Mannose-specific Adhesion Research Articles

Synthesis of regioisomeric maltose-based Man/Glc glycoclusters to control glycoligand presentation in 3D space.

The investigation of carbohydrate recognition in a natural environment suffers from the complexity of overlapping functional effects such as multivalency and heteromultivalency effects. Another key factor in carbohydrate recognition is the presentation mode of glycoligands in three-dimensional (3D) space. In order to trace out the effect of 3D ligand presentation, we utilized an oligosaccharide model to precisely control the spatial relation between a mannose ligand (Man) and a glucose moiety (Glc). A disaccharide (maltose) served as a scaffold to alternately conjugate Man and Glc at position 6 and 6' of a synthetic maltoside, resulting in a pair of regioisomeric heterobivalent glycoclusters. The biological effect of this specific structural tuning was tested in a native system employing mannose-specific adhesion of live E. coli cells. Indeed, the variable 3D presentation of the Man ligand resulted in a 2-fold difference between the regioisomeric heterobivalent glycoclusters as inhibitors of bacterial adhesion. This can be considered a remarkable effect, which could be interpreted by computer-aided modelling of the complexes between the bacterial lectin and the synthetic regioisomeric glycoligands.

Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP TG Anchor.

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

Molecular Switch Controlling Expression of the Mannose-Specific Adhesin, Msa, in Lactobacillus plantarum.

Some lactic acid bacteria, especially Lactobacillus spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic Lactobacillus plantarum strains, WCSF1 and 299v, display highly different mannose-specific adhesion, with L. plantarum 299v being superior to L. plantarum WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the msa gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger msa transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the msa gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the msa transcript. In addition, the ribosome binding site upstream of the msa gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the msa transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.IMPORTANCE Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In Lactobacillus plantarum, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the msa gene in L. plantarum strain WCFS1. Strains with increased msa expression could be valuable in the development and manufacture of improved probiotic products.

Functional Characterization of Probiotic Potential of Novel Pigmented Bacterial Strains for Aquaculture Applications.

The bioprospecting proficient of novel pigmented probiotic strains with respect to aquaculture industry was unexplored hitherto. In this study, we investigated the probiotic potential of novel pigmented bacterial strains isolated from the indigenous soil sediments in their vicinal habitats, which were screened for their antimicrobial activity against aquatic pathogens using agar well diffusion assay. The strains namely Exiguobacterium acetylicum (S01), Aeromonas veronii (V03), and Chryseobacterium joostei (V04) were phenotypically identified and confirmed by 16S rRNA gene sequence analysis. Further characterization revealed that strains S01 and V03 survive relatively in lower pH and higher bile salt concentrations and possess good adherence ability and broad-spectrum antibiotic susceptibility. The isolate S01 exhibited the higher adhesion ability to hydrocarbons (82%) and mannose-specific adhesion (msa) gene expression. Additionally, the probiotic effects were evaluated in Artemia nauplii fed with algae supplemented with S01, V03, and V04 strains (2.7 × 107cfu/mL) for 3days under axenic environment. We observed a significant increase (p < 0.05) in the survival rate of Artemia nauplii treated with S01 (83 ± 5%) and V03 (55 ± 5%), whereas the survival rate was only 30 ± 0% in the untreated group. Moreover, the individual length (IL) was increased in treated group S01 (156.7 ± 2.2μm), V03 (146.1 ± 3.4μm), and V04 (134.4 ± 2.5μm) compared with untreated group (116.0 ± 4.8μm). Our results revealed that E. acetylicum S01 exhibits desirable functional probiotic attributes compared to A. veronii and C. joostei and it would be a promising probiotic strain, which can be efficiently used in the aquaculture applications.

Characterization of fructophilic lactic microbiota of Apis mellifera from the Caucasus Mountains

Microbial symbionts of honeybee colony are considered as promising tools to support the honeybee population welfare. The majority of existing honeybee microbiota studies is focused on genetic description of the honeybee-associated microbiome fingerprints. The lack of a deeper knowledge on the bacterial community colonizing the honeybee niche, which may be helpful in encouraging industrial applications of this microbiota, led us to undertake this study. The biodiversity of the cultivable fructophilic lactic acid bacteria (FLAB) isolated from adult honeybee intestine and beebread samples was studied. Phenotypic properties of probiotic interest, such as the adhesive potential using in vitro models and adhesion determinants, were also investigated. Antibiotic resistance profiles as reliable markers to evaluate the impact of long-term and current exposure of honeybees to antibiotics were phenotypically determined on the isolated lactic acid bacteria (LAB). The mannose-specific adhesion and high cell surface hydrophobicity found in the studied FLAB isolates sheds light on the effective adaptation of microbiota to specific ecologic niche. It is the first report of phenotypically detected antibiotic resistance profiles of honeybee endogenous bacteria and the first account of minimum inhibitory concentration (MIC) values for four antibiotics used in beekeeping practice.

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.

Differentiation of Crohn's Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force.

Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn’s disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.

Are multivalent cluster glycosides a means of controlling ligand density of glycoarrays?

Bacterial adhesion to the glycocalyx of human host cells is of biological and medicinal importance. This process is often initiated by the interaction of bacterial lectins and specific carbohydrate ligands. Thus, adhesion of bacterial cells to glycosylated surfaces is a suitable model system to study various parameters of lectin-mediated carbohydrate recognition. Glycoarrays have become important tools to study such lectin-mediated carbohydrate recognition. However, it is difficult to adjust the characteristics of a specific glycoarray regarding its carbohydrate density or the clustering of sugar ligands, respectively. Thus, we have made an attempt to use synthetic cluster glycosides of different valencies to vary carbohydrate density on a polystyrene surface. A series of mono-, di- and trivalent mannosides were synthesised for immobilisation on pre-functionalised polystyrene microtiter plates and the resulting glycoarrays were tested as adhesive surfaces in mannose-specific adhesion of Escherichia coli. Our measurements give first promising hints about the potential of this approach to alter ligand density of glycoarrays in a systematic way.

En route to photoaffinity labeling of the bacterial lectin FimH.

Mannose-specific adhesion of Escherichia coli bacteria to cell surfaces, the cause of various infections, is mediated by a fimbrial lectin, called FimH. X-ray studies have revealed a carbohydrate recognition domain (CRD) on FimH that can complex α-D-mannosides. However, as the precise nature of the ligand–receptor interactions in mannose-specific adhesion is not yet fully understood, it is of interest to identify carbohydrate recognition domains on the fimbrial lectin also in solution. Photoaffinity labeling serves as an appropriate methodology in this endeavour and hence biotin-labeled photoactive mannosides were designed and synthesized for photoaffinity labeling of FimH. So far, the photo-crosslinking properties of the new photoactive mannosides could be detailed with the peptide angiotensin II and labeling of FimH was shown both by MS/MS studies and by affino dot–blot analysis.

Study on adhesion of Lactobacillus acidophilus to mice Peyer's patches and inhibition of pathogen invasion

Objective To investigate the adhesion mechanism of Lactobacillus acidophilus FN001外to Peyer's patches. Methods Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion and effect of sugars on in- hibition of adhesion. The presence of lectin-like proteins in the cell surface was determined by hemagglutina- tion. Effect of L. acidophilus FN001 on inhibition of adhesion of pathogens to Peyer's patches was also stud- ied. Results The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-ct-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the ad- hesive capacity indicating that cell surface proteins are involved in adhesion to Peyer's patches. L. acidophi- lus FN001 could agglutinate rabbit red cell in mannose specific manner and protease pretreatment could de-crease hemagglutinin, suggesting that L. acidophilus FN001 has mannose specific lectin (s). In adherence inhibition assay, L. acidophilus NF001 could significantly inhibit adhesion of E. coli ATCC25922 to Peyer's patches when L. acidophilus NF001 were applied to Peyer's patches first or at the same time with pathogen. Conclusion It was concluded that a mannose-specific protein mediated adhesion of L. acidophilus FN001 to the Peyer's patches, and L. acidophilus FN001 could inhibit adhesion of pathogen with similar lectins speci- ficity to Peyer's patches. Key words: Lactobacillus; Peyer's patches; Mannose-specific adhesion; Adherence inhibition

Sublethal concentrations of antibiotics and bacterial adhesion.

Various antibiotics in sublethal concentrations markedly impair adhesion of Streptococcus pyogenes and Escherichia coli to human cells. In streptococcal cells penicillin G caused an enhances loss of lipoteichoic acid, the ligand (adhesion) that binds the organism to host cells, with consequent loss of their adhesive properties. In E coli sublethal concentrations of penicillin prevented the surface expression of the mannose-specific adhesion by distorting cell wall biosynthesis. In contrast to streptococci, E coli cells could not be made to lose their adhesions once their adhesions once they had been formed. Streptomycin in subinhibitory concentration similarly suppressed the acquisition of mannose-binding and adhesive activities in several strains of antibiotic-sensitive E. coli but not in isogenic derivatives with ribosomal mutation to high-level streptomycin resistance, rpsL, or in bacteria in the stationary phase of growth, suggesting that streptomycin exerted its sublethal suppressive effects by classic mechanisms of action on the bacterial ribosome. Strain VL2, derived from one streptomycin-resistant mutant, retained a high level (1000 microgram/ml) of resistance to streptomycin but reacquired sensitivity to the sublethal effect; growth in 30 microgram streptomycin/ml suppressed mannose-sensitive haemagglutination (less than 1% of control) as well as mannose-sensitive adhesion to epithelial cells (42%) or leucocytes (7%). Although these streptomycin-treated bacteria demonstrated an unaltered degree of fimbriation their fimbriae were significantly longer than those on the untreated bacteria. Furthermore, in contrast to the untreated bacteria, the fimbriae isolated from the drug-treated bacteria were found to lack mannose-binding activity as measured by haemagglutination. It therefore, appears that streptomycin can cause even resistant bacteria to produce an aberrant fimbrial protein, presumably by causing misreading in "competent" ribosomes. These studies indicate that the use of sublethal doses of certain antibiotics whose mode of action is well known may shed light on the genetic and chemical modulation of bacterial factors involved in mucosal colonization.

Factors involved in binding of Lactobacillus plantarum Lp6 to rat small intestinal mucus

To investigate the adhesion determinants of Lactobacillus plantarum Lp6, a dairy isolate. Small intestinal mucus extracted from rats was used as a substrate for adhesion. Adhesion determinants were studied by physical, chemical and enzymatic pretreatments of the bacteria, and adhesion inhibition assay. The mannose-specific adhesins were explored by studying the effect of d-mannose on adhesion and the yeast-agglutinating ability of the bacteria. It was found that adhesion decreased after bacteria were treated with sodium metaperiodate, protease K, trypsin, lithium chloride and trichloroacetic acid. However, adhesion did not decrease after trypsin-treated bacteria were incubated with cell surface protein extract. Cell surface bound exopolysaccharides were found to inhibit the adhesion. D-mannose inhibited the adhesion in a dose-dependent manner. The bacteria could significantly agglutinate yeast and lost this ability after protease K treatment. Adhesion was mainly mediated by the mannose specific adhesins, which might be proteins that reversibly bind to the cell surface components. Cell surface-bound exopolysaccharides were also involved in adhesion. The mannose-specific adhesion of Lact. plantarum Lp6 to rat mucus might be important for competing with pathogens-binding sites in gut, which may be used to resist the colonization of the pathogens.

Clonal analysis reveals high rate of structural mutations in fimbrial adhesins of extraintestinal pathogenic Escherichia coli

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintestinal E. coli are under strong positive selection (Dn/Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.

Trivalent Cluster Mannosides with Aromatic Partial Structure as Ligands for the Type 1 Fimbrial Lectin of Escherichia coli

Mannose-specific adhesion of E. coli bacteria to their host cells is mediated by so-called type 1 fimbriae containing lectin domains present on the type 1 fimbrial FimH protein. The crystal structure of a FimH-FimC(chaperone) protein complex revealed a number of amino acids in the carbohydrate binding site with aromatic side chains. This finding is in keeping with earlier results showing high inhibitory potencies of aryl mannosides when tested as inhibitors of type 1 fimbriae-mediated bacterial adhesion. In addition, clustering of mannosyl moieties also led to favourable effects, as in the case of trivalent cluster mannosides such as (1). In order to combine both, i.e. the clustering approach and the advantage of an aromatic moiety, the herein presented study has emphasized the synthesis of three cluster mannosides (2), (3), and (4), as ligands for the type 1 fimbrial lectin, which contain a phenyl partial structure in different proximity to the core of the molecule. The inhibitory potencies of the new cluster mannosides were determined in enzyme-linked immunosorbent assays (ELISAs).

Cluster Mannosides as Inhibitors of Type 1 Fimbriae-Mediated Adhesion ofEscherichia coli: Pentaerythritol Derivatives as Scaffolds

Pentaerythritol derivatives were used as core molecules for the synthesis of two cluster α-D-mannosides, which were designed as oligomannoside mimetics. The problem of glycosyl orthoester formation, which frequently occurs in oligo-mannosylations, was solved. The clusters were tested for their capacity to block binding of Escherichia coli to yeast mannan in vitro and were found to be more than 200 times more potent in inhibiting mannose-specific adhesion than methyl α-D-mannoside.

Sequential Steps in Synaptic Targeting of Sensory Afferents Are Mediated by Constitutive and Developmentally Regulated Glycosylations of CAMs

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell–cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell–cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell–cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell–matrix to cell–cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.

Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae.

Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both the major fimbrial subunit gene, fimA, and the fimH gene in combination with either the fimF or the fimG gene were required for mannose-specific adhesion. The fimF, fimG and fimH gene products were likewise shown to play a major role in the fimbrial morphology as longitudinal modulators. The amount of FimF, FimG and FimH proteins appeared to control the length and number of the fimbriae. The DNA sequence of a 2050 bp region containing the three genes was determined. The corresponding protein sequences all exhibited homology with the fimbrial subunit protein, FimA.