Escherichia coli 83972 expressing a P fimbriae oligosaccharide receptor mimic impairs adhesion of Uropathogenic E. coli

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a significant health concern, exacerbated by the rapid emergence of multidrug resistant strains refractory to antibiotic treatment. P fimbriae are strongly associated with upper urinary tract colonization due to specific binding to α-D-galactopyranosyl-(1-4)-β-D-galactopyranoside receptors in the kidneys. Thus, inhibiting P-fimbrial adhesion may reduce the incidence of UPEC-mediated UTI. E. coli 83972 is an asymptomatic bacteriuria isolate successfully used as a prophylactic agent to prevent UTI in human studies. We constructed a recombinant E. coli 83972 strain displaying a surface-located oligosaccharide P fimbriae receptor mimic that bound to P-fimbriated E. coli producing any of the 3 PapG adhesin variants. The recombinant strain, E. coli 83972:: lgtCE, impaired P fimbriae–mediated adhesion to human erythrocytes and kidney epithelial cells. Additionally, E. coli 83972::lgtCE impaired urine colonization by UPEC in a mouse UTI model, demonstrating its potential as a prophylactic agent to prevent UTI.

Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains

NaYF4:Yb3+, Er3+ nanoparticles were successfully prepared by a polyol process using diethyleneglycol (DEG) as solvent. After being functionalized with SiO2-NH2 layer, these NaYF4:Yb3+, Er3+ nanoparticles can conjugate with activated avidin molecules (activated by the oxidation of the oligosaccharide chain). The as-formed NaYF4:Yb3+, Er3+ nanoparticles, NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups, avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), UV/Vis absorption spectra, and up-conversion luminescence spectra, respectively.

Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains

NaYF4:Yb3+, Er3+ nanoparticles were successfully prepared by a polyol process using diethyleneglycol (DEG) as solvent. After being functionalized with SiO2-NH2 layer, these NaYF4:Yb3+, Er3+ nanoparticles can conjugate with activated avidin molecules (activated by the oxidation of the oligosaccharide chain). The as-formed NaYF4:Yb3+, Er3+ nanoparticles, NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups, avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), UV/Vis absorption spectra, and up-conversion luminescence spectra, respectively. The biofunctionalization of the NaYF4:Yb3+, Er3+ nanoparticles has less effect on their luminescence properties, i.e., they still show the up-conversion emission (from Er3+, with S-4(3/2) -> I-4(15/2) at similar to 540 nm and F-4(9/2) -> I-4(15/2) at similar to 653 nm), indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.

Preparation, structural characterization and in vitro antitumor activity of kappa-carrageenan oligosaccharide fraction from Kappaphycus striatum

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum to compare the antitumor activity with carrageenan polysaccharides. Oligosaccharide fractions were isolated by gel permeation chromatography and the structure of fraction 1 (F1) was studied by using negative- ion electrospray ionization-mass spectrometry (ESI-MS), and H-1 and C-13-NMR spectrometry. The in vitro antitumor effects in three human neoplastic cell lines (KB, BGC, and Hela) of polysaccharides and F1 were investigated. The bioassay results showed that F1 exhibited relatively higher antitumor activity against the three cancer cells than polysaccharides.

Capillary zone electrophoresis investigation of interactions between granulocyte-colony stimulating factor and dextran sulfate/carrageenan oligosaccharide

The interactions between granulocyte-colony stimulating factor (G-CSF) and dextran sulfate/kappa-carrageenan oligosaccharide were studied by capillary zone electrophoresis. Dextran sulfate could strongly interact with G-CSF and the complex was detected. The binding constant and stoichiometry were determined to be 1.2x10(6) (mol/L)(-1) and 3:1, respectively. However, the interaction between K-carrageenan oligosaccharide and G-CSF was not found.

Biosynthesis and structure of the Burkholderia cenocepacia K56-2 lipopolysaccharide core oligosaccharide:truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin B

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-alpha-mannosidase from Penicillium citrinum lost 40% and alpha-mannosidase from almonds lost 60% activity at 55 degreesC over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard. reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27-53% relative to the control. (C) 2004 Elsevier B.V. All rights reserved.

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact(R) L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40degreesC and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40degreesC, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAEPAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal(R)GOS). (C) 2004 Wiley Periodicals, Inc.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.