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Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, 37007 Salamanca, Spain
2To whom correspondence should be addressed. E-mail: phueso{at}gugu.usal.es.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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KEY WORDS: • human milk • oligosaccharides • Escherichia coli
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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). Specific adhesion is mediated by bacterial proteins termed adhesins, which may or may not be associated with fimbriae (2
). These proteins are lectins that recognize and bind to a carbohydrate receptor, which is both host and tissue specific (3
). A whole repertoire of adhesins of uropathogenic Escherichia coli (UPEC)3
and enterotoxigenic E. coli (ETEC) has been characterized in detail (4
). Most adhesins agglutinate erythrocytes from one or more species because they recognize and bind cell surface glycans. Thus, several mono- or oligosaccharides may inhibit this hemagglutination (5
).
ETEC strains are the most common cause of diarrhea in children in developing countries and are responsible annually for 800,000 deaths in children <5 y old (6
). Once they have recognized their carbohydrate receptor and have became attached to the small intestine, ETEC produce the enterotoxins that cause the symptoms of diarrhea. Although the different human ETEC adhesins show geographical variations (7
), colonization factor antigens (CFA)/I, CFA/II and CFA/IV occur most frequently (8
). E. coli is also one of the most frequent causes of urinary tract infections (UTI). The most common fimbriae on UPEC are the P and the type 1 fimbriae (1
).
There is considerable epidemiologic evidence to support the notion that breast-fed infants have lower incidences of several diseases, such as diarrhea and otitis media, than bottle-fed children (9
). Human milk contains large amounts of complex carbohydrates, both free and conjugated. These molecules have been proposed to be a source of protection for breast-feeding infants (10
). They would act as soluble receptor analogs of the cell surface carbohydrates, inhibiting bacterial adhesion to the epithelial surface and preventing the beginning of the infective process (11
). Human milk contains mainly neutral fucosylated oligosaccharides although there is also a fraction of acidic oligosaccharides, characterized by the presence of one or more sialic acid residues (12
).
The aim of this study was to determine the ability of human milk oligosaccharides to bind two human ETEC strains (expressing CFA/I and CFA/II fimbriae) and to attempt to clarify to what extent this binding could be due to the acidic fraction. Previously, the acidic oligosaccharide composition of three stages of lactation was studied (unpublished results).
Urine from breast-fed infants contains sialyloligosaccharides that are not present in bottle-fed infants, and a nutritional origin (the milk) has been proposed for them (13
,14
). Therefore, the binding capacity of human milk oligosaccharides to two UPEC strains (expressing P and P-like fimbriae) was also examined.
The inhibition capacity of bovine milk oligosaccharides was also tested for both the ETEC and the UPEC human strains. If these molecules functioned as good inhibitors, bovine milk could be an alternative and available source, if formula supplementation with bioactive oligosaccharides were to be considered.
Because most fimbriae agglutinate erythrocytes, the inhibition of this hemagglutination by free milk oligosaccharides can be used as an indirect measure of their binding capacity (15
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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Two human ETEC strains, and two human UPEC strains, expressing four different fimbriae, were kindly provided by the Laboratorio de Referencia de E. coli (Facultad de Veterinaria, Lugo, Spain). The characteristics of each strain are listed in Table 1
. Bacteria were grown in Mueller-Hinton broth (Difco, Detroit, MI) and incubated for 5–7 d at 37°C. When grown in this medium, the bacteria lacked fimbriae. For fimbriae expression, all of the strains were incubated on CFA agar plates at 37°C for 16 h, as previously reported by Evans et al. (16
) This medium is appropriate for fimbriae expression in all of the strains tested here.
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Human milk was obtained from 12 healthy volunteer Spanish donors who had delivered at term. Colostrum (d 1–4), transitional (d 12–17) and mature milk (d 28–32) samples were collected. Bovine milk was obtained from six Spanish-Brown cows on d 2 (colostrum), d 7 (transitional milk), d 90 (mature milk) and d 270 (late lactation milk) after calving. Isolation of total oligosaccharides was performed as described by Kobata (17
) with some modifications. Briefly, samples were defatted by centrifugation (3000 x g, 30 min, 4°C) and filtered through glass wool. The filtrates were mixed with 2 volumes of ethanol and allowed to stand overnight at 4°C. Most proteins and lactose precipitated and were removed by centrifugation at 0°C for 20 min, 3000 x g, and ethanol evaporated. The crude oligosaccharide fraction was further purified by molecular exclusion chromatography (Sephadex G-25, Pharmacia, Uppsala, Sweden) to eliminate residual proteins and peptides. Ninhydrin-positive fractions were discarded, and orcinol-positive fractions were collected. Oligosaccharides were mainly free of proteins and lipids. They were quantified using the phenol-H2SO4 method (18
).
Neuraminidase treatment.
Neuraminidase (EC 3.2.1.18) from Clostridium perfringens was from Sigma Chemical (St. Louis, MO). Milk oligosaccharide samples were treated with neuraminidase in 50 mmol/L sodium acetate buffer, pH 5.5, for 16 h at 37°C. After treatment, neuraminidase was removed and the absence of sialyloligosaccharides was monitored by HPLC.
Standard oligosaccharides and monosaccharides.
Mannose (Man), N-acetylneuraminic acid (NeuAc), N-glycolylneuraminic acid (NeuGc) and disialyllactose (DSL) were purchased from Sigma (St. Louis, MO). Lactose was from Merck (Darmstad, Germany). 3'-Sialyllactose (3'SL), 6'-sialyllactose (6'SL), 3'-sialyl-3-fucosyllactose (3'S3FL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc) and disialyllacto-N-tetraose (DSLNT), from human milk, and 3'-sialyllactosamine (3'SLN) and 6'-sialyllactosamine (6'SLN), from bovine colostrum, were purchased from Glyko (Upper Heyford, UK).
HPLC.
Sialylated oligosaccharide contents were analyzed by HPLC according to the method previously described by Michalski (19
). The analyses were performed on a Waters apparatus (Waters, Milford, MA) using a Waters NH2-bound silica column (Carbohydrate Analysis, 300 x 3.9 mm). HPLC elution of milk oligosaccharide samples was performed using a gradient of acetonitrile and 15 mmol/L KH2PO4 adjusted to pH 5.2 as follows: isocratic elution with 25% 15 mmol/L KH2PO4 for 10 min, linear gradient to 50% 15 mmol/L KH2PO4 for 50 min, and these conditions were kept for 15 min. Flow rate was 1 mL/min. Oligosaccharides were detected at 206 nm and identified by comparison of their retention times with those of standard oligosaccharides injected previously.
Hemagglutination assays.
Calf and human erythrocytes were obtained at our laboratory. Calf blood samples were provided by Matosa S.A. (Salamanca, Spain) and the human blood (O-type) was from three different donors. A 5% erythrocyte suspension in PBS (pH 7.2, 150 mmol/L) was used. Bacterial growth corresponding to one Petri dish was recovered with 1 mL PBS, and the colony-forming units (cfu) present in this solution were calculated. From this original bacterial suspension, several dilutions were made to find the minimum amount of cfu required to agglutinate erythrocytes. The dilution corresponding to that minimum was then always used for the assays (shown in Table 2
) Hemagglutination tests were carried out at 4°C on sterile V-shaped 96-well plates (Nalge Nunc International, Roskilde, Denmark). Bacteria were also resuspended in PBS. PBS (25 µL) or PBS mannose (10 g/L) was added to 25 µL of bacterial suspension and gently mixed. In the hemagglutination inhibition tests, plain PBS was replaced by different oligosaccharide dilutions (sialylated or not). After 5 min, 25 µL of erythrocyte suspension was added to each well. The plates were kept at 4°C for 2 h after which agglutination was read by the naked eye.
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Differences in oligosaccharide contents among stages of lactation were tested by one-way ANOVA. Differences in hemagglutination due to the stage of lactation were also tested by one-way ANOVA. The Scheffé test was used when the F-test was significant. Differences between oligosaccharide amounts required to inhibit hemagglutination were tested by a Student’s t test. The SPSS 9.0.1 program for Windows (SPSS, Chicago, IL) was used. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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We assayed blood from three different donors. No differences among individuals were observed. All strains agglutinated erythrocytes in both the presence and absence of mannose. CFA/I, and P fimbriae agglutinated O-type human erythrocytes best, whereas CFA/II and P-like fimbriae worked best with calf erythrocytes. FVL25 was the strain that required the most cfu to produce hemagglutination. When nonfimbriated bacteria were used, all strains failed to agglutinate erythrocytes.
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Milk sialylated oligosaccharides |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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). Only five peaks were identified in bovine milk, and these were present at each stage: 3'SLN, 3'SL, 6'SLN, 6'SL and DSL. Most of the species decreased during lactation (P < 0.05). The data obtained were generally consistent with previous reports (12
,20
).
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Several concentrations of human milk oligosaccharides (25–1200 µg/well) were used instead of plain PBS (negative control) in the hemagglutination assays. The purpose was to determine whether milk oligosaccharides inhibited hemagglutination and to determine the minimum amount of oligosaccharide required for inhibition. Oligosaccharides from colostrum, transitional milk and mature milk were assayed separately (Table 2)
but did not differ from one another.
For ETEC strains, native (nondesialylated) oligosaccharides inhibited (P < 0.01) hemagglutination more efficiently than desialylated oligosaccharides; 
50 µg of total oligosaccharides was sufficient to inhibit the hemagglutination of H4 (CFA/I-expressing strain). However, after desialylation, from 8- to 25-fold (depending on the lactational stage) more oligosaccharides were required to produce the same degree of inhibition. The inhibition of hemagglutination by strain 23 (CFA/II-expressing strain) also required more than twice the amount of oligosaccharides after desialylation (P < 0.01). Total oligosaccharides inhibited the hemagglutination of FVL3 well. Sialylation could even seem to be a handicap because, when desialylated, oligosaccharides from transitional milk were even slightly more effective (P < 0.05) at inhibiting hemagglutination. For FVL25, acidic oligosaccharides may be involved because a similar pattern to that found in the ETEC strains was observed, although differences were not so marked (P < 0.05).
Inhibition of hemagglutination by bovine milk oligosaccharides.
Several concentrations of native (nondesialylated) bovine milk oligosaccharides (25–1200 µg/well) were tested as inhibitors of the hemagglutination of the four human strains considered (Table 3
). They inhibited the hemagglutination of the two UPEC strains, although they were not as effective as human oligosaccharides. Oligosaccharides from milk at the 4 stages of lactation tested did not differ. However, bovine oligosaccharides were not very effective at inhibiting the two ETEC human strains because large amounts of oligosaccharides were necessary for inhibition, with the transitional milk oligosaccharides being the least effective (P < 0.05).
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Inhibition of hemagglutination by standard oligosaccharides and monosaccharides |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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). For this reason, we also tested the inhibitory capacity of lactose (Table 4
). To complete the set, two types of sialic acids were tested (Table 5
). The results varied among strains, but were in accordance with those for total oligosaccharides. Thus, none of the sialylated oligosaccharides tested inhibited the hemagglutination produced by FVL3, nor did lactose or sialic acids. Indeed, when sialylated oligosaccharides with a longer carbohydrate chain (3'S3FL, LSTa and DSLNT) were assayed, the degree of agglutination was greater than in the control (PBS).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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). In recent years, many studies establishing the importance of oligosaccharides in microbial recognition have been published. Human milk oligosaccharides have been shown to inhibit the attachment of Streptococcus pneumoniae and Haemophilus influenzae to host cells (23
). In the same sense, the fucosyloligosaccharide fraction of human milk has been found to protect infants from the stable toxin of E. coli (24
) and to prevent the binding of Campylobacter jejuni to its receptor in human epithelial cells (25
). Also, sialic acid-containing oligosaccharides are important in some recognition processes; for example, 3'SL inhibits the binding of Helicobacter pylori to the gastrointestinal epithelium (26
).
In this work, we tested the binding ability of milk oligosaccharides to ETEC and UPEC strains and therefore their probable role in preventing these infections in neonates. Hemagglutination due to the P-fimbriated strain (FVL3) was well inhibited when oligosaccharides were desialylated; in some of the lactational stages considered, this occurred to a greater extent. This suggests a major role of the nonsialylated fraction in the binding of this particular fimbria and even attributes a slight hindering effect to sialic acids. A UTI involving P-fimbriated E. coli is one of the best-characterized bacterial infection systems; several studies suggest that the Gal
1,4Gal-binding property of P-fimbriae is essential for pyelonephritis to occur (27
). Sialic acids do not seem to be involved in the recognition process, and indeed, no inhibition was observed when the hemagglutination test for the P-fimbriated strain was carried out in the presence of Neu Ac.
Nevertheless, the results obtained here suggest that the acidic fraction of milk oligosaccharides is responsible in part for the inhibition of hemagglutination mediated by the three other strains studied. In this case, NeuAc partially inhibited hemagglutination. Additionally, the type of sialic acid seems to be important because NeuGc, which is not present in humans under normal conditions, was not inhibitory. Although acidic oligosaccharides may be important in this inhibition process, desialylated oligosaccharides are also able to inhibit hemagglutination, although much greater quantities are needed. All of these results suggest that even though the acidic residue is involved in recognition, it is not the only compound responsible for inhibition. Thus, other parts of the oligosaccharide structure could also participate in the binding process and therefore, analog structures should produce inhibition to some extent. It was reported previously that hemagglutination mediated by adhesin CFA/I partially disappears after treating erythrocytes with neuraminidase (28
).
Native oligosaccharides from bovine milk were also tested against human strains. In this case, desialylation was not achieved. Bovine milk contains lower amounts of free oligosaccharides and, unlike human milk, these are mainly sialylated species (29
). Despite this, they seem to be much less effective at inhibiting hemagglutination of ETEC strains. In fact, the composition in acidic milk oligosaccharides of the two species is markedly different. The acidic species of human milk identified in this study are mainly consistent with those reported previously (12
), although the proportions of individual oligosaccharides vary. The three main species we identified were LSTa, 3'S3FL and DSLNT. When these oligosaccharides were tested individually, they showed partial but significant inhibition for all strains except the one expressing the P fimbria. The other sialylated species tested were 3'SL and 6'SL, which are present in both human and bovine milk. 6'SL showed a low inhibition capacity for FVL 25 and 23, but was quite a good inhibitor of H4. Conversely, 3'SL inhibited hemagglutination mediated by FVL25 and 23 well, but not that mediated by H4. Thus, the 2,6-linked sialic acid seems to be less efficient at inhibiting hemagglutination mediated by P-like and CFA/II adhesins, which show a stronger affinity for 3'sialylated forms (3'SL, 3'S3FL, and LSTa). The presence of a second sialic acid in DSLNT seems to strengthen the binding capacity because when tested, this oligosaccharide inhibited hemagglutination of the three strains well. The inhibitory capacity of 6'SLN, which is one of the main acidic species in bovine milk but which is not present in human milk (20
), was also tested. The degree of hemagglutination was similar to the PBS control when this oligosaccharide was used with any of the strains studied. This could in part explain the lower inhibitory capacity observed in bovine milk oligosaccharides compared with their human counterparts. Moreover, the greater complexity of sugar chains and the presence of fucose in human oligosaccharides must also be considered.
To conclude, human milk oligosaccharides may have a bacterial blocking effect, preventing the invasive step of E. coli expressing CFA/I or CFA/II. Sialic acid-containing oligosaccharides possibly participate in this recognition process. Human milk oligosaccharides are not digested by the different enzymes present in the alimentary tract of neonates (30
), and some of the oligosaccharides present in human milk can also be found in the urine of breast-fed neonates (13
). It is thus probable that they would continue to exert their protective effect in the urinary tract. In hemagglutination tests, P-fimbriated E. coli (responsible for pyelonephritis) and P-like fimbriated E. coli (responsible for some types of cystitis) were inhibited by the milk oligosaccharides studied here. In the case of the P-like fimbria, acidic oligosaccharides also seem to be involved in the process. It is tempting to speculate that breast-fed neonates would be better protected against infections by these bacteria. Antiadhesive carbohydrates have recently been proposed as a new generation of drugs for the treatment of bacterial disease (31
). In fact, the costs of production are rapidly diminishing and engineered bacteria are being studied as possible large-scale producers (32
). Further experiments would help to clarify the structural specificities involved in these recognition processes between bacteria and soluble oligosaccharides.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CFA, colonization factor antigen; cfu, colony-forming unit; DSL, disialyllactose; DSLNT, disialyllacto-N-tetraose; ETEC, enterotoxigenic Escherichia coli; Lac, lactose; LSTa, sialyllacto-N-tetraose a; LSTb, sialyllacto-N-tetraose b; LSTc, sialyllacto-N-tetraose c; Man, mannose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; PBS, phosphate buffer saline; 3'S3FL, 3'-sialyl-3-fucosyllactose; 3'SL, 3'-sialyllactose; 3'SLN, 3'-sialyllactosamine; 6'SL, 6'-sialyllactose; 6'SLN, 6'-sialyllactosamine; UPEC, uropathogenic Escherichia coli; UTI, urinary tract infection.
Manuscript received 20 March 2002. Initial review completed 30 April 2002. Revision accepted 20 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMilk sialylated oligosaccharidesInhibition of hemagglutination...DISCUSSIONLITERATURE CITED |
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