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Tuning Cellular Biological Functions Through the Controlled Release of NO from a Porous Ti‐MOF
Laboratoire Institut des Matériaux Poreux de Paris - Rosana V. Pinto Dr. Sujing Wang Dr. Sergio R. Tavares Prof. João Pires Prof. Fernando Antunes Dr. Alexandre Vimont Prof. Guillaume Clet Prof. Marco Daturi
Angew. Chem. Int. Ed - 59(13) - https://doi.org/10.1002/anie.201913135 - 2020
Materials for the controlled release of nitric oxide (NO) are of interest for therapeutic applications. However, to date, many suffer from toxicity and stability issues, as well as poor performance. Herein, we propose a new NO adsorption/release mechanism through the formation of nitrites on the skeleton of a titanium‐based metal–organic framework (MOF) that we named MIP‐177, featuring a suitable set of properties for such an application: (i) high NO storage capacity (3 μmol mg−1solid), (ii) excellent biocompatibility at therapeutic relevant concentrations (no cytotoxicity at 90 μg mL−1 for wound healing) due to its high stability in biological media (<9 % degradation in 72 hours) and (iii) slow NO release in biological media (≈2 hours for 90 % release). The prospective application of MIP‐177 is demonstrated through NO‐driven control of mitochondrial respiration in cells and stimulation of cell migration, paving the way for the design of new NO delivery systems for wound healing therapy.
Encapsulation of Microperoxidase-8 in MIL-101(Cr)-X Nanoparticles: Influence of Metal–Organic Framework Functionalization on Enzymatic Immobilization and Catalytic Activity
Laboratoire Institut des Matériaux Poreux de Paris - Effrosyni Gkaniatsou, Rémy Ricoux*, Kalani Kariyawasam, Ingrid Stenger, Benhui Fan, Narjès Ayoub, Samanta Salas, Gilles Patriarche, Christian Serre, Jean-Pierre Mahy, Nathalie Steunou, and Clémence Sicard
ACS Publications - 3(4) 3233–3243 - https://doi.org/10.1021/acsanm.9b02464 - 2020
Microperoxidase 8 (MP8) was immobilized within MIL-101(Cr) bearing terephthalate linkers with functionalized groups (-NH2 and -SO3H). A synthesis protocol for MIL-101(Cr)-SO3H that avoids the use of toxic Cr(VI) and HF was developed. The electrostatic interactions between the MP8 molecules and the MOF matrices were found to be crucial for a successful immobilization. Raman spectroscopy revealed the dispersion of the immobilized MP8 molecules in MIL-101(Cr)-X matrices as monomers without aggregation. The presence of functional groups resulted in higher amounts of immobilized MP8 in comparison to the bare MIL-101(Cr). The catalytic activity of MP8@MIL-101(Cr)-NH2 per material mass was higher than that for MP8@MIL-101(Cr). The presence of free amino groups can thus improve the immobilization efficiency, leading to a higher amount of catalytically active species and improving the subsequent catalytic activity of the heterogeneous biocatalysts. MP8@MIL(Cr)-X also successfully catalyzed the selective oxidation of thioanisole derivatives into sulfoxides
Toward a Rational Design of Titanium Metal-Organic Frameworks
Laboratoire Institut des Matériaux Poreux de Paris - Author links open overlay panelSujingWang128HelgeReinsch3NicolasHeymans4MohammadWahiduzzaman5CharlotteMartineau-Corcos67GuyDe Weireld4GuillaumeMaurin5ChristianSerre1
MATTER - 2(2) 440-450 - https://doi.org/10.1016/j.matt.2019.11.002 - 2020
Rational design and synthesis of metal-organic frameworks (MOFs) is of particular interest in fine-tuning the crystalline structures for given targeting applications. Considerable advance of this topic has been achieved for MOFs built with a large number of metal species but not titanium. The complex and unpredictable titanium chemistry in solution not only leads to the difficulty of isolating crystalline Ti-MOFs via direct synthesis but also results in the challenge of maintaining control over ordered structures. We demonstrated a Ti-O cluster guided green scalable preparation of a Ti-MOF (MIP-207) in a controlled manner with both post-synthetic and one-pot reaction routes. The chemical environment and functionality of the MOF structural void could be easily tuned by adopting the mixed-linker strategy, which finally resulted in an adjustable performance in CO2 capture over N2. This provides a new avenue for the rational design of Ti-MOFs in energy- and environment-related applications.
Highly Porous Hybrid Metal–Organic Nanoparticles Loaded with Gemcitabine Monophosphate: a Multimodal Approach to Improve Chemo‐ and Radiotherapy
Laboratoire Institut des Matériaux Poreux de Paris - Dr. Xue Li Dr. Erika Porcel Dr. Mario Menendez‐Miranda Jingwen Qiu Xiaomin Yang Dr. Christian Serre Alexandra Pastor Dr. Didier Desmaële
ChemMedChem - 15(3) - https://doi.org/10.1002/cmdc.201900596 - 2020
Nanomedicine recently emerged as a novel strategy to improve the performance of radiotherapy. Herein we report the first application of radioenhancers made of nanoscale metal‐organic frameworks (nanoMOFs), loaded with gemcitabine monophosphate (Gem‐MP), a radiosensitizing anticancer drug. Iron trimesate nanoMOFs possess a regular porous structure with oxocentered Fe trimers separated by around 5 Å (trimesate linkers). This porosity is favorable to diffuse the electrons emitted from nanoMOFs due to activation by γ radiation, leading to water radiolysis and generation of hydroxyl radicals which create nanoscale damages in cancer cells. Moreover, nanoMOFs act as “Trojan horses”, carrying their Gem‐MP cargo inside cancer cells to interfere with DNA repair. By displaying different mechanisms of action, both nanoMOFs and incorporated Gem‐MP contribute to improve radiation efficacy. The radiation enhancement factor of Gem‐MP loaded nanoMOFs reaches 1.8, one of the highest values ever reported. These results pave the way toward the design of engineered nanoparticles in which each component plays a role in cancer treatment by radiotherapy.
Methanol and Humidity Capacitive Sensors Based on Thin Films of MOF Nanoparticles
Laboratoire Institut des Matériaux Poreux de Paris - Miguel A. Andrés, Mani Teja Vijjapu, Sandeep G. Surya, Osama Shekhah, Khaled Nabil Salama, Christian Serre, Mohamed Eddaoudi, Olivier Roubeau, and Ignacio Gascón
ACS Publications - 12(3) 4155–4162 - https://doi.org/10.1021/acsami.9b20763 - 2020
The successful development of modern gas sensing technologies requires high sensitivity and selectivity coupled to cost effectiveness, which implies the necessity to miniaturize devices while reducing the amount of sensing material. The appealing alternative of integrating nanoparticles of a porous metal–organic framework (MOF) onto capacitive sensors based on interdigitated electrode (IDE) chips is presented. We report the deposition of MIL-96(Al) MOF thin films via the Langmuir–Blodgett (LB) method on the IDE chips, which allowed the study of their gas/vapor sensing properties. First, sorption studies of several organic vapors like methanol, toluene, chloroform, etc. were conducted on bulk MOF. The sorption data revealed that MIL-96(Al) presents high affinity toward water and methanol. Later on, ordered LB monolayer films of MIL-96(Al) particles of ∼200 nm were successfully deposited onto IDE chips with homogeneous coverage of the surface in comparison to conventional thin film fabrication techniques such as drop-casting. The sensing tests showed that MOF LB films were selective for water and methanol, and short response/recovery times were achieved. Finally, chemical vapor deposition (CVD) of a porous thin film of Parylene C (thickness ∼250–300 nm) was performed on top of the MOF LB films to fabricate a thin selective layer. The sensing results showed an increase in the water selectivity and sensitivity, while those of methanol showed a huge decrease. These results prove the feasibility of the LB technique for the fabrication of ordered MOF thin films onto IDE chips using very small MOF quantities.
Hexane isomers separation on an isoreticular series of microporous Zr carboxylate metal organic frameworks
Laboratoire Institut des Matériaux Poreux de Paris - Adriano Henrique, Tanmoy Maity, Hengli Zhao, de Pedro F. Brântuas, Alírio E. Rodrigues, Farid Nouar, Aziz Ghoufi, Guillaume Maurin, José A. C. Silva and Christian Serre
journal of materials chemistry A - 8 17780-17789 - https://doi.org/10.1039/D0TA05538G - 2020
A series of isoreticular Zr carboxylate MOFs, MIL-140A, B and C, exhibiting 1D microporous triangular shaped channels and based on different aromatic dicarboxylate ligands (1,4-BDC, 2,6-NDC and 4,4′-BPDC, respectively), were investigated by chromatographic breakthrough experiments regarding their ability to separate hexane isomers (nC6/2MP/3MP/23DMB/22DMB). Both single and equimolar multicomponent experiments were performed at the temperatures 343, 373, and 423 K and a total hydrocarbon pressure up to 50.0 kPa using the MIL-140B form. The elution order is similar to that of the normal boiling point of the compounds nC6 > 2MP > 3MP > 23DMB > 22DMB. It is noteworthy that this material enables separation of the hexane isomers by class, linear > mono-branched > di-branched, with a selectivity (linear + mono-branched isomers/di-branched isomers) up to 10 at 343 K, decreasing, however, as the temperature increases. Grand canonical Monte Carlo simulations were further performed to gain insight into the adsorption/separation mechanisms, highlighting the crucial need to consider a tiny tilting of the organic linkers for capturing the experimental observations. The impact of the pore size was finally assessed through the comparison with MIL-140A and MIL-140C, respectively, based on multicomponent experiments at 343 K. We evidenced a significant decrease of the selectivity (about 2) in both cases while the loadings were decreased or increased for MIL-140A and MIL-140C, respectively. Additionally, MIL-140C was demonstrated to exhibit an uncommon shift in the elution order occurring between nC6 and 3MP, 3MP being the last compound to saturate in the column.
First Example of Protonation of Ruddlesden–Popper Sr 2 IrO 4 : A Route to Enhanced Water Oxidation Catalysts
Laboratoire Institut des Matériaux Poreux de Paris - Ronghuang Zhang, Paul E. Pearce, Vanessa Pimenta, Jordi Cabana, Heifang Li, Daniel Alves Dalla Corte, Artem M. Abakumov, Gwenaëlle Rousse, Domitille Giaume, Michael Deschamps, and Alexis Grimaud
Chemistry of Materials, American Chemical Society - 32 (8) 3499-3509 - https://doi.org/10.1021/acs.chemmater.0c00432 - 2020
Water electrolysis is considered to be a promising way to store and convert excess renewable energies into hydrogen, which is of high value for many chemical transformation processes such as the Haber-Bosch process. The main challenge to promote the deployment of the polymer electrolyte membrane water electrolysis (PEMWE) technology lies in the design of robust catalysts for the oxygen evolution reaction (OER) under acidic conditions, since most of the transition metal-based oxides undergo structural degradation under these harsh acidic conditions. To broaden the variety of candidate materials as OER catalysts, a cation-exchange synthetic route was recently explored to reach crystalline pronated iridates with unique structural properties and stability. In this work, a new protonated phase H3.6IrO4·3.7H2O, prepared via Sr2+/H+ cation exchange at room temperature starting from the parent Ruddlesden–Popper Sr2IrO4 phase, is described. This is the first discovery of crystalline protonated iridate forming from a perovskite-like phase, adopting a layered structure with apex-linked IrO6 octahedra. Furthermore, H3.6IrO4·3.7H2O is found to possess not only an enhanced specific catalytic activity, superior to that of other perovskite-based iridates, but also a mass activity comparable to that of nanosized IrOx particles, while showing an improved catalytic stability owing to its ability to reversibly exchange protons in acid.
Revealing the Impact of Electrolyte Composition for Co-Based Water Oxidation Catalysts by the Study of Reaction Kinetics Parameters
Laboratoire Institut des Matériaux Poreux de Paris - Yan Duan, Nicolas Dubouis, Jiaqiang Huang, Daniel Alves Dalla Corte, Vanessa Pimenta, Zhichuan J. Xu, and Alexis Grimaud
ACS Publications - 7 4160–4170 - https://doi.org/10.1021/acscatal.0c00490 - 2020
Recent studies have revealed the critical role played by the electrolyte composition on the oxygen evolution reaction (OER) kinetics on the surface of highly active catalysts. While numerous works were devoted to understand the effect of the electrolyte composition on the physical properties of the catalysts’ surface, very little is known yet about its exact impact on the OER kinetics parameters. In this work, we reveal that the origin for the electrolyte-dependent OER activity for Co-based catalysts originates from two different effects. Increasing the alkaline electrolyte concentration for La1–xSrxCoO3−δ perovskites with x > 0 and for amorphous CoOOH increases the pre-exponential factor, which can be explained either by an increase of the concentration of active sites or by a change in the entropy of activation. However, changing the alkali cation results in a decrease of the apparent activation enthalpy for Fe-containing amorphous films, traducing a change in intermediates’ binding energies.
Machine Learning to Improve the Sensing of Biomolecules by Conical Track-Etched Nanopore
Laboratoire Institut des Matériaux Poreux de Paris - Nathan Meyer, Jean-Marc Janot , Mathilde Lepoitevin , Michaël Smietana ,Jean-Jacques Vasseur ,Joan Torrent and Sébastien Balme
Biosensors - 10(10) 140 - https://doi.org/10.3390/bios10100140 - 2020
Single nanopore is a powerful platform to detect, discriminate and identify biomacromolecules. Among the different devices, the conical nanopores obtained by the track-etched technique on a polymer film are stable and easy to functionalize. However, these advantages are hampered by their high aspect ratio that avoids the discrimination of similar samples. Using machine learning, we demonstrate an improved resolution so that it can identify short single- and double-stranded DNA (10- and 40-mers). We have characterized each current blockade event by the relative intensity, dwell time, surface area and both the right and left slope. We show an overlap of the relative current blockade amplitudes and dwell time distributions that prevents their identification. We define the different parameters that characterize the events as features and the type of DNA sample as the target. By applying support-vector machines to discriminate each sample, we show accuracy between 50% and 72% by using two features that distinctly classify the data points. Finally, we achieved an increased accuracy (up to 82%) when five features were implemented.
Metabolic cost of rapid adaptation of single yeast cells
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Gabrielle Woronoff, Philippe Nghe, Jean Baudry, Laurent Boitard, Erez Braun, Andrew D. Griffiths, and Jérôme Bibette
PNAS - 117 (20) 10660-10666 - https://doi.org/10.1073/pnas.1913767117 - 2020
Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.
High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Annabelle Gérard, Adam Woolfe, […]Colin Brenan
Nat Biotechnol. - 38 715–721 - https://doi.org/10.1038/s41587-020-0466-7 - 2020
Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.
Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Marco Molari, Klaus Eyer, Jean Baudry, Simona Cocco, Rémi Monasson
e-Life - 1 619 - DOI: 10.7554/eLife.55678 - 2020
Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modeling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.
Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Marco Molari, Klaus Eyer, Jean Baudry, Simona Cocco, Rémi Monasson
e-Life - 1 619 - DOI: 10.7554/eLife.55678 - 2020
Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modeling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.
The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Magda Rybczynska, Jean Baudry, Eyer Klaus
ELSEVIER - 38 5337-5342 - https://doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.
Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Nature Protocols - 15 2920–2955 - https://www.nature.com/articles/s41596-020-0354-0 - 2020
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Klaus Eyer, Carlos Castrillon, Guilhem Chenon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths and Jean Baudry
Nat Immunol. - 9 205 - doi.org/10.4049/jimmunol.2000112 - 2020
One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.
Ultrafast photomechanical transduction through thermophoretic implosion
Laboratoire Micromégas - N. Kavokine, S. Zou, R. Liu, H. Zhong, A. Nigues, B. Zou and L. Bocquet
Nature Communications - 11(1) 50 - doi.org/10.1038/s41467-019-13912-w - 2020
Since the historical experiments of Crookes, the direct manipulation of matter by light has been both a challenge and a source of scientific debate. Here we show that laser illumination allows to displace a vial of nanoparticle solution over centimetre-scale distances. Cantilever-based force measurements show that the movement is due to millisecond-long force spikes, which are synchronised with a sound emission. We observe that the nanoparticles undergo negative thermophoresis, and ultrafast imaging reveals that the force spikes are followed by the explosive growth of a bubble in the solution. We propose a mechanism accounting for the propulsion based on a thermophoretic instability of the nanoparticle cloud, analogous to the Jeans’s instability that occurs in gravitational systems. Our experiments demonstrate a new type of laser propulsion and a remarkably violent actuation of soft matter, reminiscent of the strategy used by certain plants to propel their spores.
Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST.
Laboratoire Physique des biomolécules - Chekli, Yankel; Peron-Cane, Caroline; Dell'Arciprete, Dario; Allemand, Jean-François; Li, Chenge; Ghigo, Jean-Marc; Gautier, Arnaud; Lebreton, Alice; Desprat, Nicolas; Beloin, Christophe
journal article - 10(1) 15791 - 10.1038/s41598-020-72498-2 - 2020
Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation 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 used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.
Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells.
Laboratoire Physique des biomolécules - Peron-Cane, Caroline; Fernandez, José-Carlos; Leblanc, Julien; Wingertsmann, Laure; Gautier, Arnaud; Desprat, Nicolas; Lebreton, Alice
journal article - 16(10) e1009001 - 10.1371/journal.ppat.1009001 - 2020
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.s.
Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking
Laboratoire Physique des biomolécules - Martin Rieu; Valle-Orero, Jessica; Ducos, Bertrand; Allemand, Jean-François; Croquette, Vincent
bioRxiv - - 10.1101/2020.09.30.321232 - 2020
Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.Competing Interest StatementThe authors have declared no competing interest.

515 publications.