Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.     

Oligosaccharide structural specificity of the soluble agglutinin released from guinea pig colonic epithelial cells

Abstract Guinea pig colonic epithelial cells release a soluble lectin capable of agglutinating numerous strains of Shigella and Escherichia coli as well as other bacteria. Using pure oligosaccharides and glycopeptides with well-defined structures to inhibit the agglutination of Shigella flexneri 1b by the soluble intestinal lectin, we have been able to demonstrate that the latter recognises different structural types. Inhibition by human milk glucoprotein glycopeptides with biantennary glycans of the N -acetyllactosamine type was dependent on the simultaneous presence of unsubstituted terminal non-reducing galactose residues and of a fucose residue α-1,6-linked to the asparagine-conjugated N -acetylglucosamine residue. Unsubstituted terminal non-reducing galactose was also determinant for inhibition by human milk oligosaccharides. Finally oligosaccharides possessing the Man (α1–2) Man structure inhibited more effectively than those with a Man(α1–3)Man sequence. The fact that these different structural motifs were all inhibitory raises the problem of the possible existence of a multispecific lectin or of several different lectins in the guinea pig colonic mucosa mediating bacterial adherence.     

Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.     

Utilization of natural fucosylated oligosaccharides by three novel alpha-L-fucosidases from a probiotic Lactobacillus casei strain

Three putative α-L-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2'-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3'-, 4'-, and 6'-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa.     

Identification of a new alpha1,2-fucosyltransferase involved in O-antigen biosynthesis of Escherichia coli O86:B7 and formation of H-type 3 blood group antigen

Escherichia coli O86 possesses high human blood group B activity because of its O-antigen structure, sharing the human blood group B epitope. In this study, the wbwK gene of E. coli O86:B7 was expressed and purified as the GST fusion protein. Thereafter, the wbwK gene was biochemically identified to encode an alpha1,2-fucosyltransferase through radioactivity assays, as well as mass spectrometry and NMR spectroscopy. WbwK shows strict substrate specificity and only recognizes Gal beta1,3GalNAc alpha-OR (T-antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen. In contrast to other alpha1,2-fucosyltransferases, WbwK does not display activity toward the simple substrate Gal beta-OMe. Comparison with another recently characterized alpha1,2-fucosyltransferase (WbsJ) of E. coli O128:B12 indicates a low level of amino acid identity between them; however, they share a common acceptor substrate, Gal beta1,3GalNAc alpha-OR. Domain swapping between WbwK and WbsJ revealed that the smaller variable domains located in the C-terminus determine substrate specificity, whereas the larger variable domain in the N-terminus might play a role in forming the correct conformation for substrate binding or for localization of the alpha1,2-fucosyltransferase involved in O-antigen biosynthesis. In addition, milligram scale biosynthesis of the H-type 3 blood group antigen was explored using purified recombinant WbwK. WbwK may have potential applications in masking T-antigen, the tumor antigen, in vivo.     

Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.     

Tumor necrosis factor alpha increases the expression of glycosyltransferases and sulfotransferases responsible for the biosynthesis of sialylated and/or sulfated Lewis x epitopes in the human bronchial mucosa.

There is increasing evidence that inflammation may affect glycosylation and sulfation of various glycoproteins. The present study reports the effect of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, on the glycosyl- and sulfotransferases of the human bronchial mucosa responsible for the biosynthesis of Lewis x epitope and of its sialylated and/or sulfated derivatives, which are expressed in human bronchial mucins. Fragments of macroscopically normal human bronchial mucosa were exposed to TNF-alpha at a concentration of 20 ng/ml. TNF-alpha was shown to increase alpha1,3-fucosyltransferase activity as well as expression of the two alpha1,3-fucosyltransferase genes expressed in the human airway, FUT3 and FUT4. It had no influence on alpha1,2-fucosyltransferase activity or FUT2 expression. It also increased alpha2,3-sialyltransferase activity and the expression of ST3Gal-III and, more importantly, ST3Gal-IV and both N-acetylglucosamine 6-O-sulfotransferase and galactose 3-O-sulfotransferase. These results are consistent with the observation of oversialylation and increased expression sialyl-Lewis x epitopes on human airway mucins secreted by patients with severe lung infection such as those with cystic fibrosis, whose airways are colonized by Pseudomonas aeruginosa. However, other cytokines may also be involved in this process.     

alpha(1,2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells.

E-selectin is a cytokine-inducible, calcium-dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte-endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E-selectin binds to ligands carrying the tetrasaccharide sialyl-Lewis x (NeuAcalpha2,3Galbeta1,4[Fucalpha1, 3]GlcNAc)1 or its isomer sialyl-Lewis a (NeuAcalpha2, 3Galbeta1, 3[Fucalpha1,4]GlcNAc). We examined the effect of expressing the H-type alpha(1,2)-fucosyltransferase or the alpha(2, 6)-sialyltransferase on the synthesis of sialyl-Lewis x by alpha(1, 3)fucosyltransferase. We found that H-type alpha(1, 2)-fucosyltransferase but not alpha(2,6)-sialyltransferase, strongly inhibited sialyl-Lewis x expression and E-selectin adhesion. We assume that H-type alpha(1,2)-fucosyltransferase competes with the endogenous alpha(2,3)-sialyltransferase for the N-acetyllactosamine structures assigned to further serve as acceptors for alpha(1, 3)fucosyltransferase.     

A DNA polymorphism influencing α(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion

The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.     

Glycobiology of human milk

Glycans are characteristic components of milk, and each species has unique patterns of specific carbohydrates. Human milk is unusually rich in glycans, with the major components being lactose and oligosaccharides, representing approximately 6.8 and 1% of the milk, respectively. Other sources of glycans in human milk include monosaccharides, mucins, glycosaminoglycans, glycoproteins, glycopeptides, and glycolipids. In human milk, the presence and patterns of these glycans vary depending upon the stage of lactation and the maternal genes and their genetic polymorphisms that control glycosyl transferases. The synthesis of milk glycans utilizes a significant portion of the metabolic energy that the mother expends when producing her milk, but other than lactose, these glycans contribute little to the nutritional needs of the infant. The data herein support several functions. 1) Many human milk glycans inhibit pathogens from binding to the intestinal mucosa. 2) Human milk glycans attenuate inflammation. 3) Glycans also directly stimulate the growth of beneficial (mutualist) bacteria of the microbiota (formerly considered commensal microflora of the intestine); these mutualists and their fermentation products can, in turn, (a) inhibit pathogens, (b) modulate signaling and inflammation, and (c) the fermentation products can be absorbed and utilized as a source of dietary calories. These functions can help direct and support intestinal postnatal growth, development, and ontogeny of colonization. The many functions of the milk glycans may synergistically protect infants from disease. Hence, human milk glycans and their homologs may serve as novel prophylactic or therapeutic agents for a diverse range of deleterious conditions.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

Three bovine alpha2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Galbeta1-3GalNAc acceptor substrates

To investigate the synthesis of alpha2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct alpha2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in alpha1,2 linkage to ganglioside GM(1)and galacto- N -biose, but not to the phenyl-beta-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian alpha2-fucosyltransferases. Southern blot analyses detected four related alpha2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the alpha2-fucosyltransferase activity which can also use type 1 and phenyl-beta-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four alpha2-fucosyltransferases are present, three adding a fucose on alpha1,2 linkage on type 3/4 acceptor (Galbeta1-3GalNAc) and another able to transfer also fucose on phenyl-beta-D-galactoside and type 1 (Galbeta1-3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.     

The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked alpha2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2(BBe)) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no sigma1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells was consistently associated with the T1L sigma1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 sigma1 protein with glycoconjugates containing alpha2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.     

Pathogenicity ofCampylobacter jejuni in intraperitoneally or intravenously inoculated ferrets

Ferret kits inoculated intravenously (IV) withCampylobacter jejuni after pretreatment with parenteral iron developed more severe systemic signs and more prolonged bacteremia than untreated inoculated controls. Watery diarrhea began in both groups 2–16 h after inoculation and lasted less than 48 h.C. jejuni was cultured from rectal swabs 2–8 h after inoculation, and gut colonization persisted up to 15 days, suggesting that colonization does not necessarily induce diarrhea. Gut colonization occurred as rapidly after IV inoculation of ferrets in which the common bile duct had been ligated as it did in unligated controls.C. jejuni apparently reached the intestinal lumen by mucosal invasion from the bloodstream. Bacteremia following natural infection could thus result in repeated passages ofC. jejuni across the gut wall, exposing the mucosa to both the bacterial cells and their metabolic products. Histological evidence of an inflammatory response in the mucosa, without severe epithelial damage, suggests a toxin-mediated secretory diarrhea.     

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.     

Development of biosensor-based assays to identify anti-infective oligosaccharides

It is now well accepted that milk oligosaccharides can have a direct inhibitory effect on pathogenic microorganisms by interfering with their adhesion to human cells. Many free oligosaccharides from milk are considered to be soluble receptor analogs of epithelial cell surface carbohydrates and, thus, function as receptor decoys to which pathogens can bind instead of the host. In reality, there are few rapid methods to screen for such oligosaccharides, and much of the research in this area has centered on using human cell line models of infection that are time-consuming. Therefore, a quick and sensitive method is required for detecting the binding of microorganisms to milk oligosaccharides. Our study describes a number of biosensor-based methods to achieve these aims. Our approach involved the exposure of whole bacterial cells to the well-characterized human milk oligosaccharide, 2′-fucosyllactose, immobilized to a pretreated gold chip surface. The technique was validated by screening a range of pathogenic bacteria, including Campylobacter jejuni, to which 2′-fucosyllactose is known to bind. Where binding was detected, its specificity was confirmed by preincubation studies using unlabeled 2′-fucosyllactose. The techniques described represent a quick, cost-effective, and highly reproducible detection method for identifying anti-infective oligosaccharides.