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* Department of Pharmaceutical Chemistry, University of Kuopio, FI-70211 Kuopio, Finland;
Department of Surgical and Perioperative Science, Sports Medicine, Umeå University, S-90187, Umeå, Sweden;
** Department of Odontology/Cariology, Umeå University, Umeå, Sweden; and
Faculté de Necker-Enfants Malades, INSERM U570, Paris, France
2To whom correspondence should be addressed. E-mail: jenni.hakkarainen{at}uku.fi.
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ABSTRACT |
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KEY WORDS: • Neisseria meningitidis • oligosaccharide • human milk • bovine milk • bacterial adhesion
Neisseria meningitidis, meningococcus, is a human-specific pathogen causing life-threatening septicemia and meningitis in newborns. The meningococcus may colonize the nasopharynx of asymptomatic carriers and transmit person-to-person or translocate to blood or through the blood-brain barrier causing septicemia or meningitis, respectively (1–3). Adhesion of N. meningitidis to epithelial and endothelial cells occurs through type IV pili in encapsulated strains (1,4). Type IV pili are composed of pilin subunits (5), which together with the tip-located PilC1 protein (6), are potential adhesins. Both the multimodal adhesion and human-specific character of N. meningitidis have hampered delineation of the receptors mediating its interactions with host cells.
Human milk confers a defensive system on the newborn through immunoglobulins and milk oligosaccharides and glycoconjugates (7). Adhesion-inhibiting milk oligosaccharides have been described for both gram-negative (8–14) and gram-positive bacteria (13,15). Adding oligosaccharides to infant food or formulas or adult food may therefore have benefits (16) beyond the nutritional effects (17,18); such an application could be economically reasonable by employing whey from dairy companies as a source for oligosaccharides.
The purpose of the present study was to test the binding of Neisseria meningitidis to glycoproteins and to investigate the ability of milk oligosaccharides to inhibit this binding. Using a pili binding assay, we found N. meningitidis pili to bind bovine thyroglobulin and human salivary agglutinin (19) glycoproteins. The binding to glycoproteins was inhibited effectively by human (HMO) or bovine milk oligosaccharides (BMO).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bacterial strain. Neisseria meningitidis serogroup C class I strain 8013 (20) was cultured on GC medium (Difco Laboratories) containing supplements as described by Kellogg et al. (21) in anaerobic conditions at 37°C in 5% CO2 for 18 h.
Purification of agglutinin from human saliva. Agglutinin was purified from human parotid saliva as described (22,23). Briefly, a Streptococcus mutans strain Ingbritt suspension and saliva were mixed. After pelleting of bacterial aggregates by centrifugation at 5000 x g for 15 min, salivary agglutinin was released into the supernatant by EDTA. Released agglutinin was then further purified by gel chromatography and the protein amount determined with the Bio-Rad DC Protein Assay with bovine albumin as a standard.
Isolation and biotin labeling of pili.
Isolation of the pili was carried out at 0°C. Five plates of cultivated bacteria were harvested and suspended in 20 mL of sterile 10 mmol/L Hepes buffer. The tubes were mixed vigorously on a Vortex mixer for exactly 30 s, and the mixture was centrifuged at 8000 x g at 4°C for 20 min. An aliquot of 16 mL of the supernatant was loaded onto a Biomax Ultrafree-15 centrifugal filter device (100-kDa cutoff; Millipore) and centrifuged at 100 x g at 4°C. The concentrated solution was washed twice with 15 mL of 10 mmol/L Hepes and concentrated to a volume of 1 mL by employing centrifugation at 100 x g at 4°C in the Biomax Ultrafree-15 centrifugal filter device. The biotin labeling of pili was performed in PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 12.4 mmol/L Na2HPO4 · H2O, 1.5 mmol/L KH2PO4, pH 7.4; buffer A) using D-biotinoyl-
-aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics) according to the instructions of the manufacturer. Biotin-labeled pili were stored at 4°C.
Milk oligosaccharides. Oligosaccharides were isolated from human (pooled from 3 healthy donors) and bovine milk at 4°C essentially as described by Kobata (24). The milk (250 mL) was defatted by centrifugation at 100 x g for 15 min, filtered through glass wool, followed by the addition of ethanol (96.1 volume-%) to the filtrate to give a final ethanol concentration of 68%. The mixture was incubated at 4°C overnight. The precipitate was removed by centrifugation at 26,890 x g for 15 min and washed twice with 50 mL of 67% ethanol at 0°C. The supernatants were combined and concentrated into syrup by rotary evaporation. The syrup was diluted with 25 mL of water and centrifuged at 100 x g for 15 min at 4°C to separate the insoluble material. The syrup was subject to gel chromatography on a Sephadex G-25 (Pharmacia Biotech) column (102 x 1.6 cm) and saccharides were eluted with water. The total hexose content was determined by the method of Kobata (24), and a periodate-resorcinol assay for sialic acids was performed using the method of Jourdian et al. (25). The HMO effluent was pooled into 2 fractions, acidic and neutral, after analysis of total hexose and sialic acids and freeze-dried. The BMO were pooled into acidic and neutral BMO.
Pili binding assay. The binding of purified pili to glycoproteins coated in microtiter wells was performed as follows. Aliquots of 100 µL of the glycoproteins diluted in PBS (140 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4 · H2O, 1.5 mmol/L KH2PO4, pH 7.4; buffer B) at 100 mg/L (bovine thyroglobulin or chicken ovalbumin) or 1 mg/L (human salivary agglutinin) or dry milk powder solution [5% (wt:v) dry milk powder 0.05% (v:v) Tween 20 in buffer A, pH 7.4] were incubated in polyvinylchloride microtiter plate wells (Falcon Flexible Plate, Becton Dickinson Labware) at 4°C overnight. The wells were washed 5 times with washing buffer [0.05% (v:v) Tween 20 in buffer A, pH 7.4] and the glycoprotein-coated wells were saturated with 250 µL of dry milk powder solution by incubation at room temperature for 60 min. The wells were washed 5 times with washing buffer. Biotin-labeled pili corresponding to isolated pili from 1:8 plate were further diluted 1:8 with buffer B and 100 µL of the diluted pili was added to the wells. The wells were incubated at room temperature for 60 min, and washed 5 times with washing buffer. Then, 100 µL of Streptavidin-POD conjugate (Roche Diagnostics) in dry milk powder solution (diluted 1:4000) was added and incubation was performed at room temperature for 60 min. After 5 washes with washing buffer, an aliquot of 100 µL of ABTS-substrate (Roche Diagnostics) was added and the absorbances were measured at 405 nm. The assays were carried out in triplicate.
Inhibition of pili binding. The pili binding inhibition tests were performed by preincubation of the biotin-labeled pili (corresponding to isolated pili from 1:8 plate) diluted 1:4 with buffer B with 40 g/L milk oligosaccharide fraction (mixed 1:1) for 60 min at room temperature with gentle rocking on the rocking platform. Control biotin-labeled pili (corresponding to isolated pili from 1:8 plate) were diluted 1:8 with buffer B and treated identically. After preincubation, 100 µL of the 1:1 mixed solutions and control biotin-labeled pili was added to the glycoprotein-coated microtiter plate wells and the binding assay procedure was carried out as described above.
Statistical analysis. Results are presented as means ± SD. Significance in the differences of the means was tested using a 2-tailed paired Student’s t test using Microsoft Excel. Differences with P-values < 0.01 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4-fold and human salivary agglutinin 2-fold increased binding compared with dry milk powder, which was considered as the background (control). Binding to chicken ovalbumin was not remarkable and did not differ from background. The relatively high background resulted from oligosaccharides in dry milk powder made from bovine milk (Fig. 1A). Different BMO fractions inhibited binding of pili to dry milk powder (P < 0.01; data not shown), with mainly the acidic BMO fraction inhibiting the binding. The inhibition by acidic BMO to dry milk powder fraction is seen in Fig. 1A (control).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bovine thyroglobulin contains N-linked complex and hybrid (oligomannosidic) glycans (26), and human salivary agglutinin O- or N-linked (poly)lactosamine and hybrid oligosaccharides (27), serving as potential receptors. Both chicken ovalbumin, with low-avidity pili binding and mainly high-mannose–type glycans (28), and bovine thyroglobulin are well-characterized proteins commonly used to characterize carbohydrate receptors for microorganisms (29,30). Because N. meningitidis pili did not bind to ovalbumin, we deduced that the receptor structures possibly did not contain mannose, and binding to bovine thyroglobulin could thus be mediated by a complex type of bovine thyroglobulin carbohydrate chains. This observation offers preliminary information on the carbohydrate composition of the possible receptor structure. Moreover, saliva represents the first line of defense, and human salivary agglutinin, a complex of the scavenger receptor cysteine-rich protein gp-340 and sIgA, was selected because it aggregates and adheres to a wide range of commensal and pathogenic microorganisms (27) and contains domains for multiple host innate or immune defenses. Here we report a novel interaction of N. meningitidis and human salivary agglutinin. The binding of N. meningitidis to human salivary agglutinin was inhibited up to 50% by acidic BMO. Therefore, the ability of soluble and surface-bound forms of salivary agglutinin to bind N. meningitidis (27) and the inhibition of these possible binding activities by milk oligosaccharides constitute a logical and a rational target for further studies. This is especially important when considering the possible availability of BMO as protective food additives against N. meningitidis.
Both N. subflava (31) and N. gonorrhoeae (32) use sialyl and neutral oligosaccharides, respectively, for adhesion in vitro. The corresponding adhesins, however, are not known. N. meningitidis carries 2 potential adhesins, the tip-located PilC1 (33) and pilin subunit proteins (6). At present, however, we do not know whether pili binding to bovine thyroglobulin or human salivary agglutinin involves PilC1 or pilin differentially, in particular because acidic BMO completely inhibited binding to bovine thyroglobulin although binding partially to human salivary agglutinin.
Arguments for carbohydrate receptors for N. meningitidis also come from the ability of acidic BMO and neutral HMO to inhibit pili binding even at 1–2 g/L of oligosaccharides under microtiter well assay conditions with excess amounts of bacterial pili. Human milk contains 5.0–13 g/L oligosaccharides, and even up to 22 g/L for colostrum; interference with adhesion may therefore occur at a physiologically reasonable level (7,34,35). Human milk contains diverse and complex neutral and acidic oligosaccharide structures (16), whereas bovine milk (34) is less complex and contains mainly sialylated oligosaccharides. The receptor epitope on bovine thyroglobulin may be an internal core rather than a terminal sialic acid sequence because both neutral HMO and acidic BMO inhibited pili binding effectively. Further studies including desialylation of bovine thyroglobulin and acidic BMO as well as purification and analysis of individual binding oligosaccharides are warranted to delineate the receptor active sequences for Neisseria meningitidis pili.
It was reported that HMO inhibit adhesion of pathogenic Escherichia coli (9) and that fucosylated milk oligosaccharides are capable of preventing the colonization of Campylobacter jejuni in vivo (8). Together, these reports (8–15,35,36) and the present observation with N. meningitidis suggest that milk oligosaccharides have potential use in the development of functional foods and drugs to combat infectious diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 10 May 2005. Initial review completed 9 June 2005. Revision accepted 19 July 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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