Biology subjects

Depardieu, F.

Publications and source records attributed to Depardieu, F..

10 recordsLinked to original sources

bioRxiv2026

OLD sentinel: an abortive tRNase surveys phage replication and DNA defects in RecBCD-compromised cells

OLD, an abortive immunity protein from prophage P2, consists of an ABC ATPase sensor and a TOPRIM nuclease effector -- a core architecture shared by a large protein family, including components of antiphage systems Gabija, PARIS, Septu, and Lamassu. OLD was originally identified for its lethality in recB-deficient cells and inhibition of bacteriophage {lambda} infection, but the mechanisms governing its activation have remained elusive. Here, we present the cryo-EM structure of an inactive OLD tetramer and show that destabilization into dimeric form opens the TOPRIM catalytic site, stimulating tRNA cleavage. This activity arrests translation, a phenotype rescued by phage-encoded tRNAs. We demonstrate that OLD activation is not strictly RecBCD-dependent: OLD binds aberrant DNA structures in recB-deficient cells, but activation during infection requires recognition of single-stranded DNA hairpins at the phage replication origin. Collectively, our findings reveal how host and phage DNA processing factors create a complex landscape controlling OLD-mediated immunity.

molecular biology↗

bioRxiv2026

Rapid modular evolution of antiviral repertoires in P2 phages and their P4 satellites: shuttling, swapping and mixing

Interactions between bacteria, bacteriophages, and their satellites are shaped by a myriad of defence and counter-defence mechanisms. Here, we identified and characterized the defence hotspots of thousands of P2-like phages and P4-like satellites to elucidate the origins and evolutionary dynamics of defence systems. Both P4 and P2 encode a broad diversity of recognizable defence systems. Defences are a substantial, yet likely underestimated, share of the elements pangenomes, as shown by novel antiviral functions discovered in P4 loci lacking known defence genes. Defence loci are very rapidly swapped, without pseudogenization, suggesting defences are replaced before becoming non-adaptive. This intense local recombination melds components of distinct systems into novel functional chimeras. Systems swap so rapidly that many elements with identical core genes have completely different defences. Surprisingly, despite P4 and P2s concomitant replication and packaging, they almost never exchange defence genes. In contrast, near identical defence systems can be found in distinct types of MGEs and in cryptic chromosomal locations. Our findings highlight P4 and P2 as mobile platforms driving the modular diversification of bacterial antiviral repertoires. Hence, bacterial defences change quickly by phage and satellite turnover, and by the quick swap of defences within these elements.

microbiology↗

bioRxiv2025

Specificity and Mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB effector of the PARIS defence system is a Toprim nuclease previously shown to target the E. coli tRNALys(UUU), but not a tRNALys(UUU) variant encoded by bacteriophage T5. We demonstrate here that the T5-tRNALys(UUU) is required but not sufficient to bypass PARIS immunity. Combining tRNA-sequencing, genetics, phage infection and in vitro biochemical data, we reveal that the E. coli tRNAThr(UGU), is another prime target of AriB and tRNAAsn(GUU) represents a secondary, yet biologically relevant, target of the PARIS effector. Activated AriB protein cleaves these targets in vitro, and the cleavage reaction is not dependent on the presence of specific tRNA modifications. We show that the overexpression of phage T5 tRNALys(UUU), tRNAThr(UGU) and tRNAAsn(GUU) variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose a model for tRNA recognition by the AriB dimer and provide molecular details of its nuclease activity and specificity.

microbiology↗

bioRxiv2024

Sedentary chromosomal integrons as biobanks of bacterial anti-phage defence systems

Integrons are genetic systems that accelerate bacterial adaptation by acquiring and shuffling gene cassettes. Mobile integrons spread antibiotic resistance genes among bacteria, while the sedentary chromosomal integrons contain up to hundreds of cassettes of unknown function. Here, we show that many of these cassettes encode anti-phage defence systems. We found numerous streamlined variants of known systems, which have presumably evolved to fit the small size constraints of integron cassettes recombination and genesis. Intrigued by the rarity of known systems in the sedentary chromosomal integron of the Vibrio cholerae 7th cholera pandemic strain, we tested the presence of anti-phage functions in all its cassettes of unknown function. We found that at least 16 of the strain cassettes have an anti-phage activity in V. cholerae or E. coli. This represents 18% of the tested cassettes and almost 10% of all the integron cassettes, providing at long last a key adaptive role for a significant fraction of the sedentary integrons. Most of the newly discovered systems have little or no similarity to previously known ones and our experiments show that several mediate defence through cell lysis or growth arrest. One of these systems encodes a 64 amino acids protein, which represents the smallest known protein providing autonomous phage resistance. Given the thousands of uncharacterized integron cassette families, integrons could represent an untapped treasure trove of streamlined anti-phage systems.

genetics↗

bioRxiv2024

In vivo delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a Type IV Secretion System

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a Type IV Secretion System (T4SS) into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the class 2 Cas endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from the cuts produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The gRNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using both TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides an in vivo delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells. Significance StatementWe have developed a novel approach for introducing CRISPR-Cas genetic tools into bacteria. During bacterial conjugation, the relaxase protein is transferred through the secretion system covalently attached to the transferred DNA. By fusing the Cas protein with the conjugative relaxase, we have observed functional Cas activity in the recipient cells, eliminating the need for nuclease expression in these cells. The covalently attached DNA molecule can supply gRNA and donor DNA, enabling seamless genetic modifications through recombination. We have also successfully translocated fusions of relaxases to base editors which are active in recipient cells. This method can be applied to any potential recipient cells, making it particularly interesting for wild type bacterial strains that lack available genetic tools. Furthermore, this method has the potential to be extended to eukaryotic cells.

microbiology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

Computational design of novel Cas9 PAM-interacting domains using evolution-based modelling and structural quality assessment

We present here an approach to protein design that enables to leverage (i) scarce functional information such as experimental data (ii) evolutionary information learned from a natural sequence variants and (iii) physics-grounded modeling. Using a Restricted Boltzmann Machine (RBM), we learned a sequence model of a protein family. We use semi-supervision to leverage available functional information during the RBM training. We then propose a strategy to explore the protein representation space that can be informed by external models such as an empirical force field method (FoldX). This method was applied to a domain of the Cas9 protein responsible for recognition of a short DNA motif. We experimentally assessed the functionality of 71 variants that were generated to explore a range of RBM and FoldX energies. We show how a combination of functional, structural and evolutionary information can identify functional variants with high accuracy. Sequences with as many as 50 differences (20% of the protein domain) to the wild-type retained functionality. Overall, 21/71 sequences designed with our method were functional. Interestingly, 6/71 sequences showed an improved activity in comparison with the original wild-type protein sequence. These results demonstrate the interest in further exploring the synergies between machine-learning of protein sequence representations and physics grounded modeling strategies informed by structural information.

bioinformatics↗

bioRxiv2020

Generating functional protein variants with variational autoencoders

The design of novel proteins with specified function and controllable biochemical properties is a longstanding goal in bio-engineering with potential applications across medicine and nanotechnology. The vast expansion of protein sequence databases over the last decades provides an opportunity for new approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Advances in deep generative models have led to the successful modelling of diverse kinds of high-dimensional data, from images to molecules, allowing the generation of novel, realistic samples. While deep models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, their potential for direct use in protein engineering remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase. We propose separate VAE models to work with aligned sequence input (MSA VAE) and raw sequence input (AR-VAE), and offer evidence that while both are able to reproduce patterns of amino acid usage characteristic of the family, the MSA VAE is better able to capture long-distance dependencies reflecting the influence of 3D structure. To validate the practical utility of the models, we used them to generate variants of luxA whose function was tested experimentally. As further evidence of the practicality of these methods for design, we showed that conditional variants of both models could be used to increase the solubility of luxA without disrupting function. Altogether 18/24 of the variants generated using the AR-VAE and 21/23 variants generated using the MSA VAE retained some luminescence activity, despite containing as many as 35 differences relative to any training set sequence. These results demonstrate the feasibility of using deep generative models to explore the space of possible protein sequences and generate useful variants, providing a method complementary to rational design and directed evolution approaches.

bioinformatics↗

bioRxiv2018

Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors

High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome of E. coli. We then apply the screen to discover E. coli genes required by phages {lambda}, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by {lambda} enables to identify genes required for the formation of functional {lambda} capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.

microbiology↗

bioRxiv2017

PhageTerm: a Fast and User-friendly Software to Determine Bacteriophage Termini and Packaging Mode using randomly fragmented NGS data

Bacteriophages are the most abundant viruses on earth and display an impressive genetic as well as morphologic diversity. Among those, the most common order of phages is the Caudovirales, whose viral particles packages linear double stranded DNA (dsDNA). In this study we investigated how the information gathered by high throughput sequencing technologies can be used to determine the DNA termini and packaging mechanisms of dsDNA phages. The wet-lab procedures traditionally used for this purpose rely on the identification and cloning of restriction fragment which can be delicate and cumbersome. Here, we developed a theoretical and statistical framework to analyze DNA termini and phage packaging mechanisms using next-generation sequencing data. Our methods, implemented in the PhageTerm software, work with sequencing reads in fastq format and the corresponding assembled phage genome.\n\nPhageTerm was validated on a set of phages with well-established packaging mechanisms representative of the termini diversity: 5cos (lambda), 3cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of 9 Clostridium difficile phages and 6 phages whose sequences where retrieved from the sequence read archive (SRA).\n\nA direct graphical interface is available as a Galaxy wrapper version at https://galaxy.pasteur.fr and a standalone version is accessible at https://sourceforge.net/projects/phageterm/.

microbiology↗