Biology subjects

Walter, J.-C.

Publications and source records attributed to Walter, J.-C..

11 recordsLinked to original sources

bioRxiv2025

Bad bugs, new drugs: The antimicrobial peptide C14R is active against the ESKAPE pathogens

The global rise of antimicrobial resistance among the ESKAPE pathogens represents a major challenge to public health. Here, we report the broad-spectrum antibacterial activity of the synthetic antimicrobial and pore-forming peptide C14R against all six ESKAPE species. Using a radial diffusion assay and resazurin-based viability testing, C14R exhibited potent bactericidal effect with minimum inhibitory concentrations (MICs), defined as the lowest concentration of an antimicrobial agent that completely inhibits visible growth of planktonic microorganisms, ranging from 3.4 g/mL (Enterococcus faecium, vancomycin-resistant) to 45.2 g/mL (Klebsiella quasipneumoniae, ESBL). C14R also inhibited biofilm formation by Gram-positive pathogens, with minimum biofilm inhibitory concentrations (MBICs), referring to the minimal concentration required to prevent the development of biofilms, of 15.0 g/mL (Staphylococcus aureus, MRSA) and 22.0 g/mL (E. faecium, VRE), whereas Gram-negatives biofilms showed higher tolerance. Together, these findings demonstrate that C14R retains high activity against multidrug-resistant ESKAPE strains, highlighting its potential as a lead compound for the development of next-generation antimicrobial drugs to expand the portfolio of available antibiotics and brace health systems against emerging severe infections. Author summaryAntibiotic-resistant infections are a growing threat worldwide. A small group of hospital-associated bacteria is especially problematic because they often evade multiple drugs and cause hard-to-treat infections. In this study, we tested the designed antimicrobial peptide C14R as a novel and effective way to fight these bacteria. Peptides are short protein fragments with the ability to puncture and disrupt microbial membranes. We evaluated C14R against six hospital related priority species (so called ESKAPE pathogens) and measured its ability to stop growth and to limit biofilm formation. C14R killed every species we tested and reduced biofilm of two bacteria. Our findings identify C14R as a promising lead for new treatments, particularly for difficult infections and those involving biofilms.

microbiology↗

bioRxiv2025

gFET-based aptasensors technology allows sensitive and specific quantification of the ESKAPE pathogens

The rapid rise of antimicrobial resistance among the ESKAPE pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp., requires diagnostic technologies capable of fast, simplified and sensitive microbial detection. Conventional culture-based diagnostics remain too time-consuming to guide early therapeutic interventions. Here, we present a graphene field-effect transistor (gFET) aptasensor platform enabling rapid, label-free and highly sensitive quantification of all six ESKAPE pathogens. Each gFET device was functionalized with a specific DNA aptamer selected from literature sources and immobilized via a mixed pyrene-based linker strategy on reduced graphene oxide (rGO). Exposure of the functionalized sensors to logarithmically diluted bacterial suspensions (10-105 CFU mL-) produced characteristic and concentration-dependent shifts in source-drain current ({Delta}IDS). For all pathogen-specific aptamers, {Delta}IDS correlated linearly with bacterial load (R2 = 0.90-1.00), while non-target bacteria generated only low-level, unspecific fluctuations. Limits of detection ranged from 10 to 1000 bacterial cells depending on the aptamer. Together, these results demonstrate that aptamer-functionalized rGO-FETs provide a robust, scalable and highly specific electronic biosensing architecture capable of distinguishing clinically relevant multidrug-resistant pathogens with excellent analytical performance. Author summaryAntimicrobial-resistant bacteria pose a growing threat to global health, especially the so-called ESKAPE pathogens, which frequently cause hospital-acquired infections and are increasingly difficult to treat. Current diagnostic methods can take several days, delaying the start of effective therapy. In our work, we developed a fast and highly sensitive biosensor that uses electrically conductive GO and short DNA molecules, called aptamers, to recognize specific bacteria. When a pathogen binds to its matching aptamer on the sensor surface, the electrical signal of the graphene changes in a measurable way. We tested ESKAPE species and showed that all of the investigated aptamers detect only their intended bacterial targets, even at very low concentrations. Importantly, the sensors respond within minutes and do not require any labelling or complex sample preparation. Our technology demonstrates how graphene-based aptasensors can support rapid and accurate detection of dangerous bacterial pathogens and could ultimately help clinicians make faster decisions in treating infections.

microbiology↗

bioRxiv2025

Fast assembly and in vivo coalescence of ParB biocondensates involved in bacterial DNA partition

Faithful DNA segregation in bacteria relies on ParABS systems, in which ParB assembles into condensates at centromere-like parS sites, while the ATPase ParA spatially organizes these complexes. How these ParB condensates maintain dynamic behavior without collapsing into a single structure has remained unclear. Here, we combine chromosome degradation with quantitative imaging to dissect the kinetics and physical principles governing ParB condensate dynamics in vivo. In the absence of the nucleoid, ParB condensates from the plasmid F diffuse freely and coalesce within seconds upon encounter. Strikingly, quantitative analyses indicate that condensates may operate near the fusion-separation boundary, such that minimal energy is sufficient to split them after replication, preventing irreversible coalescence. Using mutants, we demonstrate that condensate assembly is required for coalescence and uncover a dual role for ParAF: nucleoid tethering restricts condensate mobility and limits fusion, while ParAF also promotes a ParBF state competent for assembly and coalescence, likely by enhancing ParB-ParB interactions. Finally, condensates rapidly disassemble and reassemble upon 1,6-hexanediol treatment, underscoring their reversibility and the stabilizing contribution of ParB-DNA interactions. Together, our results establish ParBF complexes as bona-fide biocondensates tuned by ParAF to ensure robust DNA segregation. More broadly, these findings highlight regulated phase separation as a key organizing principle of bacterial replicons.

microbiology↗

bioRxiv2025

The ParB-CTP Cycle Activates Phase Separation in Bacterial DNA Segregation

Cell function relies on liquid-like membraneless organelles formed through phase transitions, yet the mechanisms ensuring their specificity and rapid assembly remain poorly understood. In bacterial chromosome segregation via the ParABS system, hundreds of ParB proteins are recruited around the centromere-like parS sequence forming the partition complex. Recent studies have shown that ParB binds CTP and undergoes cycles of loading and unloading near parS, however, this accounts for the recruitment of only a small fraction of ParB molecules, leaving its role unclear. Separately, a lattice gas model with fixed interaction energy has been proposed to describe ParB phase separation, but it fails to explain key experimental observations, including the absence of droplets in ParB variants. We reconcile these two perspectives by proposing that the ParB-CTP cycle acts as a molecular switch that enhances ParB-ParB interactions, triggering phase transition from vapor to liquid-like condensates. Our hypothesis is supported by numerical simulations of droplet formation and experiments showing that ParB variants disrupting the CTP cycle fail to undergo phase separation. These findings establish a mechanistic framework for ParB-CTP-mediated phase transitions and may have broader implications for understanding the spatial control of intracellular condensate formation.

cell biology↗

bioRxiv2024

In vivo Assembly of Bacterial Partition Condensates on Supercoiled and Linear DNA

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2-Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Lastly, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

molecular biology↗

bioRxiv2023

Chromatin structure from high resolution microscopy: scaling laws and microphase separation

Recent advances in experimental fluorescence microscopy allow high accuracy determination (resolution of 50 nm) of the 3D physical location of multiple (up to [~] 102) tagged regions of the chromosome. We investigate publicly available microscopy data for two loci of the human Chr.21 obtained from multiplexed FISH methods for different cell lines and treatments. Inspired by polymer physics models, our analysis centers around distance distributions between different tags, aiming to unravel the chromatin conformational arrangements. We show that for any specific genomic site, there are (at least) two different conformational arrangements of chromatin, implying coexisting distinct topologies which we refer to as phase and phase {beta}. These two phases show different scaling behaviors: the former is consistent with a crumpled globule while the latter indicates a confined, but more extended conformation, as a looped domain. The identification of these distinct phases sheds light on the coexistence of multiple chromatin topologies and provides insights into the effects of cellular context and/or treatments on chromatin structure.

biophysics↗

bioRxiv2022

Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we used multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc repressed genes. Finally, non-Hox Pc targets are proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not involve clustering of multiple distant Pc genes.

genetics↗

bioRxiv2021

Relaxation time asymmetry in stator dynamics of the bacterial flagellar motor

The bacterial flagellar motor (BFM) is the membrane-embedded rotary molecular motor which turns the flagellum that provides thrust to many bacterial species. This large multimeric complex, composed of a few dozen constituent proteins, has emerged as a hallmark of dynamic subunit exchange. The stator units are inner-membrane ion channels which dynamically bind and unbind to the peptidoglycan at the rotor periphery, consuming the ion motive force (IMF) and applying torque to the rotor when bound. The dynamic exchange is known to be a function of the viscous load on the flagellum, allowing the bacterium to dynamically adapt to its local viscous environment, but the molecular mechanisms of exchange and mechanosensitivity remain to be revealed. Here, by actively perturbing the steady-state stator stoichiometry of individual motors, we reveal a stoichiometry-dependent asymmetry in stator remodeling kinetics. We interrogate the potential effect of next-neighbor interactions and local stator unit depletion and find that neither can explain the observed asymmetry. We then simulate and fit two mechanistically diverse models which recapitulate the asymmetry, finding stator assembly dynamics to be particularly well described by a two-state catch-bond mechanism.

biophysics↗

bioRxiv2020

Physical modeling of a sliding clamp mechanism for the spreading of ParB at short genomic distance from bacterial centromere sites

Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation have recently been shown to belong to a new class of CTP-dependent molecular switches. Strikingly, CTP binding and hydrolysis was shown to induce a conformational change enabling ParB dimers to switch between an open and a closed conformation. This latter conformation clamps ParB dimers on DNA molecules, allowing their diffusion in one dimension along the DNA. It has been proposed that this novel sliding property may explain the spreading capability of ParB over more than 10-Kb from parS centromere sites where ParB is specifically loaded. Here, we modeled such a mechanism as a typical reaction-diffusion system and compared this Clamping & sliding model to the ParB DNA binding pattern from high-resolution ChIP-sequencing data. We found that this mechanism cannot account for all the in vivo characteristics, especially the long range of ParB binding to DNA. In particular, it predicts a strong effect from the presence of a roadblock on the ParB binding pattern that is not observed in ChIP-seq. Moreover, the rapid assembly kinetics observed in vivo after the duplication of parS sites is not easily explained by this mechanism. We propose that Clamping & sliding might explain the ParB spreading pattern at short distances from parS but that another mechanism must apply for ParB recruitment at larger genomic distances.

systems biology↗

bioRxiv2019

ATP-driven separation of liquid phase condensates in bacteria

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

cell biology↗

bioRxiv2018

Robust and conserved stochastic self-assembly mechanism for dynamic ParB-parS partition complexes on bacterial chromosomes and plasmids

Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that Nucleation & caging is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the Nucleation & Caging model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

systems biology↗