Biology subjects

Desprat, N.

Publications and source records attributed to Desprat, N..

6 recordsLinked to original sources

bioRxiv2025

Virulence priming of Listeria monocytogenes inside replication-permissive endolysosomes

Listeria monocytogenes (Lm), the foodborne pathogen responsible for listeriosis, is well-known for its intracellular lifestyle. After entering epithelial cells, it can evade within minutes from its internalization vacuole into the cytosol, or transiently co-opt and replicate within endomembrane compartments termed epithelial Spacious ListeriA-containing Phagosomes (eSLAPs). Although eSLAPs display endolysosomal features, we bring evidence that they lack cathepsin D activity and are permeated. Indeed, sustained eSLAP membrane perforation by listeriolysin O is crucial for accommodating bacterial growth and vacuole expansion. Conversely, we show that Lm broad range phospholipase C PlcB is the main escape factor from eSLAPs. Unexpectedly, we find that Lm PrfA-dependent cytosolic virulence program is primed inside eSLAPs. As a consequence, the bacteria released from eSLAPs are pre-equipped with the actin assembly-inducing protein ActA and can recruit actin faster. We propose that a delayed access to the cytosol benefit eSLAP-transiting bacteria by favoring their dissemination or evasion from xenophagy.

microbiology↗

bioRxiv2023

Magnetic actuation of otoliths allows behavioral and brain-wide neuronal exploration of vestibulo-motor processing in larval zebrafish.

The vestibular system in the inner ear plays a central role in sensorimotor control by informing the brain about the orientation and acceleration of the head. However, most experiments in neurophysiology are performed using head-fixed configurations, depriving animals of vestibular inputs. To overcome this limitation, we decorated the utricular otolith of the vestibular system in larval zebrafish with paramagnetic nanoparticles. This procedure effectively endowed the animal with magneto-sensitive capacities: applied magnetic field gradients induced forces on the otoliths resulting in robust behavioral responses comparable to that evoked by rotating the animal by up to 25{degrees}. We recorded the whole-brain neuronal response to this fictive motion stimulation using light-sheet functional imaging. Experiments performed in unilaterally injected fish revealed the activation of a commissural inhibition between the brain hemispheres. This magnetic-based stimulation technique for larval zebrafish opens new perspectives to functionally dissect the neural circuits underlying vestibular processing and to develop multisensory virtual environments, including vestibular feedback. HighlightsO_LIAfter injecting a ferrofluid into the inner ear of a larval zebrafish, the ear-stones can be actuated via magnetic forces. C_LIO_LIThis method allows one to elicit vestibular-like behavioral responses without impairing physiological inner ear functions. C_LIO_LIIt is compatible with brain-scale functional imaging and thus offers a promising avenue to investigate the neural underpinnings of vestibular-driven behaviors. C_LI eTOCFerrofluid injection into zebrafish inner ear allows magnetic manipulation of ear-stones to evoke vestibular responses in static animals. This in vivo method is compatible with brain-scale imaging, offering a promising approach to investigate neural mechanisms underlying vestibular-driven behaviors.

neuroscience↗

bioRxiv2020

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

microbiology↗

bioRxiv2019

Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues. Author summaryBacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite -and in fact, thanks to-- the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host.

microbiology↗

bioRxiv2018

Experimental Evolution of Cell Shape in Bacteria

Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1,000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

microbiology↗

bioRxiv2017

Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis

Bacterial biofilms are spatially structured communities, within which bacteria can differentiate depending on environmental conditions. During biofilm formation, bacteria attach to a surface and use cell-cell contacts to convey the signals required for the coordination of biofilm morphogenesis. How bacteria can maintain both substrate adhesions and cell-cell contacts during the expansion of a microcolony is still a critical yet poorly understood phenomenon. Here, we describe the development of time-resolved methods to measure substrate adhesion at the single cell level during the formation of E. coli and P. aeruginosa microcolonies. We show that bacterial adhesion is asymmetrically distributed along the cell body. Higher adhesion forces at old poles put the daughter cells under tension and force them to slide along each other. These rearrangements increase cell-cell contacts and the circularity of the colony. We propose a mechanical model based on the microscopic details of adhesive links, which recapitulates microcolony morphogenesis and quantitatively predicts bacterial adhesion from simple time lapse movies. These results explain how the distribution of adhesion forces at the subcellular level directs the shape of bacterial colonies, which ultimately dictates the circulation of secreted signals.

biophysics↗