Biology subjects

Ducret, A.

Publications and source records attributed to Ducret, A..

11 recordsLinked to original sources

bioRxiv2025

Col6 deficiency in a zebrafish model of Bethlem myopathy leads to dysfunction of the muscle dihydropyridine receptor

Bethlem myopathy (BM) results from mutations in genes encoding one of the three chains of collagen VI (ColVI). This muscle disease is characterized by skeletal muscle weakness and wasting worsening with age. How alteration in ColVI present outside muscle fibers in the extracellular matrix induces dysfunction within muscle fibers is still misunderstood. Here we explored intracellular Ca2+ handling properties in isolated fast skeletal muscle fibers from adult zebrafish harboring a mutation (col6a1{Delta}ex14) that is the most frequently found in BM patients. Col6a1{Delta}ex14 fish muscle exhibited progressive loss of ColVI deposition, defects in basement membrane and ColVI intracellular accumulation. By combining voltage-clamp and intracellular Ca2+ measurements on isolated fibers, we showed that voltage-dependence of intramembrane charge movements produced by the activation of CaV1.1 controlling sarcoplasmic reticulum (SR) Ca2+ release and voltage-dependence of depolarization-induced SR Ca2+ release were shifted toward negative potentials in col6a1{Delta}ex14 fish. These changes in voltage-dependence gave rise to larger SR Ca2+ leak at voltages close to resting values and to higher frequency of spontaneous SR Ca2+ release elementary events in mutant fish. Trunk muscle force and swimming performance were also found to be reduced in col6a1{Delta}ex14 fish and mis-localization of CaV1.1 subunits clusters was observed in mutant fibers t-tubules. These data indicate that ColVI deficiency in BM leads to CaV1.1 dysfunction that contributes to promote a pathogenic SR Ca2+ leak responsible for progressive muscle weakness and wasting. CaV1.1 could represent the still elusive transmembrane link allowing altered myomatrix to transduce pathogenic signals within muscle.

pathology↗

bioRxiv2025

A local composition of peptidoglycan drives the division site selection by MapZ in Streptococcus pneumoniae

Accurate division site placement is essential for bacterial cells to produce viable daughter cells with proper size and appropriate functional features. In the opportunistic pathogen bacterium Streptococcus pneumoniae, the positioning of the division site has been shown to depend on both the protein MapZ and chromosome segregation. However, the nature of this interplay and the molecular determinants guiding division site localization remained unclear. Here we demonstrated that the division site is positioned at the cell equator, the widest part of the cell body, rather than at mid-cell. In addition, we observed that the localization of MapZ and/or the divisome remain unaffected even in the absence of properly segregated DNA, indicating that chromosome segregation does not contribute to division site selection. Our findings further reveal that MapZ localization depends on the activities of two PG hydrolases DacA and DacB, whose sequential recruitment to the division site during early PG synthesis drives the formation of a distinctive PG signature required for MapZ binding. These results support a model in which MapZ identifies the division site by recognizing a specific PG composition produced only during the early stages of cell division. This PG composition becomes enriched at the cell equators which will eventually serve as the division site of the daughter cells.

microbiology↗

bioRxiv2025

Human Lung Alveolar Model with an Autologous Innate and Adaptive Immune Compartment

Lung-resident immune cells, spanning both innate and adaptive compartments, preserve the integrity of the respiratory barrier, but become pathogenic if dysregulated1. Current in vitro organoid models aim to replicate interactions between the alveolar epithelium and immune cells but have not yet incorporated lung-specific immune cells critical for tissue residency2. Here we address this shortcoming by describing human lung alveolar immuno-organoids (LIO) that contain an autologous tissue-resident lymphoid compartment, primarily composed of tissue-resident memory T cells (TRMs). Additionally, we introduce lung alveolar immuno-organoids with myeloid cells (LIOM), which include both TRMs and a macrophage-rich alveolar myeloid compartment. The resident immune cells formed a stable immune-epithelial system, frequently interacting with the epithelium and promoting a regenerative alveolar transcriptomic profile. To understand how dysregulated inflammation perturbed the respiratory barrier, we simulated T-cell-mediated inflammation in LIOs and LIOMs and used single-cell transcriptomic analyses to uncover the molecular mechanisms driving immune responses. The presence of innate cells induced a shift in T cell identity from cytotoxic to immunosuppressive, reducing epithelial cell killing and inflammation. Based on insights obtained with bulk RNA-seq data from the phase 3 IMpower150 trial, we tested whether LIOM cultures could model clinically-relevant but poorly understood pulmonary side effects caused by immunotherapies such as the checkpoint inhibitor atezolizumab3. We observed a decrease in immunosuppressive T cells and identified gene signatures that matched the transcriptomic profile of patients with drug-induced pneumonitis. Given its effectiveness in capturing outcomes and mechanisms associated with a prevalent pulmonary disease, this system unlocks opportunities for studying a wide range of immune-related pathologies in the lung.

cell biology↗

bioRxiv2024

Characterization of a GpsB-associated regulator of PBP1a reveals the organization of the cell wall remodeling complex of Streptococcus pneumoniae

Class A PBPs (aPBPs) play a key role in the biosynthesis and remodeling of peptidoglycan, the main component of the bacterial cell wall. The human bacterial pathogen Streptococcus pneumoniae produces three aPBPs, which are regulated to maintain the bacteriums ovoid shape. Although their exact functions remain unclear, evidence suggests that PBP1a and PBP2a activities are closely coordinated. In this study, we elucidated the function of an unknown function protein named GarP (GpsB-associated regulator of PBP1a), in the regulation of PBP1a activity. We showed that GarP localizes to the division septum and its absence leads to morphological defects. We further identified a GpsB-binding motif in GarP as well as in PBP2a, the PG deacetylase PgdA and the muramidase MpgA. Our analysis of genetic and protein interactions, combined with cell imaging, supports a model of a molecular complex that coordinates PG remodeling during S. pneumoniae cell division.

microbiology↗

bioRxiv2024

Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors such as the ParABS system or SMC complexes, are usually essential for this process but are surprisingly dispensable in Streptococcus pneumoniae. Rather, chromosome segregation in S. pneumoniae relies on the protein RocS, although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. Specifically, we investigated the respective function of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD) and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) defined by three conserved glutamines which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP, and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria. IMPORTANCEBacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein RocS, a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation shed light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins, while broadening the repertoire of partitioning systems used in bacteria.

microbiology↗

bioRxiv2024

Bacterial motility depends on a critical flagellum length and energy-optimised assembly

The flagellum is the most complex macromolecular structure known in bacteria and comprised of around two dozen distinct proteins. The main building block of the long, external flagellar filament, flagellin, is secreted through the flagellar type-III secretion system at a remarkable rate of several tens of thousands amino acids per second, significantly surpassing the rates achieved by other pore-based protein secretion systems. The evolutionary implications and potential benefits of this high secretion rate for flagellum assembly and function, however, have remained elusive. In this study, we provide both experimental and theoretical evidence that the flagellar secretion rate has been evolutionarily optimized to facilitate rapid and efficient construction of a functional flagellum. By synchronizing flagellar assembly, we found that a minimal filament length of 2.5 {micro}m was required for swimming motility. Biophysical modelling revealed that this minimal filament length threshold resulted from an elasto-hydrodynamic instability of the whole swimming cell, dependent on the filament length. Furthermore, we developed a stepwise filament labeling method combined with electron microscopy visualization to validate predicted flagellin secretion rates of up to 10,000 amino acids per second. A biophysical model of flagellum growth demonstrates that the observed high flagellin secretion rate efficiently balances filament elongation and energy consumption, thereby enabling motility in the shortest amount of time. Taken together, these insights underscore the evolutionary pressures that have shaped the development and optimization of the flagellum and type-III secretion system, illuminating the intricate interplay between functionality and efficiency in assembly of large macromolecular structures. Significance statementOur study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.

microbiology↗

bioRxiv2023

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus

To maintain cell integrity and facilitate cell division in Staphylococcus aureus, a well-coordinated interplay between membrane biogenesis, peptidoglycan formation, and teichoic acid synthesis is crucial. However, the molecular mechanisms and regulatory pathways that underpin their coordination are still poorly understood. CozE constitute a conserved family of membrane proteins implicated in cell division via regulation of penicillin binding proteins. It has been shown that the two staphylococcal cozE genes (cozEa and cozEb) constitute a synthetic lethal gene pair. Depletion of CozEa and CozEb simultaneously in S. aureus resulted in severely defective cell division phenotypes, reminiscent of cell lacking lipoteichoic acid (LTA). Indeed, we demonstrate that there is an intricate interplay between CozE, biosynthesis of LTA, and membrane homeostasis in S. aureus. By screening for potential genetic links, we establish that there is synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. On the contrary, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozEa and CozEb was alleviated. Furthermore, by immunoblotting, we found that CozEb plays a unique role in controlling LTA polymer length and stability. Using reconstituted proteoliposomes, we also demonstrated that CozE proteins modulate the glycolipid flipping activity of LtaA in vitro. Together, the results demonstrate a new function of CozE proteins, facilitating proper membrane homeostasis and LTA biosynthesis in S. aureus.

microbiology↗

bioRxiv2023

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a new model for conjugation that revises our understanding of the DNA transport mechanism and the dissemination of drug resistance in complex bacterial communities. One-Sentence SummaryPlasmid DNA passes through the F pilus during conjugational transfer between physically distant bacteria.

microbiology↗

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↗

bioRxiv2018

RocS drives chromosome segregation and nucleoid occlusion in Streptococcus pneumoniae

Segregation of replicated chromosomes in bacteria is poorly understood outside some prominent model strains and even less is known about how it is coordinated with other cellular processes. Here we report that RocS is crucial for chromosome segregation in the opportunistic human pathogen Streptococcus pneumoniae. RocS is membrane-bound and interacts both with DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the tyrosine-autokinase CpsD required for polysaccharide capsule biogenesis, which is crucial for S. pneumoniaes ability to prevent host immune detection. Altering the RocS-CpsD interaction drastically hinders chromosome partitioning and cell division. Altogether, this work reveals that RocS is the cornerstone of an atypical nucleoid occlusion system ensuring proper cell division in coordination with the biogenesis of a protective capsular layer.

microbiology↗

bioRxiv2018

Diverse spatial organization of photosynthetic membranes among purple nonsulfur bacteria

In diverse bacteria, proper cellular physiology requires the utilization of protein- or membrane-bound compartments that afford specific metabolic capabilities. One such compartment is the light-harvesting intracytoplasmic membrane (ICM) of purple nonsulfur bacteria (PNSB). Here we reveal that ICMs are subject to differential spatial organization among PNSB. We visualized ICMs in live cells of fourteen PNSB species by exploiting the natural autofluorescence of the photosynthetic machinery. We then quantitatively characterized ICM localization using automated computational analysis of autofluorescence patterns within single cells across the population. Our studies revealed that ICMs are localized in distinct subcellular patterns that differ between species; some PNSB elaborate ICMs throughout the cell, while others spatially restrict ICM to varying degrees. The most highly-restricted ICMs were localized in a specific pattern corresponding to progression of cell growth and division. An identical pattern of ICM restriction was conserved across at least two genera. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. This discovery of new diversity in bacterial cell organization has implications for understanding both the mechanisms underpinning spatial arrangement of bacterial compartments and the potential benefits of adopting different spatiotemporal patterns.

microbiology↗