Analysis of S-layer proteins of Lactobacillus brevis

Abstract The presence of S-layer proteins in Lactobacillus brevis was examined by SDS-PAGE analysis. Thirty six out of a total of 41 L. brevis strains possessed S-layer proteins of molecular masses ranging from 38 to 55 kDa. Western blot analysis using antisera raised against whole cells of S-layer protein-carrying strains demonstrated the heterogeneity of L. brevis S-layer proteins. No clear relationship was observed between the presence of S-layer proteins or their immunological characteristics and the physiological activity of L. brevis as a beer spoilage organism.     

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.     

The cell surface of Lactobacillus reuteri ATCC 55730 highlighted by identification of 126 extracellular proteins from the genome sequence

Bioinformatical analyses of a draft genome sequence of the commensal bacterium Lactobacillus reuteri ATCC 55730 revealed 126 genes encoding putative extracellular proteins. The function, localization and distribution in bacterial species were predicted. Interestingly, few proteins possessed LPXTG motifs or C-terminal transmembrane anchors. Instead eight proteins were putatively anchored by GW repeats and several secreted proteins were likely to be re-associated to the surface. The majority of the extracellular proteins were widely distributed, i.e., found universally or in gram-positive bacteria, but 24 were only detected in L. reuteri. Further, the number of transporters was lower, while the number of enzyme was higher than in related species.     

Binding of extracellular matrix proteins to the surface of Bacteroides spp

The binding of fibronectin an vitronectin to 207 Bacteroides strains and the binding of collagen and sialoproteins to 55 Bacteroides strains were investigated by means of latex agglutination tests. The binding of fibronectin, collagen and lactoferrin to the same 55 strains was also tested by using 125I-labelled proteins. The 207 strains, belonging to ten Bacteroides species, were isolated from different infections (51%) and from faeces of healthy subjects (49%). Most of the strains displaying fibronectin binding belonged in the species B. fragilis or B. vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. No inhibition of the binding was observed with carbohydrates. The vitronectin binding of the strains was less common, but was always observed to accompany fibronectin binding. None of the examined 55 strains exhibited any binding to fetuin or asialofetuin. The radiolabelling method indicated a low binding to 125I-fibronectin. The binding of 125I-collagen-I and 125I-lactoferrin in the Bacteroides strains tested was higher than that of 125I-fibronectin.     

一株短乳杆菌所产细菌素的部分特性

为了研究分离自内蒙古传统发酵乳制品——焦克的短乳杆菌KLDS1.0373所产细菌素的部分生物学特性(抑菌谱,对酶、pH和温度的敏感性,作用方式)。短乳杆菌KLDS1.0373发酵液经硫酸铵沉淀和葡聚糖凝胶纯化后,测定其部分生物学特性,并采用Tricine-SDS-PAGE方法确定细菌素的分子量范围。结果表明:短乳杆菌KLDS1.0373所产细菌素的抑菌活性对热和pH不敏感,在100°C或121°C处理30 min后抑菌活力略有增强,可被多种蛋白酶失活,但对α-淀粉酶不敏感。该细菌素分子量约为3.8 kD,对多种革兰氏阳性和阴性菌有抑制作用,作用方式为杀菌。     

Primary structure of selected archaeal mesophilic and extremely thermophilic outer surface layer proteins

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, sulfate reducers and sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras. Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth. The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail. The most common archaeal cell envelope is composed of a single crystalline protein or glycoprotein surface layer (S-layer), which is associated with the outside of the cytoplasmic membrane. The S-layers are directly exposed to the extreme environment and can not be stabilized by cellular components. Therefore, from comparative studies of mesophilic and extremely thermophilic S-layer proteins hints can be obtained about the molecular mechanisms of protein stabilization at high temperatures. First crystallization experiments of surface layer proteins under microgravity conditions were successful. Here, we report on the biochemical features of selected mesophilic and extremely archaeal S-layer (glyco-) proteins.     

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation

ABSTRACT

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.     

Sequential deposition of latent TGF-β binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts

Latent TGF-beta binding proteins (LTBPs) mediate the targeting of latent TGF-beta complexes into ECM structures, which is important for TGF-beta activation and functions. LTBPs-1, -3 and -4 associate with and regulate the bioavailability of TGF-betas. We investigated whether LTBP-3 and -4 are associated with pericellular fibrillar structures of human lung fibroblast ECM, and which of their domains are important for this function. Immunoblotting analyses of isolated insoluble matrices as well as immunofluorescence analyses and confocal microscopy indicated that both LTBP-3 and -4 get assembled into the ECM. Interestingly, LTBP-4 was not detected until 7-10 days of culture and LTBP-3 until 14 days of culture. This was a major difference from the deposition kinetics of LTBP-1, which was detected already within 2 days of culture. Expression analyses by real time RT-PCR indicated that the slow appearance of LTBP-3 and -4 was due to the low expression levels soon after subculture. Recombinant N-terminal fragments of LTBP-3 and -4 bound readily to fibroblast ECM. The C-terminal domain of LTBP-4, but not of LTBP-3, also associated with the matrix structures. The levels of ECM-associated latent complexes of TGF-beta1 increased in parallel with the increased production and deposition of the LTBPs. The amount of active TGF-beta in the conditioned medium decreased during extended culture. Our results suggest that ECM is an important site of deposition also for LTBP-3 and -4 and that the temporal and spatial targeting of the TGF-beta complexes are associated with ECM maturation.     

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.     

Novel surface display system for heterogonous proteins on Lactobacillus plantarum

Aims: To establish a novel cell surface display system that would enable the display of target proteins on Lactobacillus plantarum. Methods and Results: Blast P analysis of the amino acids sequence data revealed that the N‐terminus of the putative muropeptidase MurO from L. plantarum contained two putative lysin motif (LysM) repeat regions, implying that the MurO was involved in bacterial cell wall binding. To investigate the potential of MurO for surface display, green fluorescent protein (GFP) was fused to MurO at its C‐terminus and the resulting fusion protein was expressed in Escherichia coli. After being mixed with L. plantarum cells in vitro, GFP was successfully displayed on the surfaces of L. plantarum cells. Increases in the fluorescence intensities of chemically pretreated L. plantarum cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for MurO. SDS sensitivity assay showed that the GFP fluorescence intensity was reduced after being treated with SDS. To demonstrate the applicability of the MurO‐mediated surface display system, β‐galactosidase from Bifidobacterium bifidium, in place of GFP, was functionally displayed on the surface of L. plantarum cells via MurO. Conclusions: The MurO was a novel anchor protein for constructing a surface display system for L. plantarum. Significance and Impact of Study: The success in surface display of GFP and β‐galactosidase opened up the feasibility of employing the cell wall anchor of MurO for surface display in L. plantarum.     

The fibrous system in the extracellular matrix of hydromedusae

The ultrastructure and the histochemistry of the fibrous system in the mesogloeal extracellular matrix (ECM) of two hydromedusae (Polyorchis penicillatus and Aglanlha digitale) has been examined. There is a fundamental difference in the architecture of the fibrous system between the two species. In Polyorchis, 60-150 A thick, striated fibrils with periodicities of 60-65 A form a three-dimensional network which fills in the entire ECM of outer and inner mesogloea. In the outer mesogloea vertical fibres (up to 1.8 mum in diameter) penetrate the threedimensional network and branch near the exumbrellar and subumbrellar side. These branches impinge on a dense matrix covering the exumbrellar and subumbrellar surface. In Aglantha the branches of thick vertical fibres anchor at the subumbrellar side in a dense plexus (0.2-0.3 mum in thickness) which consists of two types of fibrils (35-40 and 80-100 nm in diameter). Towards the exumbrellar side the vertical fibres branch and intermingle with a meshwork of non-striated fibrils with uniform diameter (35-40 nm). These fibrils form a laminated structure (about 1 mum in thickness) so that fibrils of each layer course in the same direction but fibrils of adjacent layers run perpendicularly to each other. The banded pattern with periodicities of 600-640 A observed in the electron microscope and by histochemical methods confirm the thick vertical fibres and their branches to be a collagen. There is also strong evidence that the laminated structure in Aglantha represents layers of collagen fibrils.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

Synergistically regulated spontaneous calcium signaling is attributed to cartilaginous extracellular matrix metabolism

Ca²⁺ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca ²⁺] _i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca ²⁺ signal of in-situ porcine chondrocytes was [Ca ²⁺] _o dependent, and mediated by [Ca ²⁺] _i store release. T-type voltage-dependent calcium channel (T-VDCC) mediated [Ca ²⁺] _o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5-trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca ²⁺ release and store-operated calcium entry significantly abolished spontaneous [Ca ²⁺] _i signaling of in-situ chondrocytes. Moreover, blocking ER Ca ²⁺ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca ²⁺] _i signaling of in-situ porcine chondrocytes was tightly regulated by [Ca ²⁺] _o influx, InsP3Rs mediated [Ca ²⁺] _i store release, and Orais mediated calcium release-activated calcium channels activation. Both T-VDCC mediated [Ca ²⁺] _o influx and InsP3Rs mediated ER Ca ²⁺ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.     

Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells

In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.     

乳酸杆菌S-层蛋白的特性及功能性研究进展

文章综述了S-层蛋白的性质和功能,重点介绍了S-层蛋白对乳酸杆菌表面性质和黏附性的影响以及调节肠道功能的作用,包括减少病原菌引起的细胞凋亡、调节免疫细胞的活性、与SIGNR3相互作用参与肠道免疫反应、通过TLRS-My D88-NF-κB途径发挥生物学功能以及调控肠道黏膜相关蛋白表达。由此证明了S-层蛋白对于乳酸杆菌发挥免疫调节功能具有重要的作用。     

A three-dimensional collagen-fiber network model of the extracellular matrix for the simulation of the mechanical behaviors and micro structures

The extracellular matrix (ECM) provides structural and biochemical support to cells and tissues, which is a critical factor for modulating cell dynamic behavior and intercellular communication. In order to further understand the mechanisms of the interactive relationship between cell and the ECM, we developed a three-dimensional (3D) collagen-fiber network model to simulate the micro structure and mechanical behaviors of the ECM and studied the stress–strain relationship as well as the deformation of the ECM under tension. In the model, the collagen-fiber network consists of abundant random distributed collagen fibers and some crosslinks, in which each fiber is modeled as an elastic beam and a crosslink is modeled as a linear spring with tensile limit, it means crosslinks will fail while the tensile forces exceed the limit of spring. With the given parameters of the beam and the spring, the simulated tensile stress–strain relation of the ECM highly matches the experimental results including damaged and failed behaviors. Moreover, by applying the maximal inscribed sphere method, we measured the size distribution of pores in the fiber network and learned the variation of the distribution with deformation. We also defined the alignment of the collagen-fibers to depict the orientation of fibers in the ECM quantitatively. By the study of changes of the alignment and the damaged crosslinks against the tensile strain, this paper reveals the comprehensive mechanisms of four stages of ‘toe’, ‘linear’, ‘damage’ and ‘failure’ in the tensile stress–strain relation of the ECM which can provide further insight in the study of cell-ECM interaction.     

Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi

SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 μg mg⁻¹) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3^−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.     

A line of rat ovarian surface epithelium provides a continuous source of complex extracellular matrix

Summary A spontaneously immortalized, yet non-tumorigenic rat ovarian surface epithelial (ROSE 199) cell line, deposits large amounts of extracellular matrix (ECM) in response to crowding. The characteristics and components of ROSE 199-derived cell-free ECM were compared after three different preparative techniques: treatment with 20 mM ammonium hydroxide, with 1% sodium deoxycholate, or by repeated freeze-thaws. The ECMs were analyzed by histochemistry, immunofluorescence, electron microscopy, and Western immunoblotting. Components of ROSE 199 ECM included laminin, fibronectin, and collagen types I and III. Even though ROSE 199 is an epithelial cell line, striated collagen fibers formed a major part of its matrix. Thus, ROSE 199 matrix consists of both basement membrane and stromal matrix components. This matrix supported the adhesion, spreading, and growth of several cell types without altering their morphology or growth pattern, and enhanced the attachment of some cell types that spread on plastic only with difficulty. Immunofluorescence, electron microscopy, and dry weight determinations indicated that a greater proportion of matrix was retained in preparations obtained by ammonium hydroxide or freeze thaw techniques than after sodium deoxycholate treatment. Ammonium hydroxide and freeze-thaw treated matrices were also superior to sodium deoxycholate preparations as evidenced by enhanced initial cellular adhesion and spreading compared to cells plated on plastic. Residual nuclear material did not seem to affect the biological activity of this matrix. ROSE 199 extracellular matrix provides a novel, complex substratum for cell culture and for studies of matrix functions and synthesis.