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The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The methanogenic strain Mx-05 T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales . It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis , the only member of this order previously isolated. The cells of Mx-05 T were non-motile cocci, with a diameter range of 0.4-0.7 μm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H 2 as an electron donor. H 2 /CO 2 , formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05 T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05 T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9-8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L -1 NaCl (optimum 0.12 mol.L -1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H 4 MPT Wood-Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05 T as being a novel representative of the order Methanomassiliicoccales , within the novel family Methanomethylophilaceae , for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05 T (JCM 31474T).

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design., (© 2023. Springer Nature Limited.)

Although the presence of silica in many living organisms offers advanced properties including cell protection, the different in vitro attempts to build living materials in pure silica never favoured the cells viability. Thus, little attention has been paid to host-guest interactions to modify the expected biologic response. Here we report the physiological changes undergone by Escherichia coli K-12 in silica from colloidal solution to gel confinement. We show that the physiological alterations in growing cultures are not triggered by the initial oxidative Reactive Oxygen Species (ROS) response. Silica promotes the induction of alternative metabolic pathways along with an increase of growth suggesting the existence of rpoS polymorphisms. Since the functionality of hybrid materials depends on the specific biologic responses of their guests, such cell physiological adaptation opens perspectives in the design of bioactive devices attracting for a large field of sciences., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mercedes Perullini reports financial support was provided by EULASUR., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The human commensal fungus Candida albicans can attach to epithelia or indwelling medical devices and form biofilms, that are highly tolerant to antifungal drugs and can evade the immune response. The cell surface protein Pga59 has been shown to influence adhesion and biofilm formation. Here, we present evidence that Pga59 displays amyloid properties. Using electron microscopy, staining with an amyloid fibre-specific dye and X-ray diffraction experiments, we showed that the predicted amyloid-forming region of Pga59 is sufficient to build up an amyloid fibre in vitro and that recombinant Pga59 can also adopt a cross-β amyloid fibre architecture. Further, mutations impairing Pga59 amyloid assembly led to diminished adhesion to substrates and reduced biofilm production. Immunogold labelling on amyloid structures extracted from C. albicans revealed that Pga59 is used by the fungal cell to assemble amyloids within the cell wall in response to adhesion. Altogether, our results suggest that Pga59 amyloid properties are used by the fungal cell to mediate cell-substrate interactions and biofilm formation., (© 2023. The Author(s).)

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis., (© 2022 The Authors.)

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Intermediate filaments (IFs) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49-nanometer axial repeat both in cells and in vitro. As unit-length filaments (ULFs) were measured at ~59 nanometers, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical, and structural properties in cells.

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-β. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts., (© 2022. The Author(s).)

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment., Competing Interests: The authors declare no competing interest.

Materials science has been informed by nonclassical pathways to crystallization, based on biological processes, about the fabrication of damage-tolerant composite materials. Various biomineralizing taxa, such as stony corals, deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further assemble and transform into higher-order mineral structures. Here, we examine a similar process in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of high-resolution imaging and in situ solid-state nuclear magnetic resonance spectroscopy, we reveal the underlying mechanism of the solid-state phase transformation of these amorphous nanoparticles into crystals under aqueous conditions. These amorphous nanoparticles are covered by a hydration shell of bound water molecules. Fast chemical exchanges occur: the hydrogens present within the nanoparticles exchange with the hydrogens from the surface-bound H 2 O molecules which, in turn, exchange with the hydrogens of the free H 2 O molecule of the surrounding aqueous medium. This cascade of chemical exchanges is associated with an enhanced mobility of the ions/molecules that compose the nanoparticles which, in turn, allow for their rearrangement into crystalline domains via solid-state transformation. Concurrently, the starting amorphous nanoparticles aggregate and form ordered mineral structures through crystal growth by particle attachment. Sphere-like aggregates and spindle-shaped structures were, respectively, formed from relatively high or low weights per volume of the same starting amorphous nanoparticles. These results offer promising prospects for exerting control over such a nonclassical pathway to crystallization to design mineral structures that could not be achieved through classical ion-by-ion growth.

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome., Competing Interests: The authors have declared that no competing interests exist.

Conserved across the family Herpesviridae , glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses. IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established., (Copyright © 2017 American Society for Microbiology.)

Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated. IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, "Portogloboviridae.", (Copyright © 2017 American Society for Microbiology.)

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens., Competing Interests: I have read the journal's policy and I understand that one of the authors of this manuscript (SAJ) has competing interests because of being employed by Brammer Bio LLC, 13702 Innovation Drive Alachua FL 32615.

PEGylated nanocapsules containing a liquid core of perfluorooctyl bromide (PFOB) were formulated by an emulsion-evaporation process to be further used as ultrasound contrast agents (UCAs). In an attempt to modulate their acoustic response, related to their shell thickness-to-radius ratio, the initial concentration of polymer was varied in the formulation. Indeed, thinner shells may lead to higher echogenicity. PEGylated nanocapsules morphology was studied by electron microscopy, Small Angle Neutron Scattering and (19)F NMR spectroscopy and related to their mechanical properties to allow a better understanding of their mechanism of formation. We show that the variation of polymer concentration in the formulation impacts the formation mechanism of nanocapsules, and consequently their morphology and mechanical properties. Using low concentration of Poly(ethylene glycol)-b-poly(dl-lactide-co-glycolide) (PLGA-b-PEG), it is impossible to reduce the shell thickness of the UCA, most probably due to dewetting of the polymer layer at the PFOB/water interface. This leads to the coexistence of thick shells along with free PFOB droplets. On the other hand, for high polymer concentration, PEGylated nanocapsules with thick shells were produced with high encapsulation efficiency., (Copyright © 2016. Published by Elsevier B.V.)

Bacteria use diverse signalling pathways to adapt gene expression to external stimuli. In Gram-negative bacteria, the binding of scarce nutrients to membrane transporters triggers a signalling process that up-regulates the expression of genes of various functions, from uptake of nutrient to production of virulence factors. Although proteins involved in this process have been identified, signal transduction through this family of transporters is not well understood. In the present study, using an integrative approach (EM, SAXS, X-ray crystallography and NMR), we have studied the structure of the haem transporter HasR captured in two stages of the signalling process, i.e. before and after the arrival of signalling activators (haem and its carrier protein). We show for the first time that the HasR domain responsible for signal transfer: (i) is highly flexible in two stages of signalling; (ii) extends into the periplasm at approximately 70-90 Å (1 Å=0.1 nm) from the HasR β-barrel; and (iii) exhibits local conformational changes in response to the arrival of signalling activators. These features would favour the signal transfer from HasR to its cytoplasmic membrane partners., (© 2016 The Author(s).)

The dynamics of the formation of siRNA-lipoplexes coated with hyaluronic acid (HA) and the parameters influencing their supramolecular organization were studied. The insertion of a HA-dioleylphosphatidylethanolamine (DOPE) conjugate in the liposome structure as well as subsequent complexation with siRNA increased the liposome size. Lipoplexes were around 110 nm at high ± charge ratios with a zeta potential around +50 mV and around 230 nm at low ± ratios, with a zeta potential that decreased to negative values, reaching -45 mV. The addition of the conjugate did not compromise siRNA binding to liposomes, although these nucleic acids induced a displacement of part of the HA-DOPE conjugate upon lipoplex formation, as confirmed by capillary electrophoresis. Isothermal titration calorimetry, X-ray diffraction studies, and cryo-TEM microscopy demonstrated that in addition to electrostatic interactions with siRNA a rearrangement of the lipid bilayers takes place, resulting in condensed oligolamellar vesicles. This phenomenon is dependent on the number of siRNA molecules and the degree of modification with HA. Finally, the suitable positioning of HA on the lipoplex surface and its ability to bind specifically to the CD44 receptors in a concentration-dependent manner was demonstrated by surface plasmon resonance analysis.

In the present paper, we show that the saturated form of acidic sophorolipids, a family of industrially scaled bolaform microbial glycolipids, unexpectedly forms chiral nanofibers only at pH below 7.5. In particular, we illustrate that this phenomenon derives from a subtle cooperative effect of molecular chirality, hydrogen bonding, van der Waals forces and steric hindrance. The pH-responsive behaviour was shown by Dynamic Light Scattering (DLS), pH-titration and Field Emission Scanning Electron Microscopy (FE-SEM) while the nanoscale chirality was evidenced by Circular Dichroism (CD) and cryo Transmission Electron Microscopy (cryo-TEM). The packing of sophorolipids within the ribbons was studied using Small Angle Neutron Scattering (SANS), Wide Angle X-ray Scattering (WAXS) and 2D (1)H-(1)H through-space correlations via Nuclear Magnetic Resonance under very fast (67 kHz) Magic Angle Spinning (MAS-NMR).

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.

It is well known that organic molecules from the vertebrate extracellular matrix of calcifying tissues are essential in structuring the apatite mineral. Here, we show that water also plays a structuring role. By using solid-state nuclear magnetic resonance, wide-angle X-ray scattering and cryogenic transmission electron microscopy to characterize the structure and organization of crystalline and biomimetic apatite nanoparticles as well as intact bone samples, we demonstrate that water orients apatite crystals through an amorphous calcium phosphate-like layer that coats the crystalline core of bone apatite. This disordered layer is reminiscent of those found around the crystalline core of calcified biominerals in various natural composite materials in vivo. This work provides an extended local model of bone biomineralization.

In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure., (© 2012 Blackwell Publishing Ltd.)

Known viruses build their particles using a restricted number of redundant structural solutions. Here, we describe the Aeropyrum coil-shaped virus (ACV), of the hyperthermophilic archaeon Aeropyrum pernix, with a virion architecture not previously observed in the viral world. The nonenveloped, hollow, cylindrical virion is formed from a coiling fiber, which consists of two intertwining halves of a single circular nucleoprotein. The virus ACV is also exceptional for its genomic properties. It is the only virus with a single-stranded (ss) DNA genome among the known hyperthermophilic archaeal viruses. Moreover, the size of its circular genome, 24,893 nt, is double that of the largest known ssDNA genome, suggesting an efficient solution for keeping ssDNA intact at 90-95 °C, the optimal temperature range of A. pernix growth. The genome content of ACV is in line with its unique morphology and confirms that ACV is not closely related to any known virus.

The involvement of collagen in bone biomineralization is commonly admitted, yet its role remains unclear. Here we show that type I collagen in vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. We also show that the collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales. These results call into question recent consensus in the literature on the need for Ca-rich non-collagenous proteins for collagen mineralization to occur in vivo. Our model is based on a collagen/apatite self-assembly process that combines the ability to mimic the in vivo extracellular fluid with three major features inherent to living bone tissue, that is, high fibrillar density, monodispersed fibrils and long-range hierarchical organization.

An increasing need exists for simple, bioderived, nontoxic, and up-scalable compounds with stimuli-responsive properties. Acidic sophorolipids (SL) are glucose-based biosurfactants derived from the yeast broth of Candida bombicola (teleomorph: Starmerella bombicola). The specific design of this molecule, a sophorose head with a free end-COOH group at the end of the alkyl chain, makes it a potentially interesting pH-responsive compound. We have specifically investigated this assumption using a combination of small angle neutron scattering (SANS), transmission electron microscopy under cryogenic conditions (Cryo-TEM), and nuclear magnetic resonance (NMR) techniques and found a strong dependence of SL self-assembly on the degree of ionization, α, of the COOH group at concentration values as low as 5 and 0.5 wt %. At least three regimes can be identified where the supramolecular behavior of SL is unexpectedly different: (1) at low α values, self-assembly is driven by concentration, C, and micelles are mainly identified as nonionic objects whose curvature decreases (sphere-to-rod) with C; (2) at mid α values, the formation of COO(-) groups introduces negative charges at the micellar surface inducing an increase in curvature (rod-to-sphere transition). Repulsive electrostatic long-range interactions appear at this stage. In both regimes 1 and 2, the cross-section radius of the micelles is below 25 Å. This behavior is concentration independent. (3) At α = 1, individual micelles seem to favor the formation of large netlike tubular aggregates whose size is above 100 nm. Such a complex behavior is very unique as it is generally not observed for common alkyl-based surfactants in concentration ranges below 5-10 wt %.

Some viruses of Archaea use an unusual egress mechanism that involves the formation of virus-associated pyramids (VAPs) on the host cell surface. At the end of the infection cycle, these structures open outward and create apertures through which mature virions escape from the cell. Here we describe in detail the structure and composition of VAPs formed by the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in cells of its hyperthermophilic archaeal host. We show that the VAPs are stable and autonomous assemblies that can be isolated from membranes of infected cells and purified without affecting their structure. The purified VAPs are heterogeneous in size, reflecting the dynamics of VAP development in a population of infected cells; however, they have a uniform geometry, consisting of seven isosceles triangular faces forming a baseless pyramid. Biochemical and immunoelectron microscopy analyses revealed that the 10-kDa P98 protein encoded by the SIRV2 virus is the sole component of the VAPs. The VAPs were produced in Sulfolobus acidocaldarius and Escherichia coli by heterologous expression of the SIRV2-P98 gene. The results confirm that P98 is the only constituent of the VAPs and demonstrate that no other viral protein is involved in the assembly of pyramids. P98 was able to produce stable structures under conditions ranging from moderate to extremely high temperatures (80 °C) and from neutral to extremely acidic pH (pH 2), demonstrating another remarkable property of this exceptional viral protein.

Background Information: Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts., Results and Conclusions: In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo-Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non-pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde-fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using flotation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6.CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

The analysis of the genome of Leptospira spp., a group of bacteria of the phylum of spirochetes with several unique evolutionary and morphological features, has allowed the identification of a gene encoding a coiled-coil protein, called Scc, which is completely unrelated to any other eukaryotic or prokaryotic protein. Since coiled-coil proteins are often key elements of the cytoskeleton, we analyzed the protein Scc, which is a 24-kDa protein composed of a N-terminal coiled-coil domain, a proline-rich intermediate domain, and an acidic tail. The gene scc is located in an operon which also contains the genes encoding the initiation factor IF3 and the two ribosomal proteins L20 and L35. In this study, we showed that the presence of the coiled-coil domain was responsible for the polymerization of Scc in helix-like structures, in an ATP-independent manner, in both Escherichia coli living cells and in vitro. Analysis of the Scc polymers by electron microscopy showed filaments with a width of 6 to 10 nm, similar to that of eukaryotic intermediate filaments. Scc was also found to bind both RNA and double-stranded DNA without detectable sequence specificity. By electron microscopy, we showed that Scc polymer assembly was affected by the presence of nucleic acids, giving rise to rod-shaped structures with a width ranging from 45 to 155 nm. Finally, Leptospira biflexa cells depleted in Scc form small colonies, but the morphology of their helicoidal cell body was not affected. These results provide the first insight into a unique DNA binding filament-forming coiled-coil protein that could play an important role in the subcellular architecture of the spirochetal microorganism.

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy., (Copyright 2001 Academic Press.)

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

rab6 is a ubiquitous ras-like GTPase involved in intra-Golgi transport. We have studied at both morphological and biochemical levels the behavior of Golgi resident proteins in HeLa cells overexpressing wild-type rab6 and GTP- and GDP-bound mutants of rab6 (rab6 Q72L and rab6 T27N, respectively). We show that wild-type rab6 and rab6 Q72L overexpression induces the redistribution of the trans-Golgi protein beta-1,4-galactosyltransferase into the endoplasmic reticulum (ER) and allows the addition of sialylated O-glycans on an ER-retained protein, the major histocompatibility complex class II-associated invariant chain. Remarkably, rab6 Q72L effects, which require the integrity of microtubules, were almost indistinguishable from those induced by brefeldin A, a fungic metabolite that causes a mixing of Golgi and ER membranes. In contrast, overexpression of rab6 T27N does not cause the redistribution of Golgi proteins, but inhibits basal O-glycosylation of the major histocompatibility complex class II-associated invariant chain.

Myrmecia warts induced by human papillomavirus type 1 (HPV1) are characterized by abundant eosinophilic inclusions associated with HPV1 E4 gene products. The major HPV1 E4 proteins are a 17-kilodalton (kDa) E1-E4 fusion protein and a 16-kDa species lacking the five E1 amino acids and a few E4 residues. To study the contribution of E4 proteins to the formation of myrmecia inclusions, we used a previously designed transient expression system in the rabbit VX2-R keratinocyte line. We find that the E1-E4 and an E4 protein without the E1 residues (E4-3200) form eosinophilic inclusions. Ultrastructural and immunoelectron microscopic studies show that the electron-dense, keratohyalin-like myrmecia inclusions are recognized by anti-E4 antibodies. They are associated with tonofilament bundles at their periphery in the cytoplasm or free of filaments in the nucleus. The E1-E4 inclusions formed in vitro are also homogeneously electron dense, and are usually associated with tonofilaments at their periphery in the cytoplasm and free of filaments in the nucleus. The E4-3200 inclusions are exclusively cytoplasmic and heterogeneously electron dense, with a fibrillar structure made of entangled 10-nm filaments. The expression of either protein in VX2-R cells does not result in the collapse of the cytokeratin network, as shown by immunofluorescence double-labeling experiments. This is in contrast to data reported for the HPV16 E1-E4 protein. Our findings indicate that the E1-E4 protein by itself accounts for the formation of myrmecia inclusions, and suggest that the five N-terminal E1 amino acids play a major role in the interaction of E4 proteins with intermediate filaments.