Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Structures of the P. aeruginosa FleQ-FleN master regulators reveal large-scale conformational switching in motility and biofilm control.

Pseudomonas aeruginosa can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial survival and virulence. At the cellular level, many of the physiological transitions are orchestrated by the intracellular second messenger c-di-GMP and its receptor-effector FleQ. A bacterial enhancer binding protein, FleQ acts as a master regulator of both flagellar motility and adherence factor secretion and uses remarkably different transcription activation mechanisms depending on its dinucleotide loading state, adenosine triphosphatase (ATPase) activity, interactions with polymerase sigma (σ) factors, and complexation with a second ATPase, FleN. How the FleQ-FleN tandem can exert diverse effects through recognition of a conserved FleQ binding consensus has remained enigmatic. Here, we provide cryogenic electron microscopy (cryo-EM) structures of both c-di-GMP-bound and c-di-GMP-free FleQ-FleN complexes which deepen our understanding of the proteins' (di)nucleotide-dependent conformational switching and fine-tuned roles in gene expression regulation.

Draft Genome Sequence of Oenococcus kitaharae CRBO2176, Isolated from Homemade Water Kefir.

Here, we announce the draft genome sequence of an Oenococcus kitaharae strain isolated from homemade water kefir in Bordeaux, France. O. kitaharae CRBO2176 is deposited at the Biological Resources Center Oenology (CRBO) of the Institute of Vine and Wine Science (ISVV; Villenave d'Ornon, France).

Structural determinants of REMORIN nanodomain formation in anionic membranes.

Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.

Bacterial crystalline cellulose secretion via a supramolecular BcsHD scaffold.

Cellulose, the most abundant biopolymer on Earth, is not only the predominant constituent of plants but also a key extracellular polysaccharide in the biofilms of many bacterial species. Depending on the producers, chemical modifications, and three-dimensional assemblies, bacterial cellulose (BC) can present diverse degrees of crystallinity. Highly ordered, or crystalline, cellulose presents great economical relevance due to its ever-growing number of biotechnological applications. Even if some acetic acid bacteria have long been identified as BC superproducers, the molecular mechanisms determining the secretion of crystalline versus amorphous cellulose remain largely unknown. Here, we present structural and mechanistic insights into the role of the accessory subunits BcsH (CcpAx) and BcsD (CesD) that determine crystalline BC secretion in the Gluconacetobacter lineage. We show that oligomeric BcsH drives the assembly of BcsD into a supramolecular cytoskeletal scaffold that likely stabilizes the cellulose-extruding synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly.

Cytoskeletal components can turn wall-less spherical bacteria into kinking helices.

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems.

Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA-MexB-OprM and AcrA-AcrB-TolC, from Pseudomonas aeruginosa and Escherichia coli, respectively, depict a reduced interfacial contact between OMF and PAP, making unclear the comprehension of how OMF is recruited. Here, we show that a Q93R mutation of MexA located in the α-hairpin domain increases antibiotic resistance in the MexAQ93R-MexB-OprM-expressed strain. Electron microscopy single-particle analysis reveals that this mutation promotes the formation of tripartite complexes with OprM and non-cognate components OprN and TolC. Evidence indicates that MexAQ93R self-assembles into a hexameric form, likely due to interprotomer interactions between paired R93 and D113 amino acids. C-terminal deletion of OprM prevents the formation of tripartite complexes when mixed with MexA and MexB components but not when replacing MexA with MexAQ93R. This study reveals the Q93R MexA mutation and the OprM C-terminal peptide as molecular determinants modulating the assembly process efficacy with cognate and non-cognate OMFs, even though they are outside the interfacial contact. It provides insights into how OMF selectivity operates during the formation of the tripartite complex.

A Photoactivable Natural Product with Broad Antiviral Activity against Enveloped Viruses, Including Highly Pathogenic Coronaviruses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

Chirality Induction to CdSe Nanocrystals Self-Organized on Silica Nanohelices: Tuning Chiroptical Properties.

CdSe nanocrystals (NCs) were grafted on chiral silica nanoribbons, and the mechanism of resulting chirality induction was investigated. Because of their chiral organization, these NCs show optically active properties that depend strongly on their grafting densities and sizes of the NCs. The effect of the morphology of the chiral silica templates between helical (cylindrical curvature) vs twisted (saddle like curvature) ribbons was investigated. The g-factor of NCs-silica helical ribbons is larger than that of the NCs-silica twisted ribbons. Finally, rod-like NCs (QR) with different lengths were grafted on the twisted silica ribbons. Interestingly, their grafting direction with respect to the helix surface changed from side-grafting for short QR to tip-grafting for long rods and the corresponding CD spectra switched signs.

Investigation of the effects of two major secretory granules components, insulin and zinc, on human-IAPP amyloid aggregation and membrane damage.

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the ß-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Structural characterization of the EmrAB-TolC efflux complex from E. coli.

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Optically Active Perovskite CsPbBr3 Nanocrystals Helically Arranged on Inorganic Silica Nanohelices.

Perovskite nanocrystals (PNCs) exhibit excellent absorption and luminescent properties. Inorganic silica right (or left) handed nanohelices are used as chiral templates to induce optically active properties to CsPbBr3 PNCs grafted on their surfaces. In suspension, PNCs grafted on the nanohelices do not show any detectable chiroptical properties. In contrast, in a dried film state, they show large circular dichroism (CD) and circularly polarized luminescence (CPL) signals with dissymmetric factor up to 6 × 10-3. Grazing incidence X-ray scattering, tomography, and cryo-electron microscopy (EM) have shown closely and helically packed PNCs on the dried helices and much more loosely organized PNCs on helices in suspension. Simulations based on the coupled dipole method (CDM) demonstrate that the CD comes from the dipolar interaction between PNC assembled into a chiral structure and the CD decreases with the interparticle distance.

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues.

Invadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFß in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases.

Microfluidic diffusional sizing probes lipid nanodiscs formation.

The biophysical characterisation of membrane proteins and their interactions with lipids in native membrane habitat remains a major challenge. Indeed, traditional solubilisation procedures with detergents often causes the loss of native lipids surrounding membrane proteins, which ultimately impacts structural and functional properties. Recently, copolymer-based nanodiscs have emerged as a highly promising tool, thanks to their unique ability of solubilising membrane proteins directly from native membranes, in the shape of discoidal patches of lipid bilayers. While this methodology finally set us free from the use of detergents, some limitations are however associated with the use of such copolymers. Among them, one can cite the tedious control of the nanodiscs size, their instability in basic pH and in the presence of divalent cations. In this respect, many variants of the widely used Styrene Maleic Acid (SMA) copolymer have been developed to specifically address those limitations. With the multiplication of new SMA copolymer variants and the growing interest in copolymer-based nanodiscs for the characterisation of membrane proteins, there is a need to better understand and control their formation. Among the techniques used to characterise the solubilisation of lipid bilayer by amphipathic molecules, cryo-TEM, 31P NMR, DLS, ITC and fluorescence spectroscopy are the most widely used, with a consensus made in the sense that a combination of these techniques is required. In this work, we propose to evaluate the capacity of Microfluidic Diffusional Sizing (MDS) as a new method to follow copolymer nanodiscs formation. Originally designed to determine protein size through laminar flow diffusion, we present a novel application along with a protocol development to observe nanodiscs formation by MDS. We show that MDS allows to precisely measure the size of nanodiscs, and to determine the copolymer/lipid ratio at the onset of solubilisation. Finally, we use MDS to characterise peptide/nanodisc interaction. The technique shows a promising ability to highlight the pivotal role of lipids in promoting interactions through a case study with an aggregating peptide. This confirmed the relevance of using the MDS and nanodiscs as biomimetic models for such investigations.

Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers.

With the objective to isolate phages infecting wine bacterial spoilers, we designed a method for the isolation and purification of phages infecting grape-associated bacteria. The method proved successful to isolate GC1 tectivirus infecting the acetic acid bacterium Gluconobacter cerinus. The isolated phage represents a new genus within the Tectiviridae, named "Gammatectivirus". Using a traditional technique for the concentration of phage particles involving several steps of centrifugation, further insights in the ultrastructure of GC1 could be observed by cryo electron microscopy, saving time and effort. The simple workflow presented may be applied to other viruses infecting bacteria inhabiting other vegetal niches.

Lipidic Aminoglycoside Derivatives: A New Class of Immunomodulators Inducing a Potent Innate Immune Stimulation.

Development of simple and fully characterized immunomodulatory molecules is an active area of research to enhance current immunotherapies. Monophosphoryl lipid A (MPL), a nontoxic lipidic derivative from bacteria, is the first and currently only adjuvant approved in humans. However, its capacity to induce a potent response against weak immunogenic tumoral-associated antigens remains limited. Herein, a new generation of lipidic immunomodulators to conduct a structure-activity relationship study to determine the minimal structural elements conferring immunomodulatory properties is introduced. Two lead molecules characterized by a short succinyl linker between two oleyl chains and a polar headgroup consisting of either naturally occurring tobramycin (DOST) or kanamycin (DOSK) are identified. These two lipoaminoglycosides self-assemble in very small vesicles. In a wide variety of cells including 3D human cell culture, DOST and DOSK induce the upregulation of proinflammatory cytokines and interferon-inducible proteins in a dose and time-dependent manner via a caveolae-dependent proinflammatory mechanism and phosphatidylinositol phospholipase C activation. Furthermore, after intratumoral administration, these lipoaminoglycosides induce an efficient immune response leading to significant antitumor activity in a mouse breast cancer model. Altogether, these findings indicate that DOST and DOSK are two groundbreaking synthetic lipid immunostimulators that can be used as adjuvants to enhance current immunotherapeutic treatments.

Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer.

Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to α-helical and ß-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins.

Involvement of caveolin-1 and CD36 in native LDL endocytosis by endothelial cells.

Atherosclerosis is a lipid disease characterized by accumulation of low density lipoprotein (LDL) in the artery wall. The transport of LDL across the endothelium of coronary artery is an initiating event of atherosclerosis, whose mechanism remains poorly understood. In the last decade, it has been shown that in caveolin-1 (Cav-1) deficient mice, LDL infiltration in aorta wall is decreased and CD36 expression in aortas is down-regulated, leading to regression of atherosclerotic lesions. In the present study, we show that native LDL endocytosis is decreased in endothelial cells deficient in Cav-1 or CD36. We demonstrate that Cav-1 and CD36 interact in caveolae-rich domains by different biochemical approaches. In addition, confocal microscopy reveals some colocalization of Cav-1 with CD36. These findings indicate that caveolae and CD36 are involved in native LDL endocytosis and suggest that CD36 might be a good candidate for the transport of native LDL across the endothelium, an early event in atherosclerosis.