Mucus-binding Protein Research Articles

Heat-Killed Lactobacillus paracasei SMB092 Reduces Halitosis by Stimulating the Expression of β-Defensins in Oral Keratinocytes

The purpose of this study is to evaluate Lactobacillus paracasei SMB092 as a prophylactic agent for oral pathogens. We examined the physical interaction of SMB092 with a host by identifying the presence of mucus-binding (MuB) protein domains and the capacity of the mucin binding. We determined the role of heat-killed SMB092 in host oral immunity by quantifying the mRNA levels of β-defensins (BDs), Toll-like receptors (TLRs), and their cofactors (CD14/CD36) in normal human oral keratinocytes (HOK-16B cells). To assess the clinically relevant oral health effects of heat-killed SMB092, the growth of Porphyromonas (P.) gingivalis and the production of a volatile sulfur compound (H2S) were also measured in the filtered condition media (FCM) obtained from its cultures with HOK-16B cells. SMB092 possessed 14 putative MuB protein domains and was attached to mucin. Significant amounts of hBD1/2 and TLR2/6 were expressed in heat-killed SMB092-treated HOK-16B cells. The specific neutralization of TLR2 attenuated the expression of hBD1/2 and CD14/CD36. The FCM inhibited the growth of P. gingivalis and the production of H2S. Our data indicate that heat-killed SMB092 may contribute to a healthy oral microbiome as an immune stimulant in the production of BDs via the activation of the TLR2/6 signaling pathway.

The regulation of simulated artificial oro-gastrointestinal transit stress on the adhesion of Lactobacillus plantarum S7

BackgroundOro-gastrointestinal stress in the digestive tract is the main stress to which orally administered probiotics are exposed. The regulation of oro-gastrointestinal transit (OGT) stress on the adhesion and survival of probiotics under continuous exposure to simulated salivary-gastric juice-intestinal juice was researched in this study.ResultsLactobacillus plantarum S7 had a higher survival rate after exposure to simulated OGT1 (containing 0.15% bile salt) stress and OGT2 (containing 0.30% bile salt) stress. The adhesion ability of L. plantarum S7 was significantly increased by OGT1 stress (P < 0.05) but was not changed significantly by OGT2 stress (P > 0.05), and this trend was also observed in terms of the thickness of the surface material of L. plantarum S7 cells. The expression of surface proteins of L. plantarum S7, such as the 30 S ribosomal proteins, mucus-binding protein and S-layer protein, was significantly downregulated by OGT stress (P < 0.05); meanwhile, the expression of moonlight proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycorate kinase (PGK), beta-phosphoglucomutase (PGM1), GroEL and glucose-6-phosphate isomerase (PGI), was significantly upregulated (P < 0.05). However, the upregulation of GAPDH, PGK, PGM1 and PGI mediated by OGT1 stress was greater than those mediated by OGT2 stress. The quorum sensing pathway of L. plantarum S7 was changed significantly by OGT stress compared with no OGT stress cells (P < 0.05), and the expression of Luxs in the pathway was significantly upregulated by OGT1 stress (P < 0.05). The ABC transportation pathway was significantly altered by OGT1 stress (P < 0.05), of which the expression of the peptide ABC transporter substrate-binding protein and energy-coupling factor transporter ATP-binding protein EcfA was significantly upregulated by OGT stress (P < 0.05). The glycolide metabolism pathway was significantly altered by OGT1 stress compared with that in response to OGT2 stress (P < 0.05).ConclusionL. plantarum S7 had a strong ability to resist OGT stress, which was regulated by the proteins and pathways related to OGT stress. The adhesion ability of L. plantarum S7 was enhanced after continuous exposure to OGT1 stress, making it a potential probiotic with a promising future for application.

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose.

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-336, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and d-mannose. Among four mannobioses, α-1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 μM). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or α-1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

Evaluation of Potential Probiotic Properties of a Strain of Lactobacillus plantarum for Shrimp Farming: From Beneficial Functions to Safety Assessment.

In recent years the safety of probiotics has received increasing attention due to the possible transfer and spread of virulence factors (VFs) and antibiotic resistance genes (ARGs) among microorganisms. The safety of a strain of Lactobacillus plantarum named W2 was evaluated in phenotype and genotype in the present study. Its probiotic properties were also evaluated both in vivo and in vitro, including adherence properties, antibacterial properties and beneficial effects on the growth and immunity of Pacific white shrimp, Penaeus vannamei. Hemolysis tests, antibiotic resistance tests and whole genome sequence analysis showed that W2 had no significant virulence effects and did not carry high virulence factors. W2 was found to be sensitive to chloramphenicol, clindamycin, gentamicin, kanamycin and tetracycline, and to be resistant to ampicillin and erythromycin. Most ARGs have no transfer risk and a few have transfer risk but no significant enrichment in human-associated environments. The autoaggregation of W2 was 82.6% and the hydrophobicity was 81.0%. Coaggregation rate with Vibrio parahaemolyticus (24.9%) was significantly higher than Vibrio’s autoaggregation rate (17.8%). This suggested that W2 had adhesion potential to mucosal/intestinal surfaces and was able to attenuate the adherence of V. parahaemolyticus. In addition, several adhesion-related protein genes, including 1 S-layer protein, 1 collagen-binding protein and 9 mucus-binding proteins were identified in the W2 genome. W2 had efficiently antagonistic activity against 7 aquatic pathogenic strains. Antagonistic components analysis indicated that active antibacterial substances might be organic acids. W2 can significantly promote the growth of shrimp when supplemented with 1 × 1010 cfu/kg live cells. Levels of 7 serological immune indicators and expression levels of 12 hepatopancreatic immune-related genes were up-regulated, and the mortality of shrimp exposed to V. parahaemolyticus was significantly reduced. Based on the above, L. plantarum W2 can be applied safely as a potential probiotic to enhance the growth performance, immunity capacity and disease resistance of P. vannamei.

Multiplex SYBR Green real-time PCR for Lactobacillus acidophilus group species targeting biomarker genes revealed by a pangenome approach

The Lactobacillus acidophilus group consists of seven closely related species. Among these, Lb. acidophilus, Lb. gallinarum, and Lb. helveticus help maintain gut health and are used as a starter for fermented food. However, these species are difficult to differentiate using conventional methods due to the high similarity between the 16S rRNA and housekeeping genes. Thus, in this study, we selected biomarker genes to identify and discriminate the three species via pangenome analysis, and a multiplex SYBR Green real-time PCR that can be detected simultaneously in a single tube was developed. Pangenome analysis revealed three specific target genes: mucus-binding protein precursor to detect Lb. acidophilus, an amino acid ABC superfamily ATP binding cassette transporter carrier protein to detect Lb. gallinarum, and selenocysteine lyase to detect Lb. helveticus. The specificity was robustly verified using 26 Lb. acidophilus group strains and 62 other strains. The detection limits were 101 colony-forming units (CFU)/ml in pure culture. As per our findings, the developed method satisfactorily monitored Lb. acidophilus group species in probiotic and dairy products. This result suggests that real-time PCR based on specific targets provides a promising approach for the rapid, accurate, and sensitive identification of these three species.

Adhesion Characteristics and Dual Transcriptomic and Proteomic Analysis of Lactobacillus reuteri SH23 upon Gastrointestinal Fluid Stress.

The ability to survive in the harsh gastrointestinal tract (GIT) environment is essential for Lactobacillus reuteri (L. reuteri) exhibiting beneficial effects. In this study, we found that the hydrophobicity and auto-aggregation of L. reuteri SH23 were significantly decreased and biofilm production was also significantly decreased when L. reuteri SH23 passes through the simulated GIT. Furthermore, according to the comparative transcriptome analysis, gene expression involved in the cell envelope, metabolic processes, common stress response, regulatory systems, and transporters were also affected. Meanwhile, label-free quantitative proteomics was used to identify the differential expression of surface proteins of L. reuteri in response to simulated gastrointestinal fluid. Proteins related to the ABC transporters (Lreu_0517, Lreu_0098, and Lreu_0296) and LPxTG anchor domain proteins were upregulated in the cell surface after gastrointestinal fluid treatment, which is useful for adherence and colonization of L. reuteri in the GIT. Additionally, the recombinant Mub protein could also enhance the survival ability of L. reuteri SH23 in GIT stress environment. This study provides a comprehensive understanding of the adaptation and adhesion mechanisms of L. reuteri SH23 under the gastrointestinal tract by the transcriptomics and proteomics analysis, and mucus-binding proteins were involved in the adhesion and GIT tolerance process.

Pili and other surface proteins influence the structure and the nanomechanical properties of Lactococcus lactis biofilms

Lactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globally the strains devoid of pili displayed smoother and stiffer biofilms (Young Modulus of 4–100 kPa) than those of piliated strains (Young Modulus around 0.04–0.1 kPa). Additional display of a mucus-binding protein did not affect the biofilm stiffness but made the biofilm smoother and more compact. Finally, we demonstrated the role of pili in the biofilm cohesiveness by monitoring the homotypic adhesion of bacteria to the biofilm surface. These results will help to understand the role of pili and mucus-binding proteins withstanding external forces.

Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy.

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.

Interactions between Lactobacillus plantarum NCU116 and its environments based on extracellular proteins and polysaccharides prediction by comparative analysis

Lactic acid bacteria (LAB) play a significant role in food industry and artisan fermented-food. Most of the applicable LABs were commonly obtained from natural fermented food or human gut. And Lactobacillus plantarum NCU116 was screened from a LAB-dominated traditional Chinese sauerkraut (TCS). In order to comprehend the interaction between NCU116 and its environments, comparative genomics were performed to identify genes involved in extracellular protein biosynthesis and secretion. Four secretory pathways were identified, including Sec and FPE pathways, holins and efflux ABC transporter system. Then 348 potential secretory proteins were identified, including 11 alpha-amylases responsible for degradation of macromolecules, and 8 mucus binding proteins which attribute to adherence to intestine epithelium. Besides, EPS clusters of NCU116 (EPS116) were identified and analyzed by comparing to other strains, which suggested a novel genotype of EPS clusters. These findings could be critical to extend the application of NCU116 in food and pharmaceuticals industries.

Adhesion mechanisms of lactic acid bacteria: conventional and novel approaches for testing.

Adhesion ability is a primary criterion for the selection of probiotic microorganisms. Lactic acid bacteria contribute the majority of microorganisms with probiotic properties. They have several important mechanisms for intestinal epithelial cell adhesion. In order to adhere to the intestinal cells, they generally use various structures such as flagella, pili, S layer proteins, lipoteichoic acid, exopolysaccharides and mucus binding proteins. Various in vitro experiments were designed or study models were developed to reveal the mechanisms they utilize for binding to the intestinal cells, yet, the mechanisms for their adhesion are not fully explained. The major disadvantage of conventional models is the lack of layers forming the intestinal mucosa. Besides, these models omit the presence of natural microbiota, digestive conditions and the presence of a food matrix. Because of the disadvantages of existing models, natural tissues or novel applications like 3D organ cultures, which are better able to mimic in vivo conditions, are preferred.

Glycobiology of Host‐Microbe Interactions in the Gut

The gastrointestinal (GI) tract is colonised by trillions of bacteria collectively known as the ‘gut microbiota’ which have a profound impact on human health. Bacterial adhesion is a critical step for colonization of the host. Cell surface proteins (adhesins or lectins) mediate attachment to the host and initiate colonization by interacting with host proteins or glycoconjugates. However, in contrast to pathogens, the molecular basis for adhesion of commensal bacteria or probiotics to the gut remain poorly defined.The mucus layer covering the GI tract is the first point contact between the gut microbiota and the host. There is an emerging paradigm that mucin glycans are key molecular determinants of gut homeostasis by providing binding sites and/or nutrients to the bacteria which have adapted to the mucosal environment.Here we provide structural and functional insights into how gut bacteria adapt to the GI tract by focusing on the adhesion strategies of two inhabitant species of the GI tract, Ruminococcus gnavus1,2 and Lactobacillus reuter,3,4,5. The presentation will discuss relevant mechanistic insights into the nature, structure and function of bacterial adhesins involved in the interaction between these gut symbionts and the host glycoconjugates. Our results showed that adhesion strategy is species‐ and strain‐specific and occurs via specialised mucus‐binding proteins or carbohydrate‐binding modules. Sialic acid, a terminal epitope in intestinal mucins, plays a critical role in mediating bacterial tropism along the GI tract.Exploring the molecular basis of adhesion by resident species of the gut microbiota is important for the design of microbiota‐targeted strategies to restore gut health.Support or Funding InformationThe work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), under the Institute Strategic Programme Grant for Gut Microbes and Health (BB/R012490/1).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adherence capability and safety assessment of an indigenous probiotic strain Lactobacillus rhamnosus MTCC-5897

With the growing interest in probiotic microorganisms based on their well established immense health benefits, the present investigation was aimed to assess the adhesion potential and safety of probiotic Lactobacillus rhamnosus MTCC- 5897 (LR) before it can be put into a probiotic formulations. L. rhamnosus showed an adhesion index of 166.7 ± 11, which was further confirmed by scanning electron microscopy and relative expression of mucus binding protein (Mub) and mucus adhesion promoting protein (Map-A) genes. In vitro safety assessment by tetrazolium dye reduction, neutral red and lactate dehydrogenase (LDH) release assays revealed unchanged metabolic activity of Caco-2 cells even when incubated with L. rhamnosus ranged between 106-1010 cfu/mL for 24 h. Similarly, a moderate increase in bile salt hydrolase (bsh) expression (6.84 ± 0.73 and 3.42 ± 0.39 folds in 1% and 3% bile medium respectively) further proved its safety towards normal lipid digestion and absorption. Moreover, L. rhamnosus feeding to mice (107, 109, 1011 and 1013 cfu/animal/d) repetitively for 28 days revealed no adverse effects on parameters of general animal health status including body weight, organ indices, plasma glucose, liver malondialdehyde (MDA), serum aspartate amino transaminase (AST), cholesterol, triglycerides, high-density lipoprotein (HDL). Similarly, significant (p ≤ 0.05) reduced activities of serum alanine amino transaminase (ALT) and LDH on continuous probiotic feeding were also indicative of normal liver/kidney functions as they were in normal range for mice. Further, insignificant changes in macrophage chemoattractant protein (MCP-1) in intestinal fluid irrespective of bacterial dose fed along with significant reduction (p ≤ 0.05) of tumor necrosis factor-α (TNF-α) at much higher dose (1013 cfu/animal/d) also confirmed safe response of probotic L.rhamnosus against inflammation. To conclude, the results obtained under in vitro and in vivo studies has established the Lactobacillus rhamnosus as safe and non-toxic to weaning mice as well as human epithelial cells and thus may be used as a safe food additive.

Structure and Immunomodulatory Activity of a Recombinant Mucus-Binding Protein of Lactobacillus Acidophilus

The role of mucus-binding protein (MUB) on the adhesion activity and immunomodulatory effect of Lactobacillus acidophilus. The current research mainly focuses on the adhesion and immune function of MUB from L. acidophilus. The structural characteristics and adhesion properties of MUB were analyzed in the intestinal cell models. MUB can promote the aggregation and formation of a membrane-like morphology in L. acidophilus, which could increase the survival rate of L. acidophilus in gastrointestinal tract (GIT). Furthermore, MUB could trigger immune regulation and intestinal protection through the Toll-like receptor 4 (TLR4) signaling pathway and inhibit the activation of mitogen-activated protein kinase (MAPK) signaling pathway. MUB of L. acidophilus is an important component involved in bacterial-mucus interactions and immunomodulatory effect in gastrointestinal tract.

Mechanistic insights into the host-microbe interaction and pathogen exclusion mediated by the Mucus-binding protein of Lactobacillus plantarum

Surface adhesins of pathogens and probiotics strains are implicated in mediating the binding of microbes to host. Mucus-binding protein (Mub) is unique to gut inhabiting lactic acid bacteria; however, the precise role of Mub proteins or its structural domains in host-microbial interaction is not well understood. Last two domains (Mubs5s6) of the six mucus-binding domains arranged in tandem at the C-terminus of the Lp_1643 protein of Lactobacillus plantarum was expressed in E. coli. Mubs5s6 showed binding with the rat intestinal mucus, pig gastric mucins and human intestinal tissues. Preincubation of Mubs5s6 with the Caco-2 and HT-29 cell lines inhibited the binding of pathogenic enterotoxigenic E. coli cells to the enterocytes by 68% and 81%, respectively. Pull-down assay suggested Mubs5s6 binding to the host mucosa components like cytokeratins, Hsp90 and Laminin. Mubs5s6 was predicted to possess calcium and glucose binding sites. Binding of Mubs5s6 with these ligands was also experimentally observed. These ligands are known to be associated with pathogenesis suggesting Mub might negotiate pathogens in multiple ways. To study the feasibility of Mubs5s6 delivery in the gut, it was encapsulated in chitosan-sodium tripolyphosphate microspheres with an efficiency of 65% and release up to 85% in near neutral pH zone over a period of 20 hours. Our results show that Mub plays an important role in the host-microbial cross-talk and possesses the potential for pathogen exclusion to a greater extent than mediated by L. plantarum cells. The functional and technological characteristics of Mubs5s6 make it suitable for breaking the host-pathogen interaction.

Evaluation of adhesion properties of lactobacilli probiotic candidates

Bacterial adhesion is a complex phenomenon implicated in the host-bacterial interaction that is pivotal for probiotic activity. Eight probiotic lactobacilli candidates (Lactobacillus reuteri, L. plantarum, L. mucosae, L. murinus) were screened for their ability to adhere to abiotic and biotic surfaces in vitro. Adhesion to hydrocarbons was used for hydrophobicity assessment. Three strains of L. reuteri and L. murinus C were evaluated as hydrophobic, others as intermediate. All tested strains were able to form the biofilm on polystyrene. L. mucosae D and L. reuteri E were tested for adhesion to epithelial cell lines (HeLa and Caco-2). Both were more adherent to HeLa than to Caco-2. The adhesivity degree in HeLa reached the highest value after 8 h of co-cultivation in both lactobacilli tested, then decreased. In Caco-2, adhesion was increased within 24 h from the beginning of the co-cultivation. Mucus-binding protein gene, implicated in adhesion, was detected in L. mucosae D. Therefore, the involvement of proteinaceous substances in binding process was investigated. Cells of L. mucosae D were digested by three proteolytic enzymes (proteinase K, pronase E, trypsin) and evaluated for time-dependent adhesivity changes to HeLa, Caco-2, and L929 cell lines. Results confirmed that proteins are most likely to play an important role in binding of lactobacilli to eukaryotic cells. One hour after treatment, L. mucosae D was able to overcome the effect of proteolytic cleavage. We assume that it was due to the restoration of its cell-surface binding structures. Co-cultivation of HeLa and L. mucosae D led to protuberance and communication channels formation in eukaryotic cells.

Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco-adhesion in the Gastrointestinal Tract.

Food and probiotic bacteria, in particular lactic acid bacteria, are ingested in large amounts by humans and are part of the transient microbiota which is increasingly considered to be able to impact the resident microbiota and thus possibly the host health. The lactic acid bacterium Lactococcus lactis is extensively used in starter cultures to produce dairy fermented food. Also because of a generally recognized as safe status, L. lactis has been considered as a possible vehicle to deliver in vivo therapeutic molecules with anti-inflammatory properties in the gastrointestinal tract. One of the key factors that may favor health effects of beneficial bacteria to the host is their capacity to colonize transiently the gut, notably through close interactions with mucus, which covers and protects the intestinal epithelium. Several L. lactis strains have been shown to exhibit mucus-binding properties and bacterial surface proteins have been identified as key determinants of such capacity. In this review, we describe the different types of surface proteins found in L. lactis, with a special focus on mucus-binding proteins and pili. We also review the different approaches used to investigate the adhesion of L. lactis to mucus, and particularly to mucins, one of its major components, and we present how these approaches allowed revealing the role of surface proteins in muco-adhesion.

Expression of recombinant truncated domains of mucus-binding (Mub) protein of Lactobacillus plantarum in soluble and biologically active form

Mucins amount to 70% of total proteins present in mammalian mucus and serve as important substrata for bacterial adhesion. In probiotic bacteria such as Lactobacillus plantarum, surface adhesion proteins mediate its adhesion to mucus and adhesion is pivotal in bi-directional host-microbe interactions. Mucus binding (Mub) proteins are a group of bacterial surface adhesion proteins that bind to mucin proteins. The structural framework and functional role of these proteins needs immediate attention but is poorly understood because of their large size, low yield and lack of highly purified protein. The lp_1643 gene of L. plantarum encodes a large Mub protein of 240 kDa and has six mucus binding (Mub) domains in tandem. In this study, the fragment of lp_1643 containing the last two domains with their preceding spacers herein referred to as Mubs5s6 was cloned and expressed in E. coli for probing its functional role in the adhesion of L. plantarum.The protein was expressed with a solubility enhancing maltose binding protein (MBP) fusion tag, yet the MBP-Mubs5s6 protein expressed majorly (>90%) as biologically insoluble inclusion bodies. Thus, extensive optimization of culture conditions was carried out to achieve high level soluble expression (∼70%) of Mubs5s6 protein from its initial low level of solubility. The recombinant protein was purified up to homogeneity by affinity chromatography. Recombinant MBP-Mubs5s6 protein showed strong adhesion potential by binding with human intestinal tissue sections. Our results show a step-by-step hierarchical approach to improve the solubility of difficult-to-express extracellular surface proteins while retaining high functional viability.

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins.

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism.

ABSTRACTVancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections.IMPORTANCE Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcalmol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management.