Biology subjects

Petit, J.

Publications and source records attributed to Petit, J..

14 recordsLinked to original sources

bioRxiv2026

Deep learning extracts MoA-specific signatures from high-throughput images of chemically and genetically perturbed Corynebacteria

Tuberculosis (TB) is the worldwide leading infectious killer due to a single pathogen and increasing antimicrobial resistance (AMR) makes it imperative to discover and develop new drugs with novel modes of action (MoAs) to treat TB infections. Phenotypic screening of chemical libraries has proven effective at identifying new compounds against bacterial pathogens. However, a major limitation of standard screens is their inability to uncover the MoA of hits thereby preventing targeted selection of compounds with novel MoAs. Linking drug perturbations to mutants from images could potentially enable to predict the targets of compounds that act through novel MoAs. Here, we develop a deep learning (DL)-based method to screen drug-treated Corynebacterium glutamicum (Cglu), a surrogate model for Mycobacterium tuberculosis (Mtb). Our DL model is based on a convolutional neural network architecture that takes high throughput images as input and is trained to distinguish between different MoAs. We show that our approach can robustly differentiate between the MoAs of established antibiotics and correctly recognise the MoA of antibiotics that were not previously seen by the DL model. We also show that inhibitors with the same and previously unseen MoA cluster together and apart from all other reference drugs, allowing for new MoA discovery. Importantly, we show that our model links images of chemical (drugs) and genetic (mutants) perturbations targeting similar pathways, thus paving the way towards mutant-based target prediction of compounds that act through novel MoAs, directly from high-content images. Finally, we explore the phenotypes induced by genetic disruption of pathways and demonstrate that features extracted with our DL model recover known biological relationships from high-throughput images alone using the cell cycle of Cglu as a case study, a finding with promising potential for fundamental mechanistic studies.

microbiology↗

bioRxiv2026

Automated Optimization of Bacterial Tracking Pipelines with TrackMate 8

Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, Microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacteria morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacteria images in TrackMate, (2) a TrackMate-Helper extension for parameter optimization, and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging.

microbiology↗

bioRxiv2025

Functional dissection of Wag31 domains for septal recruitment and polar distribution during the cell cycle

Bacterial cell morphogenesis is controlled by the synthesis and organization of peptidoglycan and driven by multi-protein complexes such as the divisome and elongasome. Here we investigate the role of the Corynebacterium glutamicum DivIVA homologue, Wag31, the elongasome scaffold essential for polar growth in Corynebacteriales. Conditional depletion of Wag31 results in viable but coccoid-shaped cells, showing that Wag31 is essential for rod shape maintenance. Our structural phylogenetic analyses of DivIVA homologues revealed that in Actinobacteria, unlike Firmicutes, an intrinsically disordered region spatially separates the N-terminal lipid-binding domain (LBD) from the C-terminal coiled-coil domain (CCD). We show that the LBD is necessary and sufficient for septum localization, independent of its membrane-binding properties, while the CCD domain mediates self-interaction and polar accumulation. Our findings suggest that Wag31 is recruited specifically to the septum through protein-protein interactions, priming the future pole and allowing for a timely divisome-elongasome transition at cytokinesis. Once the pole is formed the self-aggregative properties of the C-terminal CCD dominate and form a stable structure that likely organizes the pole for cell wall biosynthesis.

microbiology↗

bioRxiv2025

Role of the tomato MARS1/ROUGH gene encoding a LYSINE-SPECIFIC HISTONE DEMETHYLASE 1 in adventitious root and fruit skin formation

In contrast to animals, plants have a high regenerative capacity, and they can form new organs and even complete individuals from a few cells present in adult tissues, either in response to injury or to the alteration of their environment. In this study, we describe the isolation and characterization of the more adventitious roots1-1 (mars1-1) mutant, which exhibits enhanced regenerative potential upon wounding in tomato hypocotyl explants. Additionally, the mars1-1 fruits exhibited a rough surface due to the ectopic proliferation of subepidermal cells, which formed callus-like structures on the cuticle. The MARS1/ROUGH gene encodes a conserved lysine-specific histone demethylase, SlLSD1, which regulates a variety of processes in metazoans, including cell proliferation, stem cell pluripotency, and embryogenesis. Two CRISPR/Cas9 null alleles, mars1-2 and mars1-3, were generated and their pleiotropic phenotype was characterized. We found elevated levels of H3K4me1 in mars1/rough seedlings, which suggests that SlLSD1 is required for the demethylation of this histone mark. To ascertain the impact of altered epigenetic marks in the mars1/rough mutants on gene expression regulation, we conducted a transcriptome analysis using a variety of RNA-Seq studies on tomato hypocotyls. By employing specific bioinformatic workflows and leveraging on the resolution of directional RNA-Seq data, we have identified over several dozen distinct genomic regions that exhibit de novo expression in the mars1/rough mutants. One such region includes a novel B-type cyclin gene, which is upregulated in the mars1/rough mutants and may account for the observed phenotypes. Our findings indicate that SlLSD1 plays a role in the establishment and maintenance of silencing in specific genomic regions that are essential for tissue-specific reprogramming.

plant biology↗

bioRxiv2025

Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA in Mycobacterium tuberculosis.

D,L-endopeptidase RipA is the major PG hydrolase required for daughter cell separation in Mycobacterium tuberculosis (Mtb), as RipA defects severely hinder cell division and increase antibiotic vulnerability. Despite extensive studies, the mechanisms governing Mtb RipA regulation remain controversial and poorly understood. Here, we report an integrative structural and functional analysis of the SteAB system, a regulatory complex that has been shown to modulate cell separation in the model organism Corynebacterium glutamicum (Cglu) and is conserved across Mycobacteriales. Although Mtb SteB was previously proposed to act as a mycobacterial outer membrane copper transporter, the crystal structures of the homodimeric protein, alone and in complex with the RipA coiled-coil (CC) domain, rule out this hypothesis. Instead, the high-affinity SteB-RipA interaction, together with computational and biophysical data, strongly supports the role of SteB as a direct RipA activator that releases enzyme autoinhibition upon complex formation. In addition, crystallographic characterization of the cytoplasmic core of SteA revealed a homodimeric organization harboring a conserved functional pocket similar to the phosphonucleotide-binding site of thiamine pyrophosphokinase. These data, coupled with the in vivo phenotypic analysis of a steAB knockout mutant of Cglu, support a model in which the transmembrane SteAB heterotetramer, driven by cytoplasmic ligand binding, orchestrates the productive periplasmic positioning of RipA, leading to PG hydrolysis activation. These findings shed new light on the regulation of mycobacterial cell wall remodeling, with implications for understanding Mtb pathogenesis and identifying novel antimicrobial targets.

microbiology↗

bioRxiv2025

Deep learning recognises antibiotic modes of action from brightfield images

Antimicrobial resistance is a growing public health threat predicted to cause up to 10 million deaths a year by 2050. To circumvent existing bacterial resistance mechanisms, discovering antibiotics with novel modes of action (MoAs) is crucial. While growth inhibition assays can robustly identify antibiotic molecules, they miss promising compounds with subinhibitory phenotypes and do not inform on drug MoA. Microscopy-based cytological profiling of drug-treated bacteria with hand-crafted image descriptors or more recently deep learning (DL) provides complementary information on the MoA. However, current approaches are limited by the need for fluorescent labelling and drug exposure at inhibitory concentrations. It also remains unclear if cytological profiling enables the detection of drugs with novel MoAs. Here, we demonstrate an approach based on supervised DL to identify antibiotic drug MoA from microscopy images without fluorescent labelling. We train a convolutional neural network to predict treatment conditions from brightfield images of Escherichia coli exposed to reference drugs covering multiple MoAs. Our method can detect drug exposure at subinhibitory concentrations and distinguishes individual drug treatments with high accuracy (86.2%). The learned representations implicitly capture MoA-specific phenotypes, enabling perfect MoA recognition (100%) without retraining using a model trained on only 644 images. Our approach can identify the MoA of previously unseen drugs with good accuracy (77.8{+/-}3.3%), as long as the MoA is represented by at least one of the training compounds. Finally, we show that our approach can detect if a drug has a novel MoA with an area under the curve above 0.75 for five out of six MoAs, facilitating microscopy-based identification of novel classes of antibiotics. Our methods and results pave the way towards an automated pipeline for antibiotic drug discovery based on imaging and DL.

microbiology↗

bioRxiv2024

Targeting the tomato fruit cuticle by gene overexpression and editing with the fruit epidermis-preferential nsLTP promoter

The thick cuticle covering and embedding epidermal cells of tomato (Solanum lycopersicum) fruit, a model for cuticle studies, plays important roles in fruit protection and quality. To further our understanding of the influence of cuticle components on cuticle architecture and properties, the next step is to engineer cuticle composition. Ideally, to avoid indirect effects at whole-plant level, a fruit-and epidermis-specific promoter should be used. Here, to overexpress and edit target genes in tomato, we selected a non-specific Lipid Transfer Protein 2 promoter (pronsLTP) with preferential activity in fruit epidermis, as shown by NLS-GFP fluorescence analysis. Overexpression of SlMYB75 driven by pronsLTP induced anthocyanin accumulation specifically in the growing fruit epidermis, consistent with the cuticle deposition pattern. In keeping with changes in anthocyanins, the cuticle composition in flavonoids and phenolic acids was altered, as shown by Raman microspectroscopy, as were several cuticle properties. We then edited the carotenoid PSY1 gene with a pronsLTP-driven CRISPR/Cas9 system. Different PSY1 knock-out alleles were detected predominantly in fruit exocarp and mesocarp of color-impaired pronsLTP::Cas9-Psy1 mutants, and very little in the leaf. A mutant carrying a heritable knock-out mutation in both fruit and leaf was also detected, indicating that mutants obtained with pronsLTP may require screening before being studied. Altogether, our results indicate that pronsLTP2 can efficiently result in gene overexpression and editing in fruit epidermis. Implications of these findings are important for the functional analysis and genetic engineering of cuticle-related genes in tomato.

plant biology↗

bioRxiv2024

Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or S-layer, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle core, a three-helix bundle arm, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

microbiology↗

bioRxiv2024

Exploration of a European-centered strawberry diversity panel provides markers and candidate genes for the control of fruit quality traits

Fruit quality traits are major breeding targets in cultivated strawberry (Fragaria x ananassa). Taking into account the requirements of both growers and consumers when selecting high quality cultivars is a real challenge. Here, we used an original diversity panel enriched with unique European accessions and the 50K FanaSNP array to highlight the evolution of strawberry diversity over the past 160 years, investigate the molecular basis of 12 major fruit quality traits by GWAS, and provide genetic markers for breeding. Results show that considerable improvements of key breeding targets including fruit weight, firmness, composition and appearance occurred simultaneously in European and American populations. Despite the high genetic diversity of our panel, we observed a drop in nucleotide diversity in certain chromosome regions, revealing the impact of selection. GWAS identified 71 associations with 11 quality traits and, while validating known associations (firmness, sugar), highlighted the predominance of new QTL, demonstrating the value of using untapped genetic resources. Three of the six selective sweeps detected are related to glossiness or skin resistance, two little-studied traits important for fruit attractiveness and, potentially, postharvest shelf-life. Moreover, major QTL for firmness, glossiness, skin resistance and susceptibility to bruising are found within a low diversity region of chromosome 3D. Stringent search for candidate genes underlying QTL uncovered strong candidates for fruit color, firmness, sugar and acid composition, glossiness and skin resistance. Overall, our study provides a potential avenue for extending shelf-life without compromising flavor and color as well as the genetic markers needed to achieve this goal.

genetics↗

bioRxiv2023

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

microbiology↗

bioRxiv2022

Cuticle architecture and mechanical properties: a functional relationship delineated through correlated multimodal imaging

O_LICuticle are multifunctional hydrophobic biocomposites that protect aerial organs of plants. Along plant development, plant cuticle must accommodate different mechanical constraints combining extensibility and stiffness, the corresponding structure-function relationships are unknown. Recent data showed a fine architectural tuning of the cuticle architecture and the corresponding chemical clusters along fruit development which raise the question of their impact on the mechanical properties of the cuticle. C_LIO_LIWe investigated the in-depth nanomechanical properties of tomato fruit cuticle from early development to ripening, in relation to chemical and structural heterogeneities by developing a correlative multimodal imaging approach. C_LIO_LIUnprecedented sharps heterogeneities were evidenced with the highlighting of an in-depth mechanical gradient and a soft central furrow that were maintained throughout the plant development despite the overall increase in elastic modulus. In addition, we demonstrated that these local mechanical areas are correlated to chemical and structural gradients. C_LIO_LIThis study shed light on a fine tuning of mechanical properties of cuticle through the modulation of their architecture, providing new insight for our understanding of structure-function relationships of plant cuticle and for the design of biosinpired material. C_LI

plant biology↗

bioRxiv2022

Characterization of Morreton Virus (MORV) as a Novel Oncolytic Virotherapy Platform for Liver Cancers

Morreton virus (MORV) is a novel oncolytic Vesiculovirus, genetically distinct from vesicular stomatitis virus (VSV). we report that MORV induced potent cytopathic effects in a panel of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines. In addition, high intranasal doses of MORV were not associated with significant adverse effects and were well tolerated in mice bearing liver tumor xenografts and syngeneic liver cancers. Furthermore, single intratumoral treatments with MORV (1 x 107 TCID50) triggered a robust antitumor immune response leading to substantial tumor regression and disease control in a syngeneic CCA model, using 10-fold lower dose compared to VSV (1 x 108 TCID50). In addition, MORV and VSV both induced prominent tumor growth delay in immunodeficient mice bearing Hep3B hepatocellular carcinoma (HCC) but not in mice bearing HuCCT-1 CCA xenografts. Our findings indicate that wild-type MORV is safe and can induce potent tumor regression in HCC and CCA animal models without adverse events via immune-mediated and immune-independent mechanisms. Further development and clinical translation of MORV as virotherapy for liver cancers are warranted.

immunology↗

bioRxiv2020

Surfactant-free production of biomimetic artificial cells using PDMS-based microfluidics

Microfluidic-based production of cellular mimics (e.g. giant vesicles) presents a paradigm-shift in the development of artificial cells. While encapsulation rates are high and vesicles are mono-disperse compared to swelling-based techniques, current microfluidic emulsion-based methods heavily rely on the addition of additives such as surfactants, glycerol and even ethanol to produce stable vesicles. In this work, we present a microfluidic platform designed for the production of cellular mimics in the form of giant unilamellar vesicles (GUVs). Our PDMS-based device comprises a double cross-junction and a serpentine-shaped shear inducing module to produce surfactant-free and additive-free monodisperse biomimetic GUVs. Vesicles can be made with neutral and charged lipids in physiological buffers and, unlike previous works, it is possible to produce them with pure water both inside and outside. By not employing surfactants such as block co-polymers, additives like glycerol, and long-chain poly-vinyl alcohol that are known to alter the properties of lipid membranes, the vesicles are rendered truly biomimetic. The membrane functionality and stability are validated by lipid diffusion, membrane protein incorporation, and leakage assays. To demonstrate the usability of the GUVs using this method, various macromolecules such as DNA, smaller liposomes, mammalian cells and even microspheres are encapsulated within the GUVs.

synthetic biology↗

bioRxiv2020

Novel ionic liquids-based extraction method that preserves molecular structure from cutin

The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plants outermost defensive barrier - the cuticle. Cutin influences many biological processes in planta however due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterisation of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymers are highly esterified, amorphous and have the typical monomeric composition as demonstrated by solid state NMR, complemented by spectroscopic (GC-MS), thermal (DSC) and x-ray scattering (WAXS) analyses. A systematic study by solution-state NMR of cryogenically milled cutins extracted from Micro-Tom tomatoes (the wild type and the gpat6 and cus1 mutants) was undertaken. Their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters, were solved. The acquired data demonstrate the existence of free hydroxyl groups in cutin and reveal novel insights on how the mutations impact the esterification arrangement of cutin. Compared to conventional approaches, the usage of ionic liquids for the study of plant polyesters opens new avenues since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes now essential tools to fingerprint the multi-functionality and the structure of cutin in planta.

plant biology↗