Biology subjects

Corona, F.

Publications and source records attributed to Corona, F..

10 recordsLinked to original sources

bioRxiv2026

Single-site DNA methylation activates retron defence by enabling effector tetramerisation

Retrons are unique among bacterial defence systems in using DNA to sense phage infection. They achieve this by producing multicopy single-stranded DNA (msDNA) that monitors the activity of DNA-targeting phage proteins. Although we understand how extensive msDNA perturbations such as degradation activate retrons, activation by subtle chemical modifications remains enigmatic. Here, we used a structure-function approach to elucidate how single-site msDNA methylation activates Retron-Sen2. The cryo-EM structure of Retron-Sen2 features RcaT, the most prevalent retron effector, sequestered as an inactive dimer within a four-component assembly of previously undescribed architecture. Methylation by DNA adenine methyltransferase disrupts the RcaT-msDNA interaction and exposes a previously occluded interface that enables RcaT dimers to form tetramers, leading to retron disassembly. We show that RcaT dimer-dimer assembly is essential for its toxicity. To assess whether this is a conserved RcaT feature, we characterised additional homologues. RcaTs capable of tetramerisation or higher-order oligomerisation are toxic and form distinct phylogenetic clades from RcaTs that remain dimeric and are non-toxic. Together, our findings provide a paradigm for retron activation by single-site msDNA modifications and showcase how variation in an intrinsic protein property can diversify the toxicity of the same bacterial defence effector during evolution.

microbiology↗

bioRxiv2025

Elucidating the mechanisms of action and evolutionary history of phage anti-defence proteins

Phages and bacteria are locked in a molecular arms race, with phage anti-defence proteins (ADPs) enabling them to evade bacterial immune systems. To streamline access to information on ADPs, we developed the Encyclopaedia of Viral Anti-DefencE Systems (EVADES), an online resource containing sequences, structures, protein family annotations, and mechanisms of action (MoA) for 268 ADPs. Through computational structural analysis we predicted MoAs for 21 uncharacterised ADPs. We demonstrate the utility of EVADES by exploring different characteristics of ADPs: (i) DNA mimic ADPs exhibit broad-spectrum activity against bacterial defence systems; (ii) protein sharing across defence systems enables multi-defence inhibitory activity of ADPs; and (iii) eukaryotic dsDNA viruses encode ADP homologs, suggesting conserved immune evasion strategies across domains of life. These results highlight the broader relevance of phage ADPs in understanding interactions between prokaryotic or eukaryotic viruses and their hosts. EVADES is freely accessible at https://www.ebi.ac.uk/finn-srv/evades. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/658234v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@18460corg.highwire.dtl.DTLVardef@19ef231org.highwire.dtl.DTLVardef@2e91a5org.highwire.dtl.DTLVardef@15c2547_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIThe EVADES database aggregates knowledge on phage anti-defence proteins (ADPs), facilitating hypothesis generation. C_LIO_LIMechanisms of action are proposed for 21 uncharacterised ADPs. C_LIO_LIADPs that function as DNA mimics can block diverse bacterial defence systems. C_LIO_LIHomology of targets of ADPs underpin their broad counter-defence capacities and enable prediction of further targets. C_LIO_LIEukaryotic double-stranded DNA viruses employ functional homologs of ADPs to evade host immune responses. C_LI

microbiology↗

bioRxiv2024

Pervasive phosphorylation by phage T7 kinase disarms bacterial defenses

Bacteria and bacteriophages are in a constant arms race to develop bacterial defense and phage counter-defense systems. Currently known phage counter-defense systems are specific to (the activity of) the targeted bacterial defense system. Here, we uncover a mechanism by which the T7 bacteriophage broadly counteracts bacterial defenses using protein phosphorylation. We show that the T7 protein kinase (T7K), which was believed to specifically redirect the function of a few host proteins, is in fact a hyper-promiscuous, dual-specificity kinase enacting a massive wave of phosphorylation on virtually all host and phage proteins during infection. The scale of phosphorylation vastly exceeds the number of previously known phosphorylation events in E. coli, has no sequence motif specificity, and results in a higher proteome-wide phosphorylation density than that of mammalian cells which encode [~] 500 kinases. Stoichiometry analysis of phosphorylation sites revealed a strong bias of T7K activity towards nucleic acid-binding substrates, which we show is mediated by its C-terminal DNA-binding domain. This specificity for highly stoichiometric phosphorylation of nucleic acid-binding proteins enables the deactivation of DNA-targeting or - containing bacterial defense systems. We provide mechanistic insight into how T7K weakens two such defense systems, Retron-Eco9 and DarTG1, through specific phosphorylation events, with single phosphomimetic mutations in key sites of the toxins abolishing defense. Finally, by screening a large collection of E. coli strains, we provide evidence of broad counter-defense capacities for T7K in nature, as strains counteracted contain diverse bacterial defense systems.

microbiology↗

bioRxiv2024

Uncovering nitroxoline activity spectrum, mode of action and resistance across Gram-negative bacteria

Nitroxoline is a bacteriostatic quinoline antibiotic, considered a metal chelator inhibiting the activity of RNA-polymerase1. Its clinical indications are limited to uncomplicated urinary tract infections (UTIs), with a clinical susceptibility breakpoint only available for Escherichia coli2. By testing > 1,000 clinical isolates, here we demonstrate a much broader activity spectrum and species-specific bactericidal activity, including multidrug-resistant Gram-negative bacteria for which therapeutic options are limited due to resistance. By combining systematic genetic and proteomic approaches with direct measurement of intracellular metals, we dissect nitroxoline perturbation of metal homeostasis and unveil additional effects on bacterial physiology. We show that nitroxoline affects outer membrane integrity, synergizing with large-scaffold antibiotics and resensitizing colistin-resistant Enterobacteriaceae in vitro and in vivo. We further characterise resistance mechanisms across E. coli, Acinetobacter baumannii and Klebsiella pneumoniae, recapitulating known E. coli resistance determinants and uncovering novel and conserved mechanisms across species, demonstrating their common effect on nitroxoline efflux.

microbiology↗

bioRxiv2023

Approximations to the solution of the Kushner-Stratonovich equation for the stochastic chemostat

This paper concerns the inverse problem of characterising the state of a bioreactor from observations. In laboratory settings, the bioreactor is represented by a device called a chemostat. We consider a differential description of the evolution of the state of the chemostat under environmental fluctuations. We model the state evolution as a stochastic process driven by Brownian motion. Under this model, the state is described by its probability distribution in time, given the distribution of the initial state. The corresponding probability density function solves a deterministic partial differential equation (PDE), the Kolmogorov forward equation. This description is refined by incorporating observations. More formally, we seek the conditional distribution of the state given an observation process as the solution to a filtering problem, with the corresponding conditional probability density function solving a non-linear stochastic PDE, the Kushner-Stratonovich equation. In numerical studies of bioreactors, the well-posedness of these differential equations is frequently assumed rather than verified. Consequently, it remains unclear whether unique solutions exist and whether numerical approximations preserve key qualitative properties, such as positivity (e.g. non-negative chemical concentrations). This paper establishes the existence and uniqueness of solutions, ensuring well-posedness before presenting numerical approximations. Furthermore, we approximate the pathwise solution to the filtering problem by three approaches: a PDE-based approximation combining finite difference and splitting methods, a classical sequential Monte Carlo method, and a linearisation method. Our results show that the first two provide a richer probabilistic description of the state than the linearisation method, making them suitable for bioreactor control architectures.

bioengineering↗

bioRxiv2022

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs. Author SummaryFree-living bacteria are wrapped by a peptidoglycan cell wall. This envelope provides shape and protects from osmotic stress, while also triggering human immune defense. Chlamydial pathogens live inside human cells and dispensed with a cell wall in their isotonic niche. Instead, they produce a transient peptidoglycan ring that is essentially involved in cell division. Recycling of peptidoglycan is likely important for chlamydiae to maintain a complete cycle of peptidoglycan ring synthesis and cell division. Moreover, the process may help to save energy and to protect from recognition through the immune system. Despite a potentially central role in cell viability and pathogenicity almost nothing is known about recycling of peptidoglycan in chlamydiae. The minimalist organisms lack all enzymes known to catalyze the process in model organism Escherichia coli. Here, we found an NlpC/P60 enzyme to serve a critical step in decomposing peptidoglycan ring derived peptides in Chlamydia trachomatis. Our data indicate that the peptidase recycles energy cost intensive components and breaks down the minimal recognition motif of innate immune factor NOD1. We also identified a natural lead compound to inhibit the NlpC/P60 enzyme opening the way for innovative anti-chlamydial drug development at the intersection of energy recovery, cell division, and immune evasion.

microbiology↗

bioRxiv2022

Peptidoglycan maturation controls spatiotemporal organisation of outer membrane proteins in Escherichia coli

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial to the maintenance of cellular integrity and enable survival in challenging environments1-5. The functionality of the outer membrane relies on outer membrane proteins (OMPs), which are inserted by the {beta}-barrel assembly machine, BAM6, 7. Previous work has shown that growing Escherichia coli cells segregate old OMPs towards the poles by an unknown mechanism8. Here, we demonstrate that peptidoglycan underpins the spatiotemporal organisation of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little impact on its activity, leading to preferential insertion of OMPs at division sites. Synchronising OMP biogenesis to cell wall growth enables bacteria to replenish their OMPs by binary partitioning. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation. This coordination offers new possibilities for the design of antibiotics that disrupt cell envelope integrity.

microbiology↗

bioRxiv2019

The inactivation of enzymes belonging to the central carbon metabolism, a novel mechanism of developing antibiotic resistance

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate (PEP) that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from PEP to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly used in the last years, mainly for treating infections caused by Gram-negative multidrug resistant bacteria as Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to antibiotics of common use. The mechanisms of mutational resistance to fosfomycin in S. maltophilia were studied in the current work. None of the mechanisms so far described for other organisms, which include the production of fosfomycin inactivating enzymes, target modification, induction of alternative peptidoglycan biosynthesis pathway and the impaired entrance of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Rather the unique cause of resistance in the studied mutants is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants showing that neither the inactivation nor the transport of the antibiotic were involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes neither any related enzyme. Finally, the mutants did not present an increased PEP concentration that might compete with fosfomycin for its binding to MurA. Based on these results, we describe a completely novel mechanism of antibiotic resistance based on the remodeling of S. maltophilia metabolism.\n\nSignificanceAntibiotic resistance (AR) has been largely considered as a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated in mechanisms that affect the antibiotics (mutations in transporters, the activity of efflux pumps and antibiotic modifying enzymes) or their targets (i.e.: target mutations, protection or bypass). Usually, AR-associated metabolic changes were considered to be a consequence (fitness costs) and not a cause of AR. Herein, we show that strong alterations in the bacterial metabolism can also be the cause of AR. In the study here presented, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that AR can be under metabolic control, our results provide evidence that AR is intertwined with the bacterial metabolism.

microbiology↗

bioRxiv2018

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding to the cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here we have discovered critical structural features for the interaction of GpsB with peptidoglycan synthases from three different bacteria and demonstrated their importance for cell wall growth and viability. We have used these structural motifs to predict and confirm novel partners of GpsB in Bacillus subtilis, illuminating the role of this key regulator of peptidoglycan synthesis. GpsB thus functions as an adaptor, to mediate the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner. Given the importance of GpsB in pathogenic bacteria, this study has not only revealed mechanistic details of how cell wall synthesis is co-ordinated with the bacterial cell cycle but could also represent a starting point for the design of much needed new antibiotics.

microbiology↗

bioRxiv2018

Biolog phenotype microarray: a tool for the identification of multidrug resistance efflux pumps inducers

Overexpression of multidrug resistance efflux pumps is a relevant mechanism of antibiotic resistance for bacterial pathogens. These systems use to present low levels of basal expression. However, they can be induced by environmental signals or stresses which can lead to situations of phenotypic induced resistance. In contrast to efflux pumps substrates, inducers of these systems have not been thoroughly studied. In this work, we have applied a novel high-throughput methodology in order to identify inducer molecules of the Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that goal, bioreporters in which the expression of the yellow fluorescent protein is linked to the activity of either the smeVWX or the smeYZ promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. Confirmation of induction was carried out measuring YFP production along the bacterial growth and by flow cytometry; mRNA levels of smeV and smeY were also determined by real-time RT-PCR after exposure to the selected compounds. Among the 144 tested compounds, iodoacetate, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and sodium selenite were found to be smeVWX inducers, while boric acid, erythromycin, chloramphenicol and lincomycin are able to trigger the expression of smeYZ. While the presence of the inducers allowed a decrease in the susceptibility to antibiotics that are known substrates of the efflux pumps, our results indicate that these efflux pumps did not contribute to S. maltophilia resistance to the analyzed inducers.\n\nImportanceMultidrug efflux pumps constitute a category of elements involved in the cellular response to stress that is universally represented; from bacteria to human cells. Besides playing basic roles in cell physiology, these elements are critical elements in the resistance to therapeutic agents, including anti-cancer drugs, antifungals and antibiotics. Stable-inheritable resistance is achieved through mutations in regulatory elements that allow overexpression of these systems. However, much less is known on the effectors, or growing conditions, that might induce their expression, leading to a situation of transient-phenotypic resistance, not detectable by current susceptibility tests, unless the inducer in known. Herein we present a methodology amenable for the high-throughput screening of efflux pumps inducers. The use of phenotype microarrays linked to fluorescence reporters have allowed to identify a set of different inducers for smeVWX and smeYZ. Notably, induction seems to be uncoupled from the detoxification of the inducers by the corresponding efflux pumps. The mechanism of action of each of the inducers for inhibiting bacterial growth allowed us to propose that smeVWX is likely induced as a response to thiol-reactive compounds, while smeYZ is induced by ribosome-targeting antimicrobials. Although applied to a specific bacterium, this method is of application to any type of organism and efflux pump, changing the growing conditions in the case of eukaryotic cells. Since the presence of inducers may change the cell response to therapeutic drugs, the identification of these molecules is of clinical relevance.

microbiology↗