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S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.
Assuntos
Glicoproteínas de Membrana , Ácidos Teicoicos , Ácidos Teicoicos/metabolismo , Ácidos Teicoicos/química , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/química , Lactobacillus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Lactobacillus acidophilus/metabolismo , Lactobacillus acidophilus/genéticaRESUMO
In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in "Ca. Micrarchaeum harzensis A_DKE," and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated, and the micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described coculture, containing "Ca. Micrarchaeum harzensis A_DKE" and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of "Ca. Micrarchaeum harzensis A_DKE." Both organisms are clearly distinguishable by cell size, shape, and surface structure. IMPORTANCE Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member "Candidatus Micrarchaeum harzensis A_DKE," an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of "Ca. Micrarchaeum harzensis A_DKE" is an important step toward understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.
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Archaea , Genoma Arqueal , Archaea/metabolismo , Genômica , FilogeniaRESUMO
Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
Assuntos
Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Sulfolobus/metabolismo , Microscopia Crioeletrônica , Dimerização , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/ultraestruturaRESUMO
The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A Streptococcus (GAS; Streptococcus pyogenes). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, spy49_0414c, abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides.IMPORTANCE This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Percepção de Quorum , Transdução de Sinais , Streptococcus pyogenes/fisiologia , Transativadores/metabolismo , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Muramidase/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Feromônios/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/crescimento & desenvolvimento , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE: The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (â4)-ß-ManNAc-(1â4)-ß-GlcNAc-(1â6)-α-GlcNAc-(1â) with α-Gal and ß-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.
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Bacillus anthracis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Polissacarídeos Bacterianos/metabolismo , Sequência de Aminoácidos , Bacillus anthracis/citologia , Bacillus anthracis/genética , Proteínas de Bactérias/genética , Deleção de Genes , Polissacarídeos Bacterianos/genéticaRESUMO
This chapter covers the fundamental aspects of bacterial S-layers: what are S-layers, what is known about them, and what are their main features that makes them so interesting for the production of nanostructures. After a detailed introduction of the paracrystalline protein lattices formed by S-layer systems in nature the chapter explores the engineering of S-layer-based materials. How can S-layers be used to produce "industry-ready" nanoscale bio-composite materials, and which kinds of nanomaterials are possible (e.g., nanoparticle synthesis, nanoparticle immobilization, and multifunctional coatings)? What are the advantages and disadvantages of S-layer-based composite materials? Finally, the chapter highlights the potential of these innovative bacterial biomolecules for future technologies in the fields of metal filtration, catalysis, and bio-functionalization.
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Bactérias/química , Glicoproteínas de Membrana/química , Nanocompostos/químicaRESUMO
We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 µm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cálcio/química , Nanoestruturas/ultraestrutura , Análise Serial de Proteínas , Salmonella typhimurium/genética , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Moleculares , Nanoestruturas/química , Análise Serial de Proteínas/instrumentação , Salmonella typhimurium/química , Difração de Raios XRESUMO
Nanosciences are distinguished by the cross-fertilization of biology, chemistry, material sciences, and solid-state physics and, hence, open up a great variety of new opportunities for innovation. The technological utilization of self-assembly systems, wherein molecules spontaneously associate under equilibrium conditions into reproducible supramolecular structures, is one key challenge in nanosciences for life and non-life science applications. The attractiveness of such processes is due to their ability to build uniform, ultra-small functional units with predictable properties down to the nanometer scale. Moreover, newly developed techniques and methods open up the possibility to exploit these structures at meso- and macroscopic scale. An immense significance at innovative approaches for the self-assembly of supramolecular structures and devices with dimensions of a few to tens of nanometers constitutes the utilization of crystalline bacterial cell surface proteins. The latter have proven to be particularly suited as building blocks in a molecular construction kit comprising of all major classes of biological molecules. The controlled immobilization of biomolecules in an ordered fashion on solid substrates and their directed confinement in definite areas of nanometer dimensions are key requirements for many applications including the development of bioanalytical sensors, biochips, molecular electronics, biocompatible surfaces, and signal processing between functional membranes, cells, and integrated circuits.
Assuntos
Glicoproteínas de Membrana/química , Nanotecnologia/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Biotecnologia/métodosRESUMO
Biological systems are able to control the assembly and positioning of proteins with nanoscale precision, as exemplified by the intricate molecular structures within cell membranes, virus capsids, and collagen matrices. Controlling the assembly of biomolecules is critical for the use of biomaterials in artificial systems such as antibacterial coatings, engineered tissue samples, and implanted medical devices. Furthermore, understanding the dynamics of protein assembly on heterogeneous templates will ultimately enable the control of protein crystallization in general. Here, we show a biomimetic, hierarchical bottom-up approach to direct the self-assembly of crystalline S-layers through nonspecific interactions with nanostructured block copolymer (BCP) thin-film templates. A comparison between physically and chemically patterned BCP substrates shows that chemical heterogeneity is required to confine the adhesion and self-assembly of S-layers to specific BCP domains. Furthermore, we show that this mechanism can be extended to direct the formation of collagen fibers along the principal direction of the underlying BCP substrate. The dynamics of protein self-assembly at the solid-liquid interface are followed using in situ high-resolution atomic force microscopy under continuous flow conditions, allowing the determination of the rate constants of the self-assembly. A pattern of alternating, chemically distinct nanoscale domains drastically increases the rate of self-assembly compared to non-patterned chemically homogeneous substrates.
Assuntos
Colágeno/química , Nanoestruturas/química , Polietilenoglicóis/química , Poliestirenos/química , Polivinil/química , Adsorção , Materiais Biomiméticos/química , Cristalização , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
Allergen-specific immunotherapy represents the only available curative approach to allergic diseases. The treatment has proven effective, but it requires repetitive administrations of allergen extracts over 3-5 years and is often associated with adverse events. This implies the need for novel therapeutic strategies with reduced side effects and decreased treatment time, which would improve patients' compliance. Development of vaccines that are molecularly well defined and have improved safety profile in comparison to whole allergen extracts represents a promising approach. Molecular allergy vaccines are based on major allergen proteins or allergen-derived peptides. Often, such vaccines are associated with lower immunogenicity and stability and therefore require an appropriate delivery vehicle. In this respect, viruses, bacteria, and their protein components have been intensively studied for their adjuvant capacity. This article provides an overview of the microbial delivery vehicles that have been tested for use in allergy immunotherapy. We review in vitro and in vivo data on the immunomodulatory capacity of different microbial vehicles for allergens and allergen-derived peptides and evaluate their potential in development of allergy vaccines. We also discuss relevant aspects and challenges concerning the use of microbes and their components in immunotherapy of allergic diseases.
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Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the assembly and recrystallization kinetics of isolated SbpA bacterial surface proteins onto silicon dioxide substrates of different surface wettability. Surface modification by UV/ozone oxidation or by vapor deposition of 1H,1H,2H,2H-perfluorododecyltrichlorosilane yielded hydrophilic or hydrophobic samples, respectively. Time evolution of frequency and dissipation factors, either individually or combined as the so-called Df plots, showed a much faster formation of crystalline coatings for hydrophobic samples, characterized by a phase-transition peak at around the 70% of the total mass adsorbed. This behavior has been proven to mimic, both in terms of kinetics and film assembly steps, the recrystallization taking place on an underlying secondary cell-wall polymer (SCWP) as found in bacteria. Complementary atomic force microscopy (AFM) experiments corroborate these findings and reveal the impact on the final structure achieved.
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Surface layers, referred simply as S-layers, are the two-dimensional crystalline arrays of protein or glycoprotein subunits on cell surface. They are one of the most common outermost envelope components observed in prokaryotic organisms (Archaea and Bacteria). Over the past decades, S-layers have become an issue of increasing interest due to their ubiquitousness, special features and functions. Substantial work in this field provides evidences of an enormous diversity in S-layers. This paper reviews and illustrates the diversity from several different aspects, involving the S-layer-carrying strains, the structure of S-layers, the S-layer proteins and genes, as well as the functions of S-layers.
Assuntos
Células , Glicoproteínas/química , Glicoproteínas/metabolismo , Células/citologia , Células/metabolismo , Glicoproteínas/genética , HumanosRESUMO
Elucidating the building principles and intrinsic features modulating certain water-associated processes (e.g., surface roughness in the nanometer scale, surface hydration and accompanied antifouling property, etc.) of surface structures from (micro)organisms is nowadays a highly challenging task in fields like microbiology, biomimetic engineering and (bio)material sciences. Here, we show for the first time the recrystallization of the wild-type S-layer glycoprotein wtSgsE from Geobacillus stearothermophilus NRS 2004/3a and its recombinantly produced non-glycosylated form, rSgsE, on gold sensor surfaces. Whereas the proteinaceous lattice of the S-layer proteins is forming a rigid layer on the sensor surface, the glycan chains are developing an overall soft, highly dissipative film. Interestingly, to the wtSgsE lattice almost twice the amount of water is bound and/or coupled in comparison with the non-glycosylated rSgsE with the preferred region being the extending glycan residues. The present results are discussed in terms of the effect of the glycan residues on the recrystallization, the adjoining hydration layer, and the nanoscale roughness and fluidic behavior. The latter features may turn out to be one of the most general ones among bacterial and archaeal S-layer lattices.
Assuntos
Membrana Celular/química , Ouro/química , Fluidez de Membrana , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestrutura , Membranas Artificiais , Adsorção , Glicosilação , Teste de MateriaisRESUMO
Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to "mine" information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.
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The uncultivated "Candidatus Altiarchaeum hamiconexum" (formerly known as SM1 Euryarchaeon) carries highly specialized nano-grappling hooks ("hami") on its cell surface. Until now little is known about the major protein forming these structured fibrous cell surface appendages, the genes involved or membrane anchoring of these filaments. These aspects were analyzed in depth in this study using environmental transcriptomics combined with imaging methods. Since a laboratory culture of this archaeon is not yet available, natural biofilm samples with high Ca. A. hamiconexum abundance were used for the entire analyses. The filamentous surface appendages spanned both membranes of the cell, which are composed of glycosyl-archaeol. The hami consisted of multiple copies of the same protein, the corresponding gene of which was identified via metagenome-mapped transcriptome analysis. The hamus subunit proteins, which are likely to self-assemble due to their predicted beta sheet topology, revealed no similiarity to known microbial flagella-, archaella-, fimbriae- or pili-proteins, but a high similarity to known S-layer proteins of the archaeal domain at their N-terminal region (44-47% identity). Our results provide new insights into the structure of the unique hami and their major protein and indicate their divergent evolution with S-layer proteins.
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An astonishingly broad application potential in biotechnology, biomimetics, and nanotechnology is revealed by studies on the structure, chemistry, biosynthesis, genetics, self-assembly, and function of supramolecular surface layers (S layers). These are monomolecular, crystalline assemblies of protein or glycoprotein subunits and represent one of the most commonly observed surface structures of prokaryotic cell envelopes (see schematic representation of an archaebacterial cell envelope).
RESUMO
Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology.