Biology subjects

Bikard, D.

Publications and source records attributed to Bikard, D..

33 records · Page 2Linked to original sources

bioRxiv2022

Within-patient evolution of plasmid-mediated antimicrobial resistance

Antibiotic resistance (AMR) in bacteria is a major threat to public health, and one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been extensively studied in vitro, but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically-relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalised patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed, for the first time, compensatory evolution of plasmid-associated fitness cost, as well as the evolution of enhanced plasmid-mediated AMR, in bacteria evolving within the gut of hospitalised patients. Crucially, we observed that the evolution of plasmid-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.

evolutionary biology↗

bioRxiv2021

Genetic determinism of phage-bacteria coevolution in natural populations

Coevolution between bacteriophage (or phage) and their bacterial host is thought to be key for the coexistence of these antagonists. Recent studies have revealed the major role of mobile genetic elements in the emergence of phage resistant hosts but how phage escape these defenses in the wild remained to be explored. Here we show a striking parallel in phage evolving counter defenses to host defenses in natural population. We established a large collection of phages and their bacterial hosts and we explored the genetic structure of their interaction. We find that clearly delineated genomic clusters of phage are specific for distinct clades within a bacterial species, Vibrio crassostreae, yet while all phages can adsorb, only a subset of hosts are killed due to intracellular defense mechanisms. Host genomes contain multiple mobile defense genes and susceptibility to phage is negatively correlated with genome size. Phages also display extensive gene content variation, but their genome size remains conserved. We show that this gene content variation in hosts and phage is due to rapid turnover of genes involved in defense and escape, and that by exchanging anti-defense genes, phages irreversibly switch host. This could be indicative of co-evolution following the matching-allele-model of specificity and the spatial and temporal variability of phage infectivity further suggests that negative-frequency dependent selection drives phage-vibrio coevolutionary dynamics. We propose a "pan-escape system" that can be shared among phages by homologous recombination within a population that infects a bacterial host.

microbiology↗

bioRxiv2021

Improving sequence-based modeling of protein families using secondary structure quality assessment

MotivationModeling of protein family sequence distribution from homologous sequence data recently received considerable attention, in particular for structure and function predictions, as well as for protein design. In particular, Direct Coupling Analysis, a method to infer effective pairwise interactions between residues, was shown to capture important structural constraints and to successfully generate functional protein sequences. Building on this and other graphical models, we introduce a new framework to assess the quality of the secondary structures of the generated sequences with respect to reference structures for the family. ResultsWe introduce two scoring functions characterizing the likeliness of the secondary structure of a protein sequence to match a reference structure, called Dot Product and Pattern Matching. We test these scores on published experimental protein mutagenesis and design dataset, and show improvement in the detection of non-functional sequences. We also show that use of these scores help rejecting non-functional sequences generated by graphical models (Restricted Boltzmann Machines) learned from homologous sequence alignments. AvailabilitySupplementary Materials, Data and Code available at https://github.com/CyrilMa/ssqa. Contactcyril.malbranke@ens.fr O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/428964v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@38d327org.highwire.dtl.DTLVardef@aeba12org.highwire.dtl.DTLVardef@13a09a2org.highwire.dtl.DTLVardef@14e85fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2021

Prophage-encoded hotspots of bacterial immune systems

The arms race between bacteria and phages led to the emergence of a variety of genetic systems used by bacteria to defend against viral infection, some of which were repurposed as powerful biotechnological tools. While numerous defense systems have been identified in genomic regions termed defense islands, it is believed that many more remain to be discovered. Here, we show that P2- like prophages and their P4-like satellites have genomic hotspots that represent a significant source of novel anti-phage systems. We validate the defense activity of 14 systems spanning various protein domains and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Immunity hotspots are present across prophages of distant bacterial species, highlighting their biological importance in the competition between bacteria and phages.

microbiology↗

bioRxiv2020

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

In Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE ({sigma}E) in Enterobacteria. {sigma}E upregulates OMP biogenesis factors, including the {beta}-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated {sigma}E activity, however, can be detrimental for OM integrity. Here we report that DolP (YraP), a {sigma}E-upregulated OM lipoprotein important for envelope integrity, is a novel interactor of BAM and we demonstrate that OM-assembled BamA is a critical determinant of the BAM-DolP complex. Mid-cell recruitment of DolP had been previously associated to activation of septal peptidoglycan remodelling during cell division, but its role during envelope stress was unknown. We now show that DolP promotes cell fitness upon stress-induced activation of {sigma}E and opposes a detrimental effect caused by the overaccumulation of BAM in the OM. During envelope stress, DolP loses its association with the mid-cell, thus suggesting a possible link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

microbiology↗

bioRxiv2020

The impact of genetic diversity on gene essentiality within the E. coli species

Bacteria from the same species can differ widely in their gene content. In E. coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. While recent advances in bacterial genomics have enabled to unravel genes required for fitness in various experimental conditions at the genome scale, most studies have focused on model strains. As a result, the impact of this genetic diversity on core processes of the bacterial cell largely remains to be investigated. Here, we developed a new CRISPR interference platform for high-throughput gene repression that is compatible with most E. coli isolates and closely-related species. We applied it to assess the importance of [~]3,400 nearly ubiquitous genes in 3 growth media in 18 representative E. coli strains spanning most common phylogroups and lifestyles of the species. Our screens highlighted extensive variations in gene essentiality between strains and conditions. Unlike variations in gene expression level, variations in gene essentiality do not recapitulate the strains phylogeny. Investigation of the genetic determinants for these variations highlighted the importance of epistatic interactions with mobile genetic elements. In particular, we showed how mobile genetic elements can trigger the essentiality of core genes that are usually nonessential. This study provides new insights into the evolvability of gene essentiality and argues for the importance of studying various isolates from the same species in bacterial genomics.

genomics↗

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↗

bioRxiv2019

Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

microbiology↗

bioRxiv2019

Co-occurrence of multiple CRISPRs and cas clusters suggests epistatic interactions

Prokaryotes use CRISPR-Cas for adaptive immunity, but the reasons for the existence of multiple CRISPR and cas clusters remain poorly understood. We found that more than 40% of the genomes encoding a system show atypical genetic organizations. Their analysis revealed negative and positive epistatic interactions between Cas subtypes. The latter often result in one single complex locus with a shared adaptation module and diverse interference mechanisms, presumably to produce more effective immune systems. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. In contrast, under-represented co-occurrences caused by functional interference or redundancy may lead to CRISPRs distant from cas genes. To investigate the origin of atypical CRISPR-Cas organizations, we analyzed plasmids and phages. Sets of nearly 2000 phages and 10000 prophages were almost devoid of CRISPR-Cas systems, but a sizeable fraction of plasmids had them. Isolated CRISPRs in plasmids were often compatible with the chromosomal cas clusters, suggesting that plasmids use CRISPRs to subvert host immunity. These results point to an important role for the interactions between multiple CRISPR and Cas in the function and evolution of bacterial immunity.

genomics↗

bioRxiv2018

Cell Diameter in Bacillus subtilis is Determined by the Opposing Actions of Two Distinct Cell Wall Synthetic Systems

Rod shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod system moves around the cell circumference, while the class A penicillin-binding proteins (aPBPs) are unorganized. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each system affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. We find that rod diameter is not determined by MreB, rather it depends on the balance between the systems: The Rod system reduces diameter, while aPBPs increase it. RodA/PBP2A can both thin or widen cells, depending on its levels relative to MreBCD. Increased Rod system activity correlates with an increased density of directional MreB filaments, and a greater fraction of directionally moving PBP2A molecules. This increased circumferential synthesis increases the amount of oriented material within the sacculi, increasing their mechanical anisotropy and reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems build rods of different widths, a model that appears generalizable: Escherichia coli containing Rod system mutants show the same relationship between the density of directionally moving MreB filaments and cell width.

microbiology↗

bioRxiv2018

Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

The main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

synthetic biology↗

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

Inhibition of NHEJ repair by type II-A CRISPR-Cas systems

CRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. Here, we hypothesized that non-homologous end joining (NHEJ) interferes with type II CRISPR-Cas systems. We tested this idea by studying the patterns of co-occurrence of the two systems in bacterial genomes. We found that NHEJ and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We investigated experimentally the possible molecular interactions causing this negative association using the NHEJ pathway from Bacillus subtilis and the type II-A CRISPR-Cas systems from Streptococcus thermophilus and Streptococcus pyogenes. Our results suggest that the NHEJ system has no effect on type II-A CRISPR-Cas interference and adaptation. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein from type II-A CRISPR-Cas system. Our findings give insights on the complex interactions between CRISPR- Cas systems and repair mechanisms in bacteria and contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.

microbiology↗

bioRxiv2017

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

synthetic biology↗

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↗