Biology subjects

Lebreton, A.

Publications and source records attributed to Lebreton, A..

10 recordsLinked to original sources

bioRxiv2026

Intracellular accumulation of AAV6 genomes governs microglia reactivity and the inflammatory response in retinal gene therapy

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.

neuroscience↗

bioRxiv2025

Virulence priming of Listeria monocytogenes inside replication-permissive endolysosomes

Listeria monocytogenes (Lm), the foodborne pathogen responsible for listeriosis, is well-known for its intracellular lifestyle. After entering epithelial cells, it can evade within minutes from its internalization vacuole into the cytosol, or transiently co-opt and replicate within endomembrane compartments termed epithelial Spacious ListeriA-containing Phagosomes (eSLAPs). Although eSLAPs display endolysosomal features, we bring evidence that they lack cathepsin D activity and are permeated. Indeed, sustained eSLAP membrane perforation by listeriolysin O is crucial for accommodating bacterial growth and vacuole expansion. Conversely, we show that Lm broad range phospholipase C PlcB is the main escape factor from eSLAPs. Unexpectedly, we find that Lm PrfA-dependent cytosolic virulence program is primed inside eSLAPs. As a consequence, the bacteria released from eSLAPs are pre-equipped with the actin assembly-inducing protein ActA and can recruit actin faster. We propose that a delayed access to the cytosol benefit eSLAP-transiting bacteria by favoring their dissemination or evasion from xenophagy.

microbiology↗

bioRxiv2023

Alternative splicing of the stress-sensitive regulator CIRBP sharpens the cellular response to bacterial infection

Cell autonomous responses to intracellular bacteria largely depend on gene expression reorganization. To gain isoform-level resolution into these regulations, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based regulations, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of "poison exons". We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin, or aerolysin) led to the dephosphorylation of SRSF proteins via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.

molecular biology↗

bioRxiv2021

A Comprehensive Phylogenetic and Bioinformatics Survey of Lectins in the Fungal kingdom

Fungal lectins are a large family of glycan-binding proteins, with no enzymatic activity. They play fundamental biological roles in the interactions of fungi with their environment and are found in many different species throughout the fungal kingdom. In particular, their contribution to defence against feeders has been emphasized and extracellular lectins may be involved in the recognition of bacteria, fungal competitors and specific host plants. Their carbohydrate specificities and quaternary structures vary widely, but evidence for an evolutionary relationship within the different classes of lectins is provided by the high degree of amino acid sequence identity shared by the different fungal lectins. The UniLectin3D database contains 194 3D structures of fungal lectins, of which 129 are characterized with their carbohydrate ligand. UniLectin3D lectin classes from all origins were used to construct 107 lectin motifs in 26 folding configurations and to screen 1,223 species deposited in the genomic portal MycoCosm of the Joint Genome Institute. The resulting 33 485 protein sequences of putative lectins are organized in MycoLec, a publicly available and searchable database. The characterization of the lectin candidates in fungal genomes is based on systematic statistics regarding potential carbohydrate ligands, protein lengths, signal peptides, relative motif positions and amino acid compositions of fungal lectins. These results shed light on the evolution of the lectin gene families.

bioinformatics↗

bioRxiv2020

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

microbiology↗

bioRxiv2020

Engineering E. coli for magnetic control and the spatial localization of functions

The fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.

synthetic biology↗

bioRxiv2019

Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues. Author summaryBacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite -and in fact, thanks to-- the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host.

microbiology↗

bioRxiv2019

Coordination of transcriptional and translational regulations in human cells infected by Listeria monocytogenes

The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a timecourse of infection of human intestinal epithelial cells by an epidemic strain of Listeria monocytogenes, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (PABPC1) slowed down the intracellular multiplication of Listeria monocytogenes, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.

molecular biology↗

bioRxiv2019

An RNA-binding protein secreted by Listeria monocytogenes activates RIG-I signaling

Recent studies have reported on the presence of bacterial RNA within or outside extracellular membrane vesicles, possibly as ribonucleoprotein complexes. Proteins that bind and stabilize bacterial RNAs in the extracellular environment have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RNA binding protein that we named Zea. We show that Zea binds and stabilizes a subset of L. monocytogenes RNAs causing their accumulation in the extracellular medium. Furthermore, Zea binds RIG-I, the vertebrate non-self-RNA innate immunity sensor and potentiates RIG-I-signaling leading to interferon {beta} production. By performing in vivo infection, we finally show that Zea modulates L. monocytogenes virulence. Together, this study reveals that bacterial extracellular RNAs and RNA binding proteins can affect the host-pathogen crosstalk.

cell biology↗

bioRxiv2018

PEPOP: new approaches to mimic non-continous epitopes

Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits as replacing a protein difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitope predicted by bioinformatics tools are commonly used and these sequential epitopes are used as such in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools proposed so far to predict peptide sequences: Superficial and PEPOP. PEPOP can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. We have developed an improved version of PEPOP dedicated to answer to the experimentalists need for a tool able to handle proteins and to turn them into peptides. The PEPOP web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature.\n\nWe present the user-friendly, well-structured web-site of PEPOP and its validation through the use of predicted immunogenic or antigenic peptides mimicking discontinuous epitopes in different experimental ways. PEPOP proposes 35 methods of peptide design to guide experimentalists in using peptides potentially capable of replacing the cognate protein in its interaction with an Ab.

bioinformatics↗