Surface Layer Protein Research Articles

Limosilactobacillus reuteri HY7503 and Its Cellular Proteins Alleviate Endothelial Dysfunction by Increasing Nitric Oxide Production and Regulating Cell Adhesion Molecule Levels.

Endothelial dysfunction, which is marked by a reduction in nitric oxide (NO) production or an imbalance in relaxing and contracting factor levels, exacerbates atherosclerosis by promoting the production of cell adhesion molecules and cytokines. This study aimed to investigate the effects of Limosilactobacillus reuteri HY7503, a novel probiotic isolated from raw milk, on endothelial dysfunction. Five lactic acid bacterial strains were screened for their antioxidant, anti-inflammatory, and endothelium-protective properties; L. reuteri HY7503 had the most potent effect. In a mouse model of angiotensin II-induced endothelial dysfunction, L. reuteri HY7503 reduced vascular thickening (19.78%), increased serum NO levels (226.70%), upregulated endothelial NO synthase (eNOS) expression in the aortic tissue, and decreased levels of cell adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and serum cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin-6 [IL-6]). In TNF-α-treated human umbilical vein endothelial cells (HUVECs), L. reuteri HY7503 enhanced NO production and reduced cell adhesion molecule levels. In HUVECs, surface-layer proteins (SLPs) were more effective than extracellular vesicles (exosomes) in increasing NO production and decreasing cell adhesion molecule levels. These findings suggested that L. reuteri HY7503 may serve as a functional probiotic that alleviates endothelial dysfunction.

B-090 Efficient Hydrophilic Surface Coating with Low Concentrations of Bovine Gamma Globulins

Abstract Background Bovine Gamma Globulin (BGG) is a high purity immunoglobulin fraction of bovine serum. BGG is used as a blocker to prevent non-specific binding in immunoassays and, due to the mixture of immunoglobulins, its blocking capabilities differ from traditional blockers like Bovine Serum Albumin (BSA). Our objective was to evaluate the adsorption characteristics of BGG to In vitro diagnostic relevant hydrophilic surfaces compared to BSA. A bead-based immunoassay was utilized to quantify the mass of protein coating a bead surface and atomic force microscopy (AFM) to analyze physical properties of the protein-surface layer. Methods The mass of BGG bound to hydrophilic beads (Dynabeads M-270 Carboxylic Acid) was determined across varying concentrations ranging from 0.1% to 1.0% in pH 7.0 phosphate buffer. Coated beads were heated in 1x SDS loading dye to dissociate bound protein, then ran on a nonreducing SDS-PAGE gel. The total protein was measured using densitometry against a standard curve, using ImageJ, and analyzed with GraphPad Prism. This methodology was also used for BSA. AFM was used to image a topographical view of BGG binding to a silica surface. Samples were prepared at concentrations from 0.1% to 1.0% in phosphate buffer at pH 7.0. Followed by a series of washes to remove non-bound molecules, the surface was allowed to dry, then scratched with an AFM cantilever. The surface was then analyzed for roughness and thickness of BGG adsorption for each concentration. Results BGG and BSA have notable differences in their coating properties at a concentration of 1.0%. In the bead assay, the protein-surface binding is enhanced ∼4.8-fold for BGG compared to BSA. Interestingly, even at 0.1% BGG bound ∼0.8-fold more than BSA at 1.0%. Despite a 6-fold increase in mass bound to the bead surface with 1.0% BGG compared to 0.1%, AFM revealed a 1.3-fold and 1.8-fold decrease in height and roughness at the lower concentration, while maintaining effective coating. These results suggest that higher BGG concentrations result in thicker, rougher surface coating. This could lead to steric hindrance and obstruction of binding sites, affecting assay performance. Therefore, a lower concentration of BGG may lead to better surface coating and prevent reduced sensitivity and accuracy within immunoassays. Conclusions The bead binding assay demonstrates that BGG displays surface adsorption with comparable or improved results with lower concentrations compared to BSA. At 0.1%, BGG exhibits effective binding to a hydrophilic surface equivalent to that achieved by BSA at 1.0%. Additionally, AFM data suggest that even at 0.1%, BGG effectively coats the silica surface and potentially reduces steric hinderance compared to BGG at 1%. While BSA remains a reliable choice for many applications, our findings suggest that BGG can be an effective blocker for immunoassay applications. The choice of a suitable blocker varies depending on factors such as assay high background, sensitivity, and specificity. By tailoring the choice of blocker to the specific requirements of the assay, researchers can optimize assay performance by improving the accuracy and the reliability of the assay.

The Anti-obesity Effects of Postbiotics: A Systematic Review of Pre-Clinical and Clinical Studies

BackgroundThe growing prevalence of obesity has become a major concern worldwide, therefore a great number of studies are conducted every day in the field of obesity. Since postbiotics are a newly introduced term, there is not much systematic evidence about their function and impact on obesity. We designed this study to systematically review the effect of different types of postbiotics on obesity. MethodsA systematic search was conducted using PubMed, SCOPUS, and Web of Science databases up to August 2023. Both human and animal interventional studies that investigated the effects of any type of postbiotic on obesity and obesity-related factors were eligible. Screening, data extraction, and quality assessment were conducted independently by two researchers. The quality of the studies was appraised using Cochrane and Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE’s) risk of bias tool. ResultsOf the 19373 retrieved studies, finally, 49 studies were included (9 human studies and 40 animal studies). Short-chain fatty acids and heat-killed (inactivated) bacteria were the most used postbiotics. In human clinical trials, inactivated Lactobacillus amylovorus (CP1563), Bifidobacterium animalis subsp. lactis (CECT 8145) and Pediococcus pentosaceus (LP28) were administered orally as postbiotics which improved body composition and anthropometric indices. Animal studies evaluated other types of postbiotics including muramyl dipeptide, cell-free extracts, urolithin A&B, extracellular Vesicles, exopolysaccharides, and surface Layer Proteins, supporting the anti-obesity effects of postbiotics. ConclusionPostbiotics seem to be a safe intervention and the results were in favor of a reduction in adipogenesis as well as an increase in energy expenditure. Further high-quality studies are required in this relatively new topic.

Caesalpinia bonducella Seeds Extracts are Non-toxic to the Gut Bacteria Lactobacillus rhamnosus, as Substantiated by In vitro and In silico Studies

The seed kernels of Caesalpinia bonducella, a traditional medicinal plant in India, are widely used to treat various disorders, including polycystic ovary syndrome. The seed kernel possesses anti-bacterial properties against many pathogenic bacteria. However, their impact on Lactobacillus spp., a prominent gram-positive gut bacterium, has not been studied till date. The present study employed both in vitro and in silico methods to illustrate the effect of seed extract of C. bonducella against Lactobacillus rhamnosus GG. For this, disc diffusion assay was performed with 100, 500, and 1000 µg/ml of aqueous and methanolic seed extract against L. rhamnosus and E. coli, and the zone of inhibition was measured. While both the extracts inhibited the growth of E. coli, it did not show any zone of inhibition against L. rhamnosus. The latter possess surface layer proteins, SlpX and SlpA, which prevented the influx of the phytocompounds of C. bonducella, as demonstrated by molecular docking using Autodock Vina. Docking results showed that the binding of the phytocompounds to the SlpX and SlpA proteins was not in the active pockets. These findings conclude that C. bonducella seed kernel extracts are safe against the gut bacteria L. rhamnosus.

Modulation of Mouse Dendritic Cells In Vitro by Lactobacillus gasseri Postbiotic Proteins.

Different lactobacilli are probiotics for their beneficial effects that confer to the host. Recently, some of these effects were associated with released metabolic products/constituents (postbiotics). In the present study, the potential immunomodulatory capacity of the probiotic Lactobacillus gasseri OLL2809 cell-free supernatant (sup) was investigated in murine bone marrow-derived dendritic cells (DCs). Bacteria induced significantly higher expression of all examined cytokines than those induced by the stimulatory lipopolysaccharide (LPS) itself. On the contrary, sup only induced the anti-inflammatory IL-10 similarly to LPS, whereas IL-12 and IL-6 secretions were stimulated at a lower level. Moreover, sup reduced the surface expression of the analyzed co-stimulatory markers CD40, CD80, and CD86. Treatments of sup with different digestive enzymes indicated the proteinaceous nature of these immunomodulatory metabolites. Western blot and immunoadsorption analyzes revealed cross-reactivity of sup with the surface-layer proteins (SLPs) isolated from OLL2809. Therefore, we directly tested the ability of OLL2809 SLPs to stimulate specifically cytokine expression in iDCs. Interestingly, we found that all tested cytokines were induced by SLPs and in a dose-dependent manner. In conclusion, our results highlighted distinct immune properties between L. gasseri OLL2809 and its metabolites, supporting the concept that bacterial viability is not an essential prerequisite to exert immunomodulatory effects.

Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant.

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.

Time-resolved cryogenic electron tomography for the study of transient cellular processes.

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

Evaluating The Cytotoxic Activity of Lactobacillus plantarum IIA-1A5 Against MCF-7 Human Breast Cancer Cells and Identifying Its Surface Layer Protein Gene

Breast cancer is a serious global health concern, with a high mortality rate worldwide. Using natural compounds as potential cancer therapies is a promising approach to address this issue. Previous research has shown that probiotic lactic acid bacteria (LAB) and their metabolites, such as surface layer protein (slp), have a positive impact on a variety of health disorders, including cancer. The purpose of this study was to evaluate the ability of Lactobacillus plantarum IIA-1A5 to suppress the growth of the MCF-7 breast cancer cell line and detect the presence of the slp gene. Intracellular and extracellular protein fractions were isolated from L. plantarum IIA-1A5 cultures. The protein concentrations and molecular weights of the extracts were measured. The anticancer activity of the extracts was assessed using the MTT cytotoxicity test, and IC50 values were calculated. The slp gene was identified through polymerase chain reaction (PCR) amplification and nucleotide sequencing. The results demonstrated that L. plantarum IIA-1A5 had a concentration-dependent inhibitory effect on MCF-7 breast cancer cells, with IC50 values of 6.831 and 12.35 μg/mL for intracellular extracts and extracellular extracts, respectively. Additionally, PCR amplification and nucleotide sequencing confirmed the presence of the slp gene, which may contribute to the strain’s anticancer abilities. These findings suggest the potential of L. plantarum IIA-1A5 as a natural anticancer agent against MCF-7 breast cancer cells. Further research is warranted to elucidate the underlying mechanisms of L. plantarum IIA-1A5 in breast cancer treatment.

An ultrasensitive genosensor for detection of toxigenic and non-toxigenic Clostridioides difficile based on a conserved sequence in surface layer protein coding gene

Clostridioides difficile (C. difficile) is the most common agent of antibiotic-associated diarrhea, leading to intestinal infection through the secretion of two major toxins. Not all strains of this bacterium are toxigenic, but some of them cause infection via their accessory virulence factors, such as surface layer protein (SlpA). SlpA is conserved in both toxigenic and non-toxigenic strains of C. difficile. In the present work, an amplification-free electrochemical genosensor was designed for the detection of the slpA gene. A glassy carbon electrode coated with gold nanoparticle-reduced graphene oxide nanocomposite was used as the working electrode, and its surface was modified using a simple thiolated linear oligonucleotide as the bioreceptor. Moreover, the hexaferrocenium tri[hexa(isothiocyanato) iron(III)] trihydroxonium (HxFc) complex was used as an intercalator, and its redox signal was recorded using differential pulse voltammetry. Scan rate studies indicated a quasi-reversible adsorption-controlled process for the HxFc complex. This genosensor showed high sensitivity with a limit of detection of 0.2 fM, a linear response range of 0.46–1900 fM, and a satisfactory specificity toward the synthetic slpA target gene. Also, the genosensor indicated responses in the mentioned linear range toward the genome extracted from either toxigenic or non-toxigenic strains of C. difficile.

Exopolysaccharides and Surface-Layer Proteins Expressed by Biofilm-State Lactiplantibacillus plantarum Y42 Play Crucial Role in Preventing Intestinal Barrier and Immunity Dysfunction of Balb/C Mice Infected by Listeria monocytogenes ATCC 19115.

Our previous study showed that Lactiplantibacillus plantarum Y42 in the biofilm state can produce more exopolysaccharides and surface-layer proteins and showed a stronger promoting effect on intestinal barrier function than that in the planktonic state. In this study, oral administration of the live/pasteurized planktonic or biofilm L. plantarum Y42 and its metabolites (exopolysaccharides and surface-layer proteins) increased the expression of Occludin, Claudin-1, ZO-1, and MUC2 in the gut of the Balb/C mice after exposure to Listeria monocytogenes ATCC 19115 and inhibited the activation of the NLRP3 inflammasome pathway, which in turn reduced the levels of inflammatory cytokines IL-1β and IL-18 in the serum of the mice. Furthermore, oral administration of the live/pasteurized planktonic or biofilm L. plantarum Y42 and its metabolites increased the abundance of beneficial bacteria (e.g., Lachnospiraceae_NK4A136_group and Prevotellaceae_UCG-001) while reducing the abundance of harmful bacteria (e.g., norank_f__Muribaculaceae) in the gut of the mice, in line with the increase of short-chain fatty acids and indole derivatives in the feces of the mice. Notably, biofilm L. plantarum Y42 exerted a better preventing effect on the intestinal barrier dysfunction of the Balb/C mice due to the fact that biofilm L. plantarumY42 expressed more exopolysaccharides and surface-layer proteins than the planktonic state. These results provide data support for the use of exopolysaccharides and surface-layer proteins extracted from biofilm-state L. plantarum Y42 as functional food ingredients in preventing intestinal barrier dysfunction.

Postbiotics in colorectal cancer: intervention mechanisms and perspectives.

Colorectal cancer (CRC) is a common malignancy affecting the gastrointestinal tract worldwide. The etiology and progression of CRC are related to factors such as environmental influences, dietary structure, and genetic susceptibility. Intestinal microbiota can influence the integrity of the intestinal mucosal barrier and modulate intestinal immunity by secreting various metabolites. Dysbiosis of the intestinal microbiota can affect the metabolites of the microbial, leading to the accumulation of toxic metabolites, which can trigger chronic inflammation or DNA damage and ultimately lead to cellular carcinogenesis and the development of CRC. Postbiotics are preparations of inanimate microorganisms or their components that are beneficial to the health of the host, with the main components including bacterial components (e.g., exopolysaccharides, teichoic acids, surface layer protein) and metabolites (e.g., short-chain fatty acids, tryptophan metabolite, bile acids, vitamins and enzymes). Compared with traditional probiotics, it has a more stable chemical structure and higher safety. In recent years, it has been demonstrated that postbiotics are involved in regulating intestinal microecology and improving the progression of CRC, which provides new ideas for the prevention and diagnosis of CRC. In this article, we review the changes in intestinal microbiota in different states of the gut and the mechanisms of anti-tumor activity of postbiotic-related components, and discuss the potential significance of postbiotics in the diagnosis and treatment of CRC. This reviews the changes and pathogenesis of intestinal microbiota in the development of CRC, and summarizes the relevant mechanisms of postbiotics in resisting the development of CRC in recent years, as well as the advantages and limitations of postbiotics in the treatment process of CRC.

A monoclonal antibody collection for C. difficile typing ?

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. The goal of this study was to generate monoclonal antibodies recognizing various epidemic ribotypes of C. difficile. To do so, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

Effects of plant-based heat killed lactic acid bacteria and its lithium chloride-extracted cellular protein on high-fat-induced obesity

The anti-obesity action of postbiotics derived from plant-based probiotics remains unclear. The effects of heat killed lactic acid bacteria (Lactobacillus plantarum, LPHK and Lactobacillus curvatus, LCHK) along with bioactive their cell envelop components known as surface layer proteins (SLPs, LPSLP, and LCSLP) on obesity were investigated. LPHK, LCHK, LPSLP, and LCSLP significantly reduced lipid accumulation in 3T3-L1 adipocytes, concurrently regulating the expression of adipogenic genes. Notably, LPHK demonstrated significant improvements in high-fat (HF)-induced body weight gain, adipose tissue weight gain, liver weight gain, and reduced plasma concentrations of triglyceride, total-cholesterol, and LDL-cholesterol in C57BL/6J mice. Additionally, LPHK significantly regulated the expression of genes related to adipogenesis and anti-apoptosis. Moreover, while LPSLP significantly reduced liver weight, plasma triglyceride and LDL-cholesterol concentrations, and upregulated genes associated with hepatic fatty acid oxidation, it did not affect the body weight and adipose tissue weight gain. These results suggest that additional components other than SLP are necessary for the anti-obesity action of LPHK. In conclusion, non-viable plant-based LPHK can be used as a naturally occurring product for preventing obesity, especially in individuals with immunocompromised conditions.

Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function

ABSTRACTParaprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes’ formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria.

The phytopathogen Paracidovorax citrulli possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if P. citrulli formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of npdA orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by P. citrulli AAC00-1 was confirmed by gene deletion mutagenesis (ΔnpdA), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of npdA were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in P. citrulli and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the P. citrulli S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.

Effects of Lacticaseibacillus paracasei SNB-derived postbiotic components on intestinal barrier dysfunction and composition of gut microbiota

The bacterial surface components are considered as effector molecules and show the potential to support intestinal health, but the detailed mechanism of how the gut microbiota changes after the intervention of surface molecules is still unknown. In the present study, capsular polysaccharide (B-CPS) and surface layer protein (B-SLP) were extracted from Lacticaseibacillus paracasei S-NB. The protective effect of direct administration of B-CPS (100 μg/mL) and B-SLP (100 μg/mL) on intestinal epithelial barrier dysfunction was verified based on the LPS-induced Caco-2 cell model. Additionally, the B-CPS and B-SLP could be utilized as carbon source and nitrogen source for the growth of several Lactobacillus strains, respectively. The postbiotic potential of B-CPS and B-SLP was further evaluated by in vitro fermentation with fecal cultures. The B-CPS and a combination of B-CPS and B-SLP regulated the composition of gut microbiota by increasing the relative abundances of Bacteroides, Bifidobacterium, Phascolarctobacterium, Parabacteroides, Subdoligranulum and Collinsella and decreasing the abundance of pathogenic bacteria like Escherichia-Shigella, Blautia, Citrobacter and Fusobacterium. Meanwhile, the total short-chain fatty acid production markedly increased after fermentation with either B-CPS individually or in combination with B-SLP. These results provided an important basis for the application of B-CPS and B-SLP as postbiotics to improve human intestinal health.

Propionibacterium freudenreichii CIRM-BIA 129 mitigates colitis through S layer protein B-dependent epithelial strengthening.

The growing incidence of human diseases involving inflammation and increased gut permeability makes the quest for protective functional foods more crucial than ever. Propionibacterium freudenreichii (P. freudenreichii) is a beneficial bacterium used in the dairy and probiotic industries. Selected strains exert anti-inflammatory effects, and the present work addresses whether the P. freudenreichii CIRM-BIA129, consumed daily in a preventive way, could protect mice from acute colitis induced by dextran sodium sulfate (DSS), and more precisely, whether it could protect from intestinal epithelial breakdown induced by inflammation. P. freudenreichii CIRM-BIA129 mitigated colitis severity and inhibited DSS-induced permeability. It limited crypt length reduction and promoted the expression of zonula occludens-1 (ZO-1), without reducing interleukin-1β mRNA (il-1β) expression. In vitro, P. freudenreichii CIRM-BIA129 prevented the disruption of a Caco-2 monolayer induced by proinflammatory cytokines. It increased transepithelial electrical resistance (TEER) and inhibited permeability induced by inflammation, along with an increased ZO-1 expression. Extracellular vesicles (EVs) from P. freudenreichii CIRM-BIA129, carrying the surface layer protein (SlpB), reproduced the protective effect of P. freudenreichii CIRM-BIA129. A mutant strain deleted for slpB (ΔslpB), or EVs from this mutant strain, had lost their protective effects and worsened both DSS-induced colitis and inflammation in vivo. These results shown that P. freudenreichii CIRM-BIA129 daily consumption has the potential to greatly alleviate colitis symptoms and, particularly, to counter intestinal epithelial permeability induced by inflammation by restoring ZO-1 expression through mechanisms involving S-layer protein B. They open new avenues for the use of probiotic dairy propionibacteria and/or postbiotic fractions thereof, in the context of gut permeability.NEW & NOTEWORTHY Propionibacterium freudenreichii reduces dextran sodium sulfate (DSS)-induced intestinal permeability in vivo. P. freudenreichii does not inhibit inflammation but damages linked to inflammation. P. freudenreichii inhibits intestinal epithelial breakdown through S-layer protein B. The protective effects of P. freudenreichii depend on S-layer protein B. Extracellular vesicles from P. freudenreichii CB 129 mimic the protective effect of the probiotic.

Lyoprotectants for Lactobacillus brevis B144 and their capability to maintain the expression of surface layer protein

Lactobacillus brevis as a probiotic has been reported to facilitate micronutrient absorption such as minerals (zinc, calcium, magnesium) into the intestine through the surface layer protein (Slp). Negatively charged-Slp from the bacteria that adheres on the intestinal mucosa binds to the positively charged-minerals, e.g., calcium, zinc, iron, magnesium and potassium via an ionic interaction. In this study, several sugar-based lyoprotectants (glucose, galactose, and lactose) and skim milk were implemented in the L. brevis B144 InaCC freeze drying. The dried L. brevis B144 InaCC was kept in the refrigerator up to three months and monthly tested for the cell viability. The dried L. brevis B144 InaCC was re-culture and then its Slp was isolated using 8 M guanidine hydrochloride. In general, combinations of skim milk and the sugar-based lyoprotectant provided higher protection than the skim milk alone. Meanwhile, without any lyoprotectant, the L. brevis B144 InaCC lose its ability to grow. In regards to maintain the capability of the Slp expression, the L. brevis B144 InaCC which was protected with the combination of 20% skim milk and 10% galactose showed the highest protein expression level among the other lyoprotectants.

Interaction mechanism between surface layer protein and yeast mannan: Insights from multi-spectroscopic and molecular dynamics simulation analyses

Tibet kefir grain (TKG) formation is mainly dependent on the aggregation of lactobacillus and yeasts. The interaction of surface layer protein (SLP) and yeast mannan plays an important role in mediating the co-aggregation of Lactobacillus kefiri with Saccharomyces cerevisiae. The interaction mechanism of the two was researched through multispectral spectroscopy, morphology observation and silico approaches. Fluorescence spectra data revealed that mannan was bound to SLP through a spontaneous binding process. The particle size of the binding complex increased as the mannan concentration increased. Synchronous fluorescence spectroscopy and circular dichroism (CD) spectra showed the conformational and microenvironment alteration of SLP treated with mannan. Molecular docking results indicated that hydrophobic interactions played major roles in the formation of SLP-mannan complexes. These findings provide a deeper insight into the interactions of protein and polysaccharide, and this knowledge is valuable in the application of SLP and mannan in co-fermentation systems.

A Next-Generation qPlus-Sensor-Based AFM Setup: Resolving Archaeal S-Layer Protein Structures in Air and Liquid.

Surface-layer (S-layer) proteins form the outermost envelope in many bacteria and most archaea and arrange in two-dimensional quasicrystalline structures via self-assembly. We investigated S-layer proteins extracted from the archaeon Pyrobaculum aerophilium with a qPlus sensor-based atomic force microscope (AFM) in both liquid and ambient conditions and compared it to transmission electron microscopy (TEM) images under vacuum conditions. For AFM scanning, a next-generation liquid cell and a new protocol for creating long and sharp sapphire tips was introduced. Initial AFM images showed only layers of residual detergent molecules (sodium dodecyl sulfate, SDS), which are used to isolate the S-layer proteins from the cells. SDS was not visible in the TEM images, requiring more thorough sample preparation for AFM measurements. These improvements allowed us to resolve the crystallike structure of the S-layer samples with frequency-modulation AFM in both air and liquid.