abolishes the ATP burst, enabling a more reliable assessment of intracellular ATP levels with this assay. Our findings thus establish a direct link between the ATP burst and the canonical mechanism of action of cell wall inhibitors. We further demonstrate the utility of this approach as a functional readout for identifying compounds that disrupt the mycobacterial cell wall and for screening synergistic or antagonistic interactions with known cell wall-targeting agents. Together, our results elucidate the mechanistic basis of the ATP burst and provide a valuable tool for antimycobacterial drug discovery and development.

microbiology↗

bioRxiv2023

The PDIM paradox of Mycobacterium tuberculosis: new solutions to a persistent problem

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli. This can be remedied by supplementation with odd-chain fatty acids, cholesterol, or vitamin B12. We developed a much-needed facile and scalable routine assay for PDIM production and show that propionate supplementation enhances the growth of PDIM-producing bacilli and selects against PDIM-negative mutants, analogous to in vivo conditions. Our results solve a major issue in tuberculosis research and exemplify how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

microbiology↗

bioRxiv2021

Mycobacterium tuberculosis H2S functions as a sink to modulate central metabolism, bioenergetics, and drug susceptibility

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.

microbiology↗

bioRxiv2018

Synthesis and degradation of FtsZ determines the first cell division in starved bacteria

In natural environments, microbes are typically non-dividing. Such quiescent cells manage fleeting nutrients and gauge when intra- and extracellular resources permit division. Quantitative prediction of the division event as a function of nutritional status is currently achieved through phenomenological models for nutrient-rich, exponentially growing cultures. Such models, however, cannot predict the first division of cells under limiting nutrient availability. To address this, we analyzed the metabolic capability of starved Escherichia coli that were fed pulsed glucose at defined frequencies. Real-time metabolomics and microfluidic single-cell microscopy revealed unexpected, rapid protein and nucleic acid synthesis already in non-dividing cells. Additionally, the lag time to first division shortened as pulsing frequency increased. Here, we demonstrate that the first division from a non-dividing state occurs when the facilitating protein FtsZ reaches division-supporting concentration. A dynamic model quantitatively relates lag time to FtsZ synthesis from nutrient pulses and its protease-dependent degradation. Consistent with model predictions, lag time shortened when FtsZ synthesis was supplemented or protease inhibitors were added. Lag time prolonged when ftsZ was repressed or FtsZ degradation rate was increased. Thus, we provide a basis to quantitatively predict bacterial division using information about molecular determinants and the nutrient input.

microbiology↗