Bacterial Surface Layer Proteins Research Articles

A Computational Approach for Protein-Protein Interactions of Bacterial Surface Layer Protein with Human Erb3 and αIIB-β3 Receptors

Host microbial interactions had significant factors in maintains homeostasis and immune-related activity. One such interaction made by Lactobacillus sp. with Surface layer proteins (Slps) had been studied through a computational approach. Erb3 and αIIB-β3, which are epithelial surface layer receptors, are subjected to interact with the Slp homology model. Both cell surface receptors were subjected to interact through computational docking, followed by molecular dynamics simulations through the coarse-grain method to explore the conformational stability. Through the implementation of the molecular docking for the surface layer protein A, we have shown the surface layer protein A, protein-protein interactions are higher in cellular receptors with epidermal growth factor receptor at an -34.45 ΔG and -51.19 ΔG through molecular docking with Erb3 and αIIB-β3. This study shows the unique interaction of Slp with the epithelial surface receptors like Erb3 and αIIB-β3, which are multipurpose applications in microbial-based drug therapeutics.

Molecular Logic of Prokaryotic Surface Layer Structures.

Most prokaryotic cells are encased in a surface layer (S-layer) consisting of a paracrystalline array of repeating lattice-forming proteins. S-layer proteins populate a vast and diverse sequence space, performing disparate functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. This article highlights recent advances in the understanding of S-layer structure and assembly, made possible by rapidly evolving structural and cell biology methods. We underscore shared assembly principles revealed by recent work and discuss a common molecular framework that may be used to understand the structural organization of S-layer proteins across bacteria and archaea.

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly

Electrochemical Biosensors Based on S-Layer Proteins.

Designing and development of electrochemical biosensors enable molecule sensing and quantification of biochemical compositions with multitudinous benefits such as monitoring, detection, and feedback for medical and biotechnological applications. Integrating bioinspired materials and electrochemical techniques promote specific, rapid, sensitive, and inexpensive biosensing platforms for (e.g., point-of-care testing). The selection of biomaterials to decorate a biosensor surface is a critical issue as it strongly affects selectivity and sensitivity. In this context, smart biomaterials with the intrinsic self-assemble capability like bacterial surface (S-) layer proteins are of paramount importance. Indeed, by forming a crystalline two-dimensional protein lattice on many sensors surfaces and interfaces, the S-layer lattice constitutes an immobilization matrix for small biomolecules and lipid membranes and a patterning structure with unsurpassed spatial distribution for sensing elements and bioreceptors. This review aims to highlight on exploiting S-layer proteins in biosensor technology for various applications ranging from detection of metal ions over small organic compounds to cells. Furthermore, enzymes immobilized on the S-layer proteins allow specific detection of several vital biomolecules. The special features of the S-layer protein lattice as part of the sensor architecture enhances surface functionalization and thus may feature an innovative class of electrochemical biosensors.

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

Regulating colonic dendritic cells by commensal glycosylated large surface layer protein A to sustain gut homeostasis against pathogenic inflammation.

Microbial interaction with the host through sensing receptors, including SIGNR1, sustains intestinal homeostasis against pathogenic inflammation. The newly discovered commensal Propionibacterium strain, P. UF1, regulates the intestinal immunity against pathogen challenge. However, the molecular events driving intestinal phagocytic cell response, including colonic dendritic cells (DCs), by this bacterium are still elusive. Here, we demonstrate that the glycosylation of bacterial large surface layer protein A (LspA) by protein O-mannosyltransferase 1 (Pmt1) regulates the interaction with SIGNR1, resulting in the control of DC transcriptomic and metabolomic machineries. Programmed DCs promote protective T cell response to intestinal Listeria infection and resist chemically induced colitis in mice. Thus, our findings may highlight a novel molecular mechanism by which commensal surface glycosylation interacting with SIGNR1 directs the intestinal homeostasis to potentially protect the host against proinflammatory signals inducing colonic tissue damage.

S-Layer Protein Coated Carbon Nanotubes

Carbon nanotubes (CNTs) have already been considered for medical applications due to their small diameter and ability to penetrate cells and tissues. However, since CNTs are chemically inert and non-dispersible in water, they have to be chemically functionalized or coated with biomolecules to carry payloads or interact with the environment. Proteins, although often only randomly bound to the CNT surface, are preferred because they provide a better biocompatibility and present functional groups for binding additional molecules. A new approach to functionalize CNTs with a closed and precisely ordered protein layer is offered by bacterial surface layer (S-layer) proteins, which have already attracted much attention in the functionalization of surfaces. We could demonstrate that bacterial S-layer proteins (SbpA of Lysinibacillus sphaericus CCM 2177 and the recombinant fusion protein rSbpA31-1068GG comprising the S-layer protein and two copies of the IgG binding region of Protein G) can be used to disperse and functionalize oxidized multi walled CNTs. Following a simple protocol, a complete surface coverage with a long-range crystalline S-layer lattice can be obtained. When rSbpA31-1068GG was used for coating, the introduced functionality could be confirmed by binding gold labeled antibodies via the IgG binding domain of the fusion protein. Since a great variety of functional S-layer fusion proteins has already been described, our new technology has the potential for a broad spectrum of functionalized CNTs.

S-layer protein-AuNP systems for the colorimetric detection of metal and metalloid ions in water

Bacterial surface layer proteins (S-layer) possess unique binding properties for metal ions. By combining the binding capability of S-layer proteins with the optical properties of gold nanoparticles (AuNP), namely plasmonic resonance, a colorimetric detection system for metal and metalloid ions in water was developed. Eight S-layer proteins from different bacteria species were used for the functionalization of AuNP. The thus developed biohybrid systems, AuNP functionalized with S-layer proteins, were tested with different metal salt solutions, e.g. Indium(III)-chloride, Yttrium(III)-chloride or Nickel(II)-chloride, to determine their selective and sensitive binding to ionic analytes. All tested S-layer proteins displayed unique binding affinities for the different metal ions. For each S-layer and metal ion combination markedly different reaction patterns and differences in concentration range and absorption spectra were detected by UV/vis spectroscopy. In this way, the selective detection of tested metal ions was achieved by differentiated analysis of a colorimetric screening assay of these biohybrid systems. A highly selective and sensitive detection of yttrium ions down to a concentration of 1.67 × 10−5 mol/l was achieved with S-layer protein SslA functionalized AuNP. The presented biohybrid systems can thus be used as a sensitive and fast sensor system for metal and metalloid ions in aqueous systems.

Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications

The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.

S-layer protein-based biosensors

The present paper highlights the application of bacterial surface (S-) layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D) protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

S-Layer Protein-Based Biosensors.

The present paper highlights the application of bacterial surface (S-) layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D) protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

Bioinspired detection sensor based on functional nanostructures of S-proteins to target the folate receptors in breast cancer cells

Development of biosensors requires a well-organized template to enhance the sensing specificity. Biological molecules that have the ability to self-assemble into ordered structures can be exploited to construct nanostructures and nanodevices. In this regard, bacterial surface layer proteins (SbpA) have been used as an underlying matrix to bind molecules on the nanometer scale. The present study provides evidence that a gold substrate functionalized with folate-modified SbpA can be used as a sensor for breast cancer cells with superior efficacy over traditional sensors. The developed sensor uses folate to recognize folate receptors, which are highly expressed on the cell membranes of some cancer cells, such as breast cancer cells. To examine the specificity of the developed sensor, folate was used as a binding partner for folate receptors on breast cancer cells (MCF-7), and liver cancer cells (HepG2) were used as the control because they lack folate receptors. The fabricated biosensor comprised a gold surface and a recrystallized S-layer protein lattice functionalized with folate and addressed acoustically and electrochemically. Acoustic studies performed using quartz crystal microbalance with dissipation monitoring (QCM-D) showed the efficiency of the developed biosensor in distinguishing between MCF-7 and HepG2 cells. Despite the ability of QCM-D to recognize cells in situ and in real-time, this biosensor had a limited detection range (1 × 105 cells/mL). To confirm the functionality of the developed platform, electrochemical measurements were performed and the obtained results were in agreement with the acoustic sensor and proving the efficiency of the designed sensing layer. The S-layer lattice-folate modified biosensor provides a promising strategy for designing efficient sensing platforms that can be used to diagnose early stage cancers with highly expressed folate receptors.

Bacterial Surface Layer Proteins: From Moonlighting to Biomimetics: A New Horizonto Lead

The landmark discovery of moonlighting proteins embarks the significant progress in understanding the biological complexity and their closed-circuit analysis. The growing continuum in the variety of moonlighting functions paved the way for further elucidation of structural-functional aspects of protein evolution and design of proteins with novel functions. Currently, the moonlighting functions in various adhesive properties of surface layer proteins, an essential component of cell surface architecture of archaea and all phylogenetic groups of eubacteria become more prominently recognized. The remarkable credentials of surface layer proteins to self-assemble into supramolecular structures at nano-scale dimension have been exploited for the production of smart biomaterials in the form of biomimetics has been thrust area of research. The finely tuned topological features in terms of shape, size, geometry and surface chemistry of surface layer proteins are crucial for the production of biomimetics. The current developments of biomimetic lipid bilayers and composite membranes find applicability in understanding the functional dynamism of evolutionary relationship of bacterial cell envelopes and vaccine development, drug development and drug delivery. Though the development of biomimetics embraces fascination but faces with technological challenges. The plethora of literature has been available for the moonlighting aspects and nano-technological applications separately but none of the review describes towards the rhythmic transition from moonlighting functions of surface layer proteins of bacteria to biomimetics development and applications. Therefore, this review describes certain basic aspects of moonlighting functions and their mechanism of action, surface layer proteins and their moonlighting functions of commensal bacteria and their transition towards biomimetics. The recent developments of biomimetics based on surface layer proteins have been summarized and also posited different challenges and future prospects.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers.

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered—after washing away the unbound S-layer protein—by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc.

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10−9 m2 V−1 s−1) when compared with unmodified fused silica (5.9 ± 0.1 10−8 m2 V−1 s−1). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.

Influencing bacterial S-layer protein recrystallization on polymer brushes through surface charge and accessible volume: A combined AFM and QCMD analysis

S-layer recrystallization is part of, at least, two major scientific and technological challenges: a model system to study 2-D polymer crystal formation and a bottom-up approach to build biomimetic and functional interfaces. In particular, the self-assembly process of the bacterial surface layer protein (SbpA) might be influenced by two different interactions: protein/substrate and protein/protein. In this work, we are taking advantage of the versatility of already well known polymer brushes to study the self-assembly mechanism of the S-protein SbpA. In particular, the recrystallization pathway of the protein has been modified by the underlying polymer charge and accessible volume offered by the positively charged poly{[2-(methacryloyloxy)ethyl] trimethylammonium chloride} (PMETAC), the negative poly(sulfo propyl methacrylate) (PSPM) and the non-charged poly(N -isopropyl acrylamide) (PNIPAAm). The recrystallization pathways on polymer brushes have been compared with the bacterial biomimetic case represented by the secondary cell wall polymer (SCWP). Results concerning protein adsorption kinetics, viscoelastic properties, crystal lattice parameters and domain formation are presented, and have been obtained with Quartz Crystal Microbalance with Dissipation Monitoring (QCMD) and Atomic Force Microscopy (AFM).

Biointerfaces: Anisotropic Crystalline Protein Nanolayers as Multi‐Functional Biointerface for Patterned Co‐Cultures of Adherent and Non‐Adherent Cells in Microfluidic Devices (Adv. Mater. Interfaces 1/2015)

In article 1400309, Ertl and co-workers exploit the self-assembly properties of various bacterial surface layer (S-layer) proteins to readily fabricate an anisotropic multi-functional nanobiointerface within microfluidic devices. S-layer proteins with cell-adhesive (rSbsB) and cell-repellant (SbpA), as well as biomolecule-binding capabilities (rSbpA/ZZ) are re-assembled within microchannels for on-chip patterning of co-cultures of mobile with adherent cell types in order to generate multi-functional surface properties.

Characterization of a nanoscale S-layer protein based template for biomolecular patterning.

Well organized template for biomolecular conjugation is the foundation for biosensing. Most of the current devices are fabricated using lithographic patterning processes and self-assembly monolayer (SAM) methods. However, the research toward developing a sub-10 nm patterned, self-regenerated template on various types of substrates is limited, mainly due to the limited functional groups of the building material. Bacterial surface layer proteins (S-layer proteins) can self-assemble into ordered lattice with regular pore sizes of 2-8 nm on different material supports and interfaces. The ordered structure can regenerate after extreme variations of solvent conditions. In this work, we developed a nanoscale biomolecular template based on S-layer proteins on gold surface for fabrication of sensing layer in biosensors. S-layer proteins were isolated from Bacillus cereus, Lysinibacillus sphaericus and Geobacillus stearothermophilus. Protein concentrations were measured by Bradford assay. The protein purities were verified by SDS-PAGE, showing molecular weights ranging from 97-135 kDa. The hydrophilicity of the substrate surface was measured after surface treatments of protein recrystallization. Atomic force microscopic (AFM) measurement was performed on substrate surface, indicating a successful immobilization of a monolayer of S-layer protein with 8-9 nm height on gold surface. The template can be applied on various material supports and acts as a self-regenerated sensing layer of biosensors in the future.

Multidimensional Assembly of S-Layer Proteins on Mobility-Controlled Polyelectrolyte Multilayers.

Polyelectrolyte multilayers have been vastly utilized as an assembling platform for various biomaterials because of their soft and charged surface characteristics, analogous to biomembrane systems. In particular, polyelectrolyte chains with high self-diffusivity can effectively transfer the surface mobility to the assembling biomolecular species, facilitating the ordered self-assembly. Herein, highly diffusional cationic polyelectrolyte chains of linear polyethylenimine are employed to induce direct binding with negatively charged bacterial surface layer proteins, which eventually lead to large-scale two-dimensional crystals. Notably, at the elevated incubation temperature, a transitory intermediate of one-dimensional chain structure is observed. We reveal that this one-dimensional intermediate is a stable precursor toward two-dimensional crystal arrays.