The ability of probiotic bacteria to adhere to the intestinal epithelium play an important role in colonization of the gastrointestinal tract, preventing their elimination by peristalsis and providing a competitive advantage in this ecosystem. To identify bacterial traits related to adhesion the probiotic strain L. acidophilus M92 was examined for autoaggregation ability and cell surface hydrophobicity L. acidophilus M92 exhibits a strong autoaggregation phenotype and also coaggregation with some pathogen microorganisms that may form a barrier that prevents their colonization. The examined probiotic strain manifests a good degree of hydrophobicity determined by microbial adhesion to hydrocarbons. Aggregation and hydrophobicity were abolished upon exposure of the cells to pronase, which suggests the possible role of cell surface layer (S-layer) proteins, approximated at 45 kDa, in a L. acidophilus M92. The relationship between autoaggregation and adhesion ability to intestinal tissue was investigated by observing the adhesivity of L. acidophilus M92 to porcine ileal epithelial cells. Removal of the S-layer proteins by extraction with 5 M LiCl reduced autaggregation and in vitro adhesion of this strain.

Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea1–5. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 A X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 A structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 A, and is stabilized by multiple Ca2+ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging. The S-layer structure of C. crescentus is revealed by combining the X-ray crystal structure of an S-layer protein and cryo-ET of cell stalks. The resulting model shows that the S-layer is porous and stabilized by calcium ions.

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Structure of the hexagonal surface layer on Caulobacter crescentus cells

Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3–5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9–12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems. Design of a two-component protein array enables robust formation of complex large-scale ordered biologically active materials.

Design of biologically active binary protein 2D materials

Proteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3–5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9–12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems. One Sentence Summary Co-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

/pdf/design-of-biologically-active-binary-protein-2d-materials-4ksqpdaitd.pdf

Design of Biologically Active Binary Protein 2D Materials

Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.

Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly.

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.

Diversity in S-layers.

Chitosan inhibits advanced glycation end products formation in chemical models and bakery food

β-Lactoglobulin (β-LG) is a whey protein with multiple binding sites for bioactive phenolics. Encapsulation of bioactive components in β-LG could enhance their activity and stability. In this study, vanillic acid (VA), ferulic acid (FA), and quercetin (QT) were encapsulated in β-LG. The resulting complex was studied by fluorescence spectroscopy, isothermal titration calorimetry, molecular docking, and molecular dynamics simulations. The β-LG–tri-ligand complex was successfully formed via an exothermic binding process when the ligands were added in the sequence of FA, QT, and VA, or QT, FA, and VA. The stabilities of the bioactive components in the complex were greatly improved by encapsulation. Molecular dynamics simulations confirmed that the binding of ligand molecules could increase the stability of the β-LG structure and made it more rigid/inflexible. The antioxidant activity and inhibition activity of the β-LG–tri-ligand complex against advanced glycation end products were higher than those of a mixture of FA, QT, and VA. The β-LG–tri-ligand complex shows great potential in functional food applications. 

Encapsulation of phenolics in β-lactoglobulin: Stability, antioxidant activity, and inhibition of advanced glycation end products

Biochar is a low-cost adsorbent for sorptive removal of antibiotics from wastewater, but the adsorption efficiency needs to be improved. In this study, coconut-shell biochar was activated with KOH to improve the adsorption efficiency and magnetically modified with FeCl3 to enable recycling. The amount of KOH and the concentration of FeCl3 were optimized to reduce the pollution and production cost. The KOH-activated and FeCl3-magnetized biochar gave good sulfonamide antibiotic (SA) removal. The maximum adsorption capacities for sulfadiazine, sulfamethazine and sulfamethoxazole were 294.12, 400.00 and 454.55 mg g−1, respectively, i.e., five to seven times higher than those achieved with raw biochar. More than 80% of the adsorption capacity was retained after three consecutive adsorption-desorption cycles. A combination of scanning electron microscopy, Brunauer-Emmett-Teller analysis, X-ray diffraction, Fourier-transform infrared and Raman spectroscopies, and magnetic hysteresis analysis showed that KOH activation increased the specific surface area, porosity, and number of oxygen-rich functional groups. Iron oxide particles, which were formed by FeCl3 magnetization, covered the biochar surface. The SAs were adsorbed on the modified biochar via hydrogen bonds between SA molecules and -OH/-COOH groups in the biochar. Investigation of the adsorption kinetics and isotherms showed that the adsorption process follows a pseudo-second-order kinetic model and a monolayer adsorption mechanism. The adsorption capacity at low pH was relatively high because of a combination of π+-π electron-donor-acceptor, charge-assisted hydrogen-bonding, electrostatic, and Lewis acid-base interactions, pore filling, van der Waals forces and hydrophobic interactions. The results of this study show that magnetically modified biochar has potential applications as an effective, recyclable adsorbent for antibiotic removal during wastewater treatment.

/pdf/adsorption-of-sulfonamides-in-aqueous-solution-on-reusable-2xsw9qlc.pdf

Adsorption of Sulfonamides in Aqueous Solution on Reusable Coconut-Shell Biochar Modified by Alkaline Activation and Magnetization

https://doi.org/10.1016/j.fochx.2022.100369

Dao Xiao

Papers

Diversity in S-layers.

Chitosan inhibits advanced glycation end products formation in chemical models and bakery food

Encapsulation of phenolics in β-lactoglobulin: Stability, antioxidant activity, and inhibition of advanced glycation end products

Adsorption of Sulfonamides in Aqueous Solution on Reusable Coconut-Shell Biochar Modified by Alkaline Activation and Magnetization

Binding of β-lactoglobulin to three phenolics improves the stability of phenolics studied by multispectral analysis and molecular modeling