Biology subjects

De Paepe, M.

Publications and source records attributed to De Paepe, M..

10 recordsLinked to original sources

bioRxiv2026

Versatility of Campylobacter jejuni Bf extracellular vesicles in regulating adaptation and virulence under combined thermal and oxidative stress

The high prevalence of aerotolerant human Campylobacter jejuni isolates suggests a correlation between the ability to survive in aerobic conditions, virulence and resistance to harsh stress conditions. However, the mechanisms are still unclear. Here, we investigated the role of bacterial extracellular vesicles (bEVs) in the adaptation of the clinical aerotolerant C. jejuni Bf strain to thermal and oxidative stress. We show that C. jejuni Bf survives and actively multiplies under this combined stress. Stress exposure induced cell rounding and loss of motility, remodeling of membrane composition, decreased membrane fluidity, and metabolic reprogramming with increased intracellular ATP levels. Lipidomic analyses further revealed that bEVs composition is markedly different from that of the parent membranes indicating that vesicle formation is selective and regulated. Although bEVs were produced in similar amounts under both microaerophilic and stress conditions, stress exposure generated significantly larger vesicles with greater diameter and dry mass, and altered their protein and lipid profiles. bEVs derived from stressed cells showed increased toxicity toward the epithelial barrier of Caco-2 cells. Taken together, these results indicate that C. jejuni bEV secretion is part of a survival strategy that connects environmental adaptation with pathogenicity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/714464v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@17aa2aforg.highwire.dtl.DTLVardef@4eab9dorg.highwire.dtl.DTLVardef@e4fba8org.highwire.dtl.DTLVardef@146109a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A Pair of DNA Glucosyltransferases Elevate Counter-defense in Bacteriophage T4

Bacteriophages encode diverse pathways to modify their nucleobases. These modifications help phages to evade the host defense systems such as restriction-modification (RM), and type II and type V CRISPR-Cas systems. On the other hand, modifications can also serve as a target for other host defense systems, illustrating the complexity of the defense and counter-defense landscape. Bacteriophage T4 encodes two glucosyltransferases (GTs), -GT and {beta}-GT, that post-replicatively add a glucose moiety to the hydroxymethylated deoxycytosines (5-hmC) on phage DNA in the - and {beta}-conformation, respectively. Among all known phages, only six closely related phages encode both -GT and {beta}-GT. Here, through biochemical and genetic analysis, we show that {beta}-GT has higher catalytic activity, whereas -GT is more strongly expressed. During the T4 infection, these factors determine the contributions of both GTs, with -GT and {beta}-GT contributing respectively to glucosylation of 66% and 33% of all 5-hmC. Encoding a single GTs is sufficient for T4 to overcome the E. coli type I and type IV RM systems, unless the glucosylation capacity decreases below the 80% threshold. However, when encountering a host encoding DNA glycosylase Brig1 in addition to type I and type IV RM systems, a second GT is necessary to enable Brig1 escapers to resist RM systems. These results demonstrate that encoding multiple GTs with redundant functionalities provides an evolutionary advantage when simultaneously confronted with multiple antiphage defense systems.

microbiology↗

bioRxiv2025

Atypical Replication Error Pattern and Limited Repair Efficiency Contribute to Elevated Mutation Rate in Phage lambda

Understanding the rate and nature of spontaneous mutations is crucial for understanding and modulating the pace and trajectory of evolution. Yet, both remain poorly characterized in double-stranded DNA (dsDNA) phages, despite their relevance for phage-based therapies. Here, we address this gap for the dsDNA phage lambda, using four complementary approaches: mutation accumulation assay combined with whole-genome sequencing, mutation visualization assay, duplex sequencing and fluctuation assay. We find that the mutation rate of wild-type phage lambda is 4.9 {+/-} 1.8 x10-9 per base per replication, approximately 15 times lower than previously estimated and about 20 times higher than that of its host, Escherichia coli (E. coli). Inactivation of Mismatch Repair (MMR), a major conserved cellular system for mutation avoidance, increases the lambda mutation rate by only 2-to 10-fold, in contrast to the approximately 150-fold increase in E. coli, however lambda does not exhibit the characteristic mutational bias associated with MMR deficiency. Interestingly replication of the lambda genome generates an error spectrum distinct from that of E. coli, characterized by a marked increase in transversions, poorly repaired by MMR. Together, these results reveal that lambda exhibits a replication error profile that is less amenable to repair, likely contributing to its elevated mutation rate.

microbiology↗

bioRxiv2024

Differential translocation of bacteriophages across the intestinal barrier in health and Crohn's disease

Impaired intestinal barrier function is a major feature of Crohns disease (CD), leading to exacerbated inflammation in response to the microbiota. In this context, the translocation of intestinal bacteriophages (phages) and their effects on the host have been little investigated. We used phage fluorescence imaging coupled with ex-vivo and in-vitro models that mimic physiological and inflammatory conditions and found that phages can translocate across the intestinal barrier without disrupting its integrity. Although the translocation rate across the intestinal epithelium depended on phage morphology and the condition of the barrier, these factors did not influence the crossing of phage across the vascular endothelium. Virome analysis confirmed that viral sequences shared between blood and stool samples are more abundant in CD patients than healthy subjects, indicating that a barrier defect facilitates phage translocation from the gut to the bloodstream.

cell biology↗

bioRxiv2023

Purchases dominate the carbon footprint of research laboratories

Despite increasing interest for the carbon footprint of higher education institutions, little is known about the carbon footprint associated to research activities. Air travel and attendance to conferences concentrate recent data and debates but purchases have attracted little attention. Here we develop a hybrid method to estimate the greenhouse gas emissions (GHG) associated to research purchases. To do so, we combine macroe-conomic databases, research-centered companies footprints and life-cycle assesments to construct a public database of monetary emission factors (EF) for research purchases. We apply it to estimate the purchases emissions of a hundred of research laboratories in France, belonging to the Labos 1point5 network and gathering more than 20000 staff, from all disciplines. We find that purchases dominate laboratory emissions, accounting for more than 50% of emissions, with a median of 2.7 t CO2e/pers, which is 3 to 4-fold the separate contribution from travel, commutes and heating. Median electricity emissions are 5-fold lower in our dataset of laboratories using low carbon electricity but they become preponderant for high carbon electricity mixes (3.5 t CO2e/pers). Purchases emissions are very heterogeneous among laboratories and are linearly correlated with budget, with an average carbon intensity of 0.31 {+/-} 0.07 kg CO2e/{euro} and differences between research domains. Finally, we quantify the effect of a series of demand-driven mitigation strategies obtaining up to -20 % in total emissions (-40 % in purchases emissions), suggesting that effectively reducing the carbon footprint of research activities calls for systemic changes. Significance statementResearch activities are recently interrogating their contribution to global warming, mainly through the impact of air travel but neglecting the emissions embodied in scientific purchases. However, goods and services used in a research laboratory emit greenhouse gases when they are produced. Here we construct a public and robust database of emission factors to quantify purchases emissions in a laboratory and we use it to assess emissions from a hundred of laboratories in France, from all disciplines. We find that purchases emissions represent half of the of the 6.3 t CO2e/pers per year emitted on average per laboratory. Emissions, however, vary greatly between laboratories and disciplines and an analysis of mitigation strategies shows that decreasing demand may significantly reduce purchases emissions.

scientific communication and education↗

bioRxiv2022

An alternative method to multiple displacement amplification for preparing virome DNA in a way adapted for the sequencing of both double-strand and single-strand DNA viruses

Background: Bulk microbiome, as well as virome-enriched shotgun sequencing only reveals the double-stranded DNA (dsDNA) content of a given sample, unless specific treatments are applied. However, genomes of viruses often consist of a circular single-stranded DNA (ssDNA) molecule. Pre- treatment and amplification of DNA using the multiple displacement amplification (MDA) method enables conversion of ssDNA to dsDNA, but this process can lead to over-representation of these circular ssDNA genomes. A more recent alternative permits to bypass the amplification step, as library adapters are ligated to sheared and denatured DNA, after an end-modification step (xGen kit). However, the sonication step might shear ssDNA more efficiently than dsDNA, therefore introducing another bias in virome sequencing. These limitations prompted us to explore an alternative method of DNA preparation for sequencing mixed ssDNA and dsDNA viromes. Results: Using a synthetic mix of viral particles, we made use of the T7 DNA polymerase (T7pol) to convert viral circular ssDNA molecules to dsDNA, while preventing over-replication of such molecules, as is the case with the Phi29 DNA polymerase. Our findings indicate that using T7pol and a mix of degenerated primers to convert ssDNA to dsDNA prior library preparation is a good alternative to the currently used methods. It better represents the original synthetic mixtures compared to MDA or direct application of the xGen kit. Furthermore, when applied to two complex virome samples, the T7pol treatment improved both the richness and abundance in the Microviridae fraction. Conclusion: We conclude that T7pol pretreatment is preferable to MDA for the shotgun sequencing of viromes, which is easy to implement and inexpensive.

genomics↗

bioRxiv2022

Prophage taming by the adherent-invasive Escherichia coli LF82 upon macrophage infection

Adherent-invasive Escherichia coli (AIEC) strains are frequently recovered from stools of patients with dysbiotic microbiota. They have remarkable properties of adherence to the intestinal epithelium, and survive better than other E. coli in macrophages. The best studied of these AIEC is probably strain LF82, which was isolated from a Crohns disease patient. This strain contains five complete prophages, which have not been studied until now. We undertook their analysis, both in vitro and inside macrophages, and show that all of them form virions. The Gally prophage is by far the most active, generating spontaneously over 108 viral particles per mL of culture supernatants in vitro, more than 100-fold higher than the other phages. Gally is over-induced after a genotoxic stress generated by ciprofloxacin and trimethoprim. However, upon macrophage infection, Gally virion production is decreased by more than 20-fold, and the transcription profile of the prophage indicates that part of the structural module is specifically repressed while the replication module is overexpressed compared to unstressed culture conditions. We conclude that strain LF82 has evolved an efficient way to "tame" its most active prophage upon macrophage infection, which may participate to its good survival in macrophages. The results are discussed in light of the active lysogeny process. AUTHOR SUMMARYProphages are bacterial viruses stably integrated into their host, to which they can provide new functions, thus increasing their fitness in the environment. Thereby, they can participate to the virulence of bacterial pathogens. However, prophages are double-edged swords that can be awakened in response to genotoxic stresses, resulting in the death of their bacterial host. This raises the question of the effect of this type of stress in the natural environments where their bacterial hosts exert their virulence. In this study, we characterized the five active prophages present in Escherichia coli LF82, a strain belonging to the intestinal microbiota and suspected to be involved in Crohns disease via its ability to invade macrophages, a highly genotoxic environment. We show that LF82 inhibits the awakening of its prophages in macrophages, allowing it to survive there. Moreover, deletion of its most active prophage does not affect the viability of LF82 in this environment. These results show that LF82 has tamed its prophages in macrophages and also suggest that if they convey fitness advantages, they probably do so in environments differing from macrophages, and which remain to be discovered.

microbiology↗

bioRxiv2022

Comparison of interferometric light microscopy with nanoparticle tracking analysis for the study of extracellular vesicles and bacteriophages

Research on extracellular vesicles (EVs) and bacteriophages (phages) has been steadily expanding over the past decades as many of their roles in medicine, biology, and ecosystems have been unveiled. Such interest has brought about the need for new tools to quantify and determine the sizes of these biological nanoparticles. A new device based on interferometric light microscopy (ILM), the Videodrop, was recently developed for this purpose. Here, we compared this new device to two nanoparticle tracking analysis (NTA) devices, the NanoSight and the ZetaView, for the analysis of EVs and phages. We used EVs isolated from bacteria, fecal samples, bovine milk and human cells, and phages of various sizes and shape, ranging from 30 to 120 nm of diameter. While NTA instruments correctly enumerated most phages, the Videodrop detected only the largest one, indicating a lower sensitivity threshold compared to the NTA devices. Nevertheless, the performance of the Videodrop compared favorably to that of the NTA devices for the determination of the concentration of eukaryotic EV samples. The NanoSight instrument provided the most precise size distributions but the Videodrop was by far the most time-saving device, making it worthy of consideration for studies conducted on a large number of samples.

cell biology↗

bioRxiv2019

The enemy from within: a prophage of Roseburia intestinalis systematically turns lytic in the mouse gut, driving bacterial adaptation by CRISPR spacer acquisition

Despite an overall stability in time of the human gut microbiota at the phylum level, strong temporal variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses. A breakdown of prophage dormancy can evolve through the selection of ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammals microbiota has been largely unexplored.\n\nHere we studied temperate phage-bacteria coevolution in gnotoxenic mice colonised with Escherichia coli and Roseburia intestinalis, a dominant symbiont of the human gut microbiota. We show that R. intestinalis harbors two active prophages, Jekyll and Shimadzu, and observed the systematic evolution of ultravirulent Shimadzu phage mutants, leading to a collapse of R. intestinalis population. In a second step, phage infection drove the fast evolution of host phage-resistance mainly through phage-derived spacer acquisition in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize.\n\nOur results demonstrate that prophages are the potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

microbiology↗

bioRxiv2018

Viral metagenomic analysis of the cheese surface: a comparative study of rapid procedures for extracting virus-like particles

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering virus-like particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the Epoisses cheese virome. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.\n\nIMPORTANCEWhether bacterial viruses (phages) are necessary or not to maintain food ecosystem function is not clear. They could play a negative role by killing cornerstone species that are necessary for fermentation. But they might also be positive players, by preventing the overgrowth of unwanted species (e.g. food spoilers). To assess phages contribution to food ecosystem functioning, it is essential to set up efficient procedures for extracting viral particles in solid food matrix, then selectively sequence their DNA without being contaminated by bacterial DNA, and finally to find strategies to assemble their genome out of metagenomic sequences. This study, using cheese rind surface as a model, describes a comparative analysis of procedures for selectively extracting viral DNA from cheese and to efficiently characterize the genome of dominant phages with cross-sample assembly.

microbiology↗