No abstract

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

A non-exhaustive list of fields in which extensive research has been dedicated to colloidal particles during the past century includes condensed matter physics, biology, optics, materials science, and chemistry. Both our current understanding of various physical phenomena and our capability to fabricate new functional materials have been considerably enriched by the development of synthetic strategies that are capable of generating copious quantities of colloidal entities of good size uniformity. Nevertheless, most of the available monodisperse colloidal materials are spherical, as the minimization of the interfacial free energy strongly drives a particle to adopt such a shape.[1] This strongly limits the number of new structures which can be engineered by using these colloids as building blocks. For instance, the crystallization of spherical colloids into three-dimensional periodic lattices has recently allowed the emergence of a very active field of research-photonic colloidal crystals, known as artificial opals. Nevertheless, the light diffraction properties of these crystals are rather limited because of their face-centered cubic lattice, which results from the packing of spheres. It has been predicted that crystals with a lower degree of symmetry, such as the diamond lattice, can exhibit a full photonic bandgap. To build such photonic crystals, well-defined colloids with nonspherical shapes are required. Van Blaaderen recently introduced the elegant term of "colloidal molecules", [2] which takes into account that spherical colloids can be treated as if they were atoms and that molecules can form more complex materials than can atoms. Therefore, the reproducible fabrication of large amounts of colloids that have a good uniformity in chemical composition, surface properties, size, and shape is a huge challenge.[3] Intensive efforts during the last decade have led to the development of nonspherical colloids of various shapes (such as rods, [4][5][6] wires, [7] triangles, [8] prisms, [9] cubes, [10] ellipsoids, [11] octahedra, [12] tetrapods, [13] ), but few of these samples combined the requirements of a true monodispersity in size and shape and the production of sufficiently large quantities. A promising synthetic procedure involves the use of a physical template to better control these two parameters. Micropatterned charged monolayers [14][15][16][17] or relief structures [18][19][20][21][22][23] on two-dimensional substrates were used to fabricate mono or binary clusters of colloids with high-arrangement accuracies, but in very small quantities. Three-dimensional templates such as liquid [24] or emulsion droplets [25,26] were also exploited to form complex colloidal assemblies with well-controlled sizes and morphologies. For example, a clever approach based on the generation of oil-in-water emulsions containing polymer microspheres and the subsequent controlled oil evaporation was first developed by Manoharan et al., [27][28][29] and led to aggregates that contain a precise number of polymer microspheres. Th...

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.

3 pagesInternational audienceLarge amounts of regular tetrapods and hexapods made of a central silica core and four or six polystyrene satellite nodules were prepared with yields over 80% from 55 nm and 85 nm silica seeds, respectively. The robustness of the process is supported by extensive statistical analyses and large-field transmission electron microscopy images

Binary colloidal particles of polyhedral morphology, obtained by an emulsion polymerization of styrene in the presence of silica seeds, are studied. Because of kinetic effects, composite particles usually exhibit polydispersity in size, shape, and composition. Thus, accurate techniques aiming at characterizing the size and the shape, as well as the composition and surface properties of such objects, are required. In this work, we use charge detection mass spectrometry (CD-MS) as a tool for the characterization of nanometer-sized composite (clusters of) particles. CD-MS measures both the mass and the charge for each ion. This single ion mass spectrometry technique enables one to construct a histogram of mass, yielding the mass distribution. CD-MS for molar mass determination and composition of composite particles is demonstrated to be complementary to transmission electron microscopy. The study of the charging capacity of these composite particles in the gas phase also appears as a valuable approach to probe the surface area of such complex nano-objects, thus giving some insight about their structure and morphology.

No abstract

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000μM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.