Biology subjects

Bigot, S.

Publications and source records attributed to Bigot, S..

7 recordsLinked to original sources

bioRxiv2026

In vitro-generated murine monocyte-derived dendritic cells habor a unique identity compared to other mononuclear phagocytes, largely conserved with their human and putative in vivo counterparts.

Whether the cells differentiating in vitro from total murine bone marrow (BM) cultures with GM-CSF +/- IL-4 are homologous to the human cells derived in vitro from purified circulating monocytes (Mo) cultured with GM-CSF and IL-4, mostly correspond to monocyte-derived dendritic cells (moDCs) or rather to macrophages (MFs), and have in vivo counterparts, is debated. It has been proposed to define cell types based on their ontogeny, leading to the proposal of excluding from the dendritic cell (DC) family the cells that do not arise from the common DC progenitors (CDP). However, recent discoveries support defining cell types rather by functions, or, as a surrogate, their instructive molecular networks. Indeed, Langerhans cells, Rorgt+ DCs, some cDC2As and human in vitro-derived moDCs do not arise from the CDP but exert hallmark DC functions instructed by specific transcriptomic modules shared with cDCs. Actually, the study of both LCs and moDCs has been key for dissecting DC biology. Yet, the existence of moDCs is put into questions. The functions once attributed to moDCs are now proposed to be exerted by type 2B conventional DCs (cDC2Bs) or type 3 DCs (DC3s) that have been characterized recently, harbor strongly overlapping phenotypes with moDCs and a similar mixed expression of genes from both the cDC and Mo/MF lineages, but a different ontogeny. To clarify these issues, we generated a transcriptomic atlas of in vitro-derived mononuclear phagocytes (MNPs). We show that moDCs are the only DC type arising in BM cultures in the presence of GM-CSF and IL-4. They express a unique transcriptomic and phenotypic signature, different from those of cDC2Bs and DC3s that are produced in FLT3-L cultures and appear to correspond to the same DC type differentiating though two different but converging ontogenetic paths. By harnessing the phenotypic and transcriptomic signatures identified from the analysis of our in vitro MNP atlas, we are able to identify in vivo a population of putative DCs differentiated from monocytes derived from the common monocyte-granulocyte progenitor (GMP), in a respiratory allergy model, specifically in the lungs of mice sensitized to house dust mite (HDM). Using the model of thioglycolate-induced peritonitis, we also characterized the CD226+ Mo-derived cells that have been reported to be moDCs or small cavity MFs (SCM) depending on studies. We showed that these cells are mainly differentiated from monocytes derived from the MDP, and split into two populations, one resembling MFs (SCMs) and the other one closer to lung moDCs but expressing distinct genes suggesting a different functional potential. Collectively, we show that the generation and characterization of specific MNP types in vitro can be harnessed to rigorously define their in vivo counterparts based on phenotypic analysis and gene module assessment, as we demonstrate for moDCs.

immunology↗

bioRxiv2026

The CUP-SHAPED COTYLEDON 2 - MIR164A regulatory feedback loop directs leaf morphogenesis

Gene regulatory networks (GRNs) establish the spatio-temporal gene expression patterns necessary for reproducible development of organisms. MicroRNAs have emerged as important regulators of gene expression controlling either target gene levels, spatial patterns or timing. These molecular functions may vary depending on how they are integrated into GRNs. Here we used the Arabidopsis leaf as a model to study the genetic and molecular interactions between the CUP-SHAPED COTYLEDON 2 (CUC2) transcription factor and its negative regulator, miR164A. Quantitative morphometric analyses showed that miR164A acts from early stages during leaf development regulating CUC2 expression dynamic rather than the spatial distribution of CUC2. At the molecular level, CUC2 directly activates MIR164A expression, forming a single negative feedback loop. Further, using a ratiometric approach, we identified a cis-regulatory element (CRE) sufficient to drive CUC2-dependent MIR164A expression at the leaf sinus. Ordinary differential equation (ODE) modelling suggests that CUC2-CRE may control the timely accumulation of CUC2. Accordingly, quantification of CUC2 protein accumulation dynamics using a spatio-temporal mean cartography strategy in CRISPR cis-regulatory variants at the MIR164A locus revealed that CUC2-dependent MIRA164A expression is required to maintain both the precise timing of CUC2 expression and CUC2 levels. Altogether, our work provides experimental evidence for a specific role of the CUC2-MIR164A module in shaping leaf development, highlighting the contribution of network topology to GRN functions.

plant biology↗

bioRxiv2026

Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli

Background Targeted-Antibacterial Plasmids (TAPs) are engineered mobile genetic elements that use bacterial conjugation to deliver selective CRISPR/Cas9 antibacterial activity against a specific target strain. Yet, the efficiency of TAPs is typically evaluated at a single time point, whereas the success of TAP-mediated resensitization critically depends on the dynamics of plasmid transfer and the complex interactions between bacterial subpopulations. This is the first study to evaluate the efficiency of a conjugation-based antibacterial approach at the subpopulation level, using an analytical framework analogous to that used for conventional antibiotics. Here, we investigate which process limits resensitization by TAPF-dCas9-OXA48: plasmid delivery, dCas9 activity, or the emergence of refractory and escape populations. Methods We fitted a mechanistic model of five interacting subpopulations (donors, recipients, transconjugants, escapers, and recusants) to 44 longitudinal conjugation experiments and used the fitted model to explore a range of biologically relevant scenarios. Results Using longitudinal conjugation data spanning 24 h, we show that up to 24% of recipients become recusants within 24h, refractory to further conjugation via entry exclusion, while secondary transconjugant emergence stays below 0.01%. Overall resensitization efficiency reaches up to 80%. Conclusion Plasmid transfer, rather than dCas9 repression, therefore appears to be the main bottleneck limiting the efficiency of TAPF-dCas9-OXA48 efficiency. These results identify plasmid delivery as a key engineering target for improving the performance of future TAPs.

bioinformatics↗

bioRxiv2026

Plasmid-encoded host reprogramming promotes plasmid dissemination

Conjugative plasmids are major drivers of antibiotic resistance dissemination, yet how newly transferred plasmids establish in recipient cells remains poorly understood. Here we investigate YfjB, a previously uncharacterized conserved leading-region protein, which is zygotically induced immediately after plasmid entry and acts specifically during the earliest post-transfer stages. Multi-omics analyses reveal that YfjB reprograms host transcription, triggering extensive metabolic rewiring that compensates the transient fitness cost of plasmid acquisition. Structural analyses show that YfjB is a ParB-like protein containing a CTP-binding domain and a helix-turn-helix DNA-binding motif, linked to a previously uncharacterized dimerization module that forms a V-shaped clamp-like architecture compatible with DNA loading. Consistently, live-cell imaging reveals nucleoid-associated foci in transconjugants, and ChIP-seq identifies multiple chromosomal binding sites. We therefore rename the protein HerB (Host Expression Reprogrammer, ParB-like). More broadly, our findings reveal how mobile genetic elements facilitate their dissemination by transiently subverting host physiology.

microbiology↗

bioRxiv2025

Genetic Determinants of pOXA-48 Plasmid Maintenance and Propagation in Escherichia coli

Conjugative plasmids are the main drivers of antibiotic resistance dissemination contributing to the emergence and extensive spread of multidrug resistance clinical bacterial pathogens. pOXA-48 plasmids, belonging to the IncL group, have emerged as the primary vehicle for carbapenem resistance in Enterobacteriaceae. Despite the problematic prevalence of pOXA-48, most research has focused on epidemiology and genomics, leaving gaps in our understanding of the mechanisms behind its propagation. In this study, we used a transposon sequencing approach to identify genetic elements critical for plasmid stability, replication, and conjugative transfer. Our results identified a novel type I toxin-antitoxin system, new essential maintenance factors, and components of the type IV secretion system and regulatory elements crucial for conjugation. This study advances our understanding of pOXA-48 biology, providing key insights into the genetic factors underlying its successful maintenance and spread in bacterial populations.

microbiology↗

bioRxiv2022

Real-time visualisation of conjugation reveals the molecular strategy evolved by the conjugative F plasmid to ensure the sequential production of plasmid factors during establishment in the new host cell

DNA conjugation is a contact-dependent horizontal gene transfer mechanism responsible for disseminating drug resistance among bacterial species. Conjugation remains poorly characterised at the cellular scale, particularly regarding the reactions occurring after the plasmid enters the new host cell. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugation in real time. We reveal that the transfer of the plasmid in single-stranded DNA (ssDNA) form followed by its conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ss-to-dsDNA conversion is the critical step that governs the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent ss-to-dsDNA conversion turns off leading gene expression and licences the expression of the other plasmid genes under the control of conventional double-stranded promoters. This elegant molecular strategy evolved by the conjugative plasmid allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

microbiology↗

bioRxiv2020

Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR /Cas systems achieve strain-specific antibacterial activity

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

microbiology↗