Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human [alpha]1-acid glycoprotein mediates growth of Streptococcus oralis

Helen L. Byers, Edward Tarelli, Karen A. Homer1 and David Beighton

Joint Microbiology Research Unit, Faculty of Clinical Dentistry, King's College School of Medicine and Dentistry, Caldecot Road, London SE5 9RW, UK

Received on June 23, 1998; revised on August 16, 1998; accepted on August 18, 1998

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum [alpha]1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-[beta]-d-glucosaminidase, and [beta]-d-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.

Key words: [alpha]1-acid glycoprotein/glycosidases/growth/Streptococcus oralis

Introduction

Viridans streptococci are components of the normal flora of the oro-pharyngeal tract (Frandsen et al., 1991) and form a highly heterogeneous group of organisms (Hardie and Whiley, 1994). These organisms are a major cause of infective endocarditis (Douglas, 1993; Bochud et al., 1994a; Bouvet et al., 1994) and have emerged as significant pathogens of immunocompromised patients and those with malignancies (Cohen et al., 1983; Henslee et al., 1984; McWhinney et al., 1993). In neutropenic cancer patients, viridans streptococci account for almost 40% of all bacteremias (Bochud et al., 1994b; Richard et al., 1995), and are associated with serious complications including adult respiratory distress syndrome and streptococcal shock, resulting in mortality levels of up to 30%. Improvements in methods for identifying unambiguously viridans streptococci to the species level using a range of phenotypic tests have enabled the association between disease and isolation of a particular species to be demonstrated, with Streptococcus oralis emerging as a major pathogen among the viridans streptococci (Douglas et al., 1993; Beighton et al., 1991, 1994; Bouvet et al., 1994; Hardie and Whiley, 1994). Little is known regarding the virulence mechanisms of these organisms, but bacterial proliferation within the host is central to pathogenicity (Douglas, 1993; Guiney and Kagnoff, 1997). The viridans streptococci are fermentative organisms, requiring a source of carbohydrate for growth, and it has been suggested that the production of glycosidic activities by these organisms may play a role in their nutrition in vivo, by degrading the glycans of host glycoproteins and releasing sugars which are utilized as a source of fermentable carbohydrate (Beighton et al., 1994).

In the present study we have investigated the ability of the human serum glycoprotein, [alpha]1-acid glycoprotein (AGP), to support the in vitro growth of S.oralis. AGP is a highly glycosylated 41 kDa positive acute phase glycoprotein which is biosynthesized in the liver and is present in the serum of healthy individuals at a concentration of ~0.9 mg/ml (Schwick and Haupt, 1980). It possesses five complex-type, sialylated N-linked glycans (Fournet et al., 1978; Yoshima et al., 1981; Hermentin et al., 1992), some containing sialyl Lewis x (sLex) structures (de Graaf et al., 1993; van der Linden et al., 1994; van Dijk, 1995). At least 30 glycoforms (bi-, tri-, and tetra-antennary) have been detected in normal serum. The function of AGP is not known in detail but its serum level increases 2- to 6-fold as a result of trauma or bacterial infections, at which time it is also reported that there is an increased synthesis of biantennary and sLex structures (de Graaf et al., 1993). The well-characterized glycan structures of this glycoprotein can be considered representative of those found in many other serum glycoproteins. In this study we describe the manner by which pathogenic S.oralis degrades AGP, utilizing released monosaccharides to support growth, and suggest that this mechanism may be applicable to the ability of the organism to utilize glycans of other N-linked glycoproteins.

Results

Growth of S.oralis on AGP

AGP used in this study comprised (as shown after acid hydrolysis) 11.3% N-acetylneuraminic acid (NeuNAc), 0.8% fucose (Fuc), 14.9% N-acetylglucosamine (GlcNAc), 6.3% galactose (Gal), and 5.2% mannose (Man) (w/w protein). AGP was able to support in vitro growth of S.oralis strain AR3 when provided as the sole source of fermentable carbohydrate. A lag period of ~6 h was observed before the onset of exponential bacterial growth, during which period S.oralis grew with a doubling time of 4.7 h (Figure 1). After a 40 h incubation period in the AGP-supplemented media, the stationary phase of growth was attained after which the absorbance at 620 nm (A620) declined slowly, this being attributable to cell death and lysis.


Figure 1. Growth of S.oralis in AGP-supplemented minimal medium. S.oralis was cultured in minimal medium supplemented with AGP (5 mg/ml) supplied as the sole source of fermentable carbohydrate. Samples of culture (200 µl) were removed and growth measured by determination of the increase in absorbance at 620 nm (A620).

Exo-glycosidase production

S.oralis produced sialidase, N-acetyl-[beta]-d-glucosaminidase, and [beta]-d-galactosidase activities, as determined using the 4-methylumbelliferone- (4-MU-) linked substrates, when grown in AGP-containing media. [alpha]-l-Fucosidase, [alpha]-d-mannosidase, and [beta]-d-mannosidase activities were not detected in cell suspensions or culture supernatants from any of the incubation periods up to 100 h using their fluorogenic substrates. Cell-associated sialidase, N-acetyl-[beta]-d-glucosaminidase, and [beta]-d-galactosidase activities reached a maximum after 10 h of growth with specific activities of 124.3, 50.4, and 18.4 nmol 4-MU released/min/mg of cell-associated protein, respectively. Supernatant glycosidase activities reached a maximum after 17 h of growth with specific activities of 52.9, 22.8, and 3.36 nmol 4-MU released/min/mg of cell-associated protein for sialidase, N-acetyl-[beta]-d-glucosaminidase, and [beta]-d-galactosidase, respectively.

SDS-PAGE analysis of AGP during growth of S.oralis

Intact AGP had a molecular weight of 47.3 kDa by SDS-PAGE analysis, and this decreased over the growth period of S.oralis (Figure 2). During the first hour of incubation (0-6 h), little change was observed in the mean molecular weight of the residual glycoprotein (47.3-42.7 kDa) but a broadening of the protein band was noted with increasing time. After 10 and 17 h of growth there were marked reductions in the mean molecular weight of residual AGP, these being 34.0 and 25.2 kDa, respectively, and this was accompanied by extensive band broadening. Throughout the 25-100 h period of growth the residual AGP was gradually reduced down to an apparent single species of molecular weight 23.0 kDa, consistent with extensive deglycosylation of AGP with little or no accompanying proteolytic cleavage of the polypeptide backbone. SDS-PAGE analysis of the polypeptide resulting from PNGase F treatment of AGP demonstrated a species with molecular weight 22.3 kDa (data not shown), which was comparable to that obtained following growth of S.oralis on the glycoprotein within the limitations of SDS-PAGE.


Figure 2. SDS-polyacrylamide gel electrophoresis of residual AGP during growth of S.oralis. Lanes 2-11 show residual AGP purified from culture after 0, 2, 4, 6, 10, 17, 25, 43, 69, and 100 h of growth, respectively. Lanes 1 and 12 show high- and low-molecular mass protein markers, respectively, with masses indicated by arrows.

Changes in monosaccharide composition of AGP during growth of S.oralis


Figure 3. Changes in monosaccharide composition of AGP during growth of S.oralis. (A) Residual monosaccharides remaining attached to the polypeptide backbone of AGP were released by treatment with TFA. NeuNAc, solid squares; Gal, open circles; Fuc, solid circles; GlcNAc, open squares; Man, solid triangles. (B) AGP monosaccharides detected free in culture supernatant. NeuNAc, solid squares; Gal, open circles; Fuc, solid circles. Percentage values in both figures are given relative to the total monosaccharide composition of intact AGP, as determined following acid hydrolysis and HPAEC analysis.

Residual monosaccharides remaining attached to the polypeptide backbone of AGP, released by treatment with TFA, and those found as the free sugars in culture supernatants were analyzed by HPAEC throughout the growth period of S.oralis (Figure 3). NeuNAc release from the glycans of AGP was detected 2 h following inoculation of culture medium with S.oralis, by which time 12% of this monosaccharide had been released (Figure 3A). Levels of free NeuNAc in the culture supernatant increased in parallel (Figure 3B) and reached a maximum after 6 h, after which time free NeuNAc was transported from the culture supernatant by the growing bacteria. Release of the constituent NeuNAc of AGP was complete by 17 h incubation, at which time its concentration in the culture supernatant was below detectable levels. Gal was released from AGP after the initiation of NeuNAc hydrolysis, with 17% of the total Gal cleaved by 4 h after the point of inoculation (Figure 3A). Levels of free Gal reached a maximum 10 h from inoculation, at which time 77% of the total AGP-derived Gal was found in the culture supernatant (Figure 3B). All of the constituent Gal had been hydrolyzed from the polypeptide backbone of AGP after 17 h, and concentrations of the free sugar declined slowly up to 43 h when it was no longer detectable. Release of Fuc from AGP did not occur until after the initiation of Gal hydrolysis and was first detected after 6 h of incubation (Figure 3A). All of the Fuc was cleaved from AGP by 25 h of growth of S.oralis and was found in the culture supernatant (Figure 3B), its concentration remaining constant over the 25-100 h incubation period. Thus this monosaccharide, although released, was not utilized by the bacteria. Cleavage of GlcNAc from AGP glycans was first detected 6 h from the point of inoculation of cultures (Figure 3A). Further release of GlcNAc from AGP continued until 43 h of incubation of S.oralis until only 22% of the total remained attached to the polypeptide; no further release of this monosaccharide was observed over the 43-100 h incubation period, and at no time was GlcNAc detected free in the culture supernatant. Loss of Man from AGP was first noted 17 h after inoculation and all had been cleaved by 43 h (Figure 3A). Like GlcNAc, concentrations of Man in culture supernatant throughout the growth period remained below detectable levels.

The sequential release of carbohydrate demonstrated here supports the SDS-PAGE analysis of residual AGP over the growth period. In the early stages of growth NeuNAc was the only sugar released from the glycans when only minimal changes in the average molecular weight of AGP were detected, but band broadening increased. Partial NeuNAc release can increase the molecular weight range of glycoprotein species present. During the exponential phase of growth (10-17 h) a burst of deglycosylation occurred with loss of NeuNAc, Gal, Fuc, and GlcNAc, and this was reflected in the dramatic changes in the molecular weight observed on SDS-PAGE. During late exponential and early stationary phases of growth (25-43 h), it was mainly Man and GlcNAc released, resulting in slight decreases in molecular weights of residual glycoprotein and increased band resolution. On completion of growth (100 h), when only GlcNAc was associated with residual glycoprotein, a single species was observed on SDS-PAGE.

HPAEC and MALDI analysis of glycans liberated from AGP during growth of S.oralis

The glycans released by hydrazinolysis of glycoprotein in the culture medium during the growth of S.oralis were analyzed by HPAEC and MALDI. Before commencement of growth, the pool of oligosaccharides thus obtained was shown by HPAEC to comprise a complex mixture of mono-, di-, tri-, and tetra-sialylated species, with neutral oligosaccharides essentially absent (Figure 4A). This is consistent with published data which also noted the highly heterogeneous nature of the sialylated oligosaccharides of AGP, with more than 40 species identified (Hermentin et al., 1992). MALDI analysis of the oligosaccharide pool obtained after desialylation with a commercially available sialidase preparation produced a spectrum (Figure 4B) consistent with the major oligosaccharides expected to be present in desialylated AGP (Fournet et al., 1978; Yoshima et al., 1981; Hermentin et al., 1992) viz. bi-, tri-, and tetra-antennary fully galactosylated glycans together with smaller amounts of their monofucosylated analogues.


Figure 4. HPAEC-PAD and MALDI analyses of AGP glycans prior to growth of S.oralis. (A) Glycans of intact AGP analyzed by HPAEC. Oligosaccharide pools were resolved using a gradient of NaOAc (20-100 mM over 0-30 min and 100-300 mM over 30-60 min) in 250 mM NaOH. The elution positions of mono-, di-, tri-, and tetra-sialylated glycans of AGP (1, 2, 3, and 4, respectively) were calibrated by reference to the retention times of oligosaccharides released from fetuin (Townsend et al., 1989). Arrow indicates the elution position of NeuNAc. (B) Glycans released from desialylated AGP (following treatment with immobilized neuraminidase) analyzed by MALDI. Glycan pools were analyzed by MALDI with 2,5-dihydroxybenzoic acid as matrix. Gal, GlcNAc and Man in assigned structures are indicated by open circles, open squares, and solid triangles, respectively. Fucosylated analogues of the major components are indicated by arrows. Other non-assigned ions may be attributed to minor oligosaccharide species or to artefacts/contaminants present as a result of hydrazinolysis.

Changes in the relative proportions of mono-, di-, tri-, and tetra-sialylated glycans with incubation time were estimated from peak areas of HPAEC-PAD chromatograms (Figure 5). Essentially, monosialylated species increased over the first 10 h of incubation with a concomitant reduction in tri- and tetra-sialylated species, and after 25 h of incubation complete desialylation of glycans was observed. These results are consistent with the steady removal of NeuNAc from the glycan chains over this time period.


Figure 5. HPAEC-PAD analyses of sialylated AGP glycans during growth of S.oralis. Samples were removed from culture and analyzed as described in Figure 4. Mono- (solid circles), di- (open squares), tri- (solid squares), and tetra- (open circles) sialylated glycans were quantified by peak integration of detector response.

Figure 6A,B demonstrates the loss of sialylated glycans and appearance of neutral glycans after 10 and 17 h of growth, respectively. At 17 h, the chromatogram had simplified, with species eluting after 12 min virtually absent demonstrating that smaller glycans were then present (Figure 6B; Cooper and Rohrer, 1995). These changes in glycan pool compositions were paralleled in their MALDI spectra. At the 10 h time point, ions corresponding to several bi-, tri-, and tetra-antennary agalacto and agalacto species lacking GlcNAc were observed (Figure 7A). Ions corresponding to monofucosylated analogues of these species, which would have appeared 146 mass units higher, were absent demonstrating that Fuc was not associated with glycoprotein at this time. At 17 h, the pool of oligosaccharides gave rise to a major ion attributable to a biantennary agalacto species containing one GlcNAc residue (Figure 7B) and this spectrum also contained an ion at m/e 933.3 attributable to the core pentasaccharide (Man3GlcNAc2). Smaller amounts of these oligosaccharides, as indicated from lowered detector response in HPAEC, were liberated by hydrazinolysis at later time points. After 100 h incubation oligosaccharides were essentially absent (Figure 6C). These results accord with those from monosaccharide analyses and demonstrate that after the removal of NeuNAc there occurs a sequential loss of Gal, Fuc, GlcNAc, and Man from the glycoprotein. Throughout the time-course the smallest oligosaccharide associated with glycoprotein which was detected by HPAEC or MALDI analyses was the core pentasaccharide and this in only relatively small amounts. Cellulose chromatography following hydrazinolysis is reported to remove saccharides smaller than tetramers (Takasaki et al., 1982), which could explain the absence of smaller saccharides in the oligosaccharide pools. However, even when procedures following hydrazinolysis were modified so as to prevent such losses (evaporation of hydrazine under vacuum over H2SO4, N-acetylation, purification on a reverse phase C18-silica cartridge, Cu(II) hydrolysis of hydrazones and cation exchange), HPAEC failed to demonstrate the presence of tetra-, tri-, or di-saccharides in the glycoprotein fraction remaining after 25 h growth at which time about 15% of the original Man was still present.


Figure 6. HPAEC-PAD analyses of AGP glycans during growth of S.oralis. Samples of culture were removed after (A) 10 h, (B) 17 h and (C) 69 h incubation, residual AGP desalted by gel filtration and the glycans released by treatment with hydrazine. Oligosaccharide pools were resolved by HPAEC using a gradient of NaOAc (20-100 mM over 0-30 min and 100-300 mM over 30-60 min) in 250 mM NaOH. 5 indicates the elution position of neutral (desialylated) glycans.


Figure 7. MALDI analyses of neutral AGP glycans during growth of S.oralis. Residual glycans of AGP following (A) 10 h and (B) 17 h incubation with S.oralis. Samples were desalted by gel filtration and pools of oligosaccharides released by treatment with hydrazine. Glycan pools were analyzed by MALDI with 2,5-dihydroxybenzoic acid as matrix. Gal, GlcNAc, and Man in assigned structures are indicated by the symbols open circles, open squares, and solid triangles, respectively. Non-assigned ions may be attributed to minor oligosaccharide species or to artefacts/contaminants present as a result of hydrazinolysis.

Nature of the residual glycans of AGP at the end of growth of S.oralis

After 100 h of incubation when bacterial growth had ceased the only carbohydrate remaining associated with glycoprotein was GlcNAc, 22% of that originally present. HPAEC analysis of products released by hydrazinolysis of the glycoprotein remaining after this time indicated the absence of carbohydrate. However, any small saccharides present may have been removed in the preliminary washings which, in addition, contain hydrazine salts and peptide material. These washings, following re-N-acetylation and hydrolysis of hydrazones, were shown to contain material which coeluted on HPAEC with GlcNAc; no other saccharide was detected. These fractions were difficult to analyze as they contained relatively large amounts of other substances; and in order to identify more fully the nature of the residual GlcNAc and to ascertain whether it was present as a monomer, dimer (N,N-diacetylchitobiose), or as a mixture of both, the glycoprotein remaining after growth was treated with alkaline borohydride under conditions to cleave N-linked saccharides (Lee and Scocca, 1972). Following sequential peracetylation, extraction with dichloromethane to remove salts, especially borate (Harris et al., 1984), and selective O-deacetylation, a product which coeluted with N-acetylglucosaminitol (GlcNAc-ol) was detected (Figure 8). No N,N-diacetylchitobiitol was present. The amount of GlcNAc-ol recovered amounted to >60% of the residual GlcNAc present after growth (determined after acid hydrolysis) and using a similar series of reactions, GlcNAc-ol was shown to be absent from intact AGP. These observations are consistent with the presence of single GlcNAc residues linked to each of the asparagine residues originally carrying glycan chains. Further, SDS-PAGE analysis of the residual polypeptide following growth of S.oralis (molecular weight 23.0 kDa; Figure 2) was found to be comparable, within the limitations of the analytical technique, with the theoretical mass of the polypeptide backbone of AGP with 5 GlcNAc residues attached (24.6 kDa).


Figure 8. HPAEC-PAD analysis of residual glycans of AGP at the end of growth of S.oralis. S.oralis was cultured for 100 h in AGP-supplemented medium, residual glycoprotein desalted by gel filtration and treated with alkaline borohydride to release sugars as the alditol derivatives. Sugar alditols were re-N-acetylated and analyzed by HPAEC on an MA1 column using isocratic elution with 80 mM NaOH. The only sugar alditol detected coeluted with GlcNAc-ol. N,N-diacetylchitobiitol (elution position indicated by arrow) was not detected.

The growth medium from which the residual glycoprotein had been removed (this containing low molecular weight substances) was also subjected to hydrolysis by alkaline borohydride. In this case fucitol was the only sugar alcohol present and this accounted for >60% of that expected from the compositional analysis value.

Inhibition of growth of S.oralis on AGP by 2,3-dehydro-N-acetylneuraminic acid

Data on monosaccharide release from AGP during growth of S.oralis indicated that hydrolysis from glycans was sequential, and that release of neutral and amino sugars occurred following release of NeuNAc. In order to further demonstrate the key role of sialidase production in exposing additional monosaccharides to cleavage by exo-glycosidases, growth of S.oralis on AGP was monitored in the presence of the sialidase inhibitor 2,3-dehydro-N-acetylneuraminic acid (2,3-dehydro-NeuNAc; Figure 9). In the absence of the sialidase inhibitor a doubling time of 5.2 h was observed, which increased to 8.8 h and 22.9 h in the presence of 10 or 100 µM 2,3-dehydro-NeuNAc, respectively. After a 38 h period of growth the A620, representative of total bacterial cell numbers, of the untreated AGP-containing cultures was 0.352 compared with 0.280 and 0.04 in cultures containing 2,3-dehydro-NeuNAc at concentrations of 10 and 100 µM, respectively. Growth of S.oralis in minimal media supplemented with glucose at a concentration of 10 mM was not affected by the addition of the sialidase inhibitor at either concentration, with doubling times and A620 values identical to those obtained in the absence of the inhibitor (data not shown).


Figure 9. Effect of 2,3-dehydro-NeuNAc on the growth of S.oralis on AGP. S.oralis was cultured in minimal media supplemented with AGP (200 µl) and containing 0 (solid circles), 10 (open squares), and 100 (solid triangles) µM 2,3-dehydro-NeuNAc. Growth was measured by determination of the increase in absorbance at 620 nm (A620).

Discussion

We previously reported (Byers et al., 1996) that S.oralis was able to utilize for growth unconjugated NeuNAc as the sole supplement to minimal medium. Other studies, using high field NMR, demonstrated that sialidase and N-acetyl-[beta]-d-glucosaminidase production by this organism resulted in the cleavage of NeuNAc and GlcNAc from AGP and serum glycoproteins, and both of the released N-acetylated sugars then supported microbial proliferation (Homer et al., 1995, 1996). Glycosidase production is widespread amongst bacteria, and of all these enzymes most interest has focused on sialidase as a virulence determinant in pathogenic organisms. Sialidase is produced by species as diverse as Vibrio cholerae, Streptococcus pneumoniae, Salmonella typhimurium and Bacillus fragilis (Corfield, 1992; Godoy et al., 1993). It has been suggested that the primary role of sialidase production may be in releasing a source of nutrient to support bacterial nutrition, with a secondary function being damage to host tissues and the disruption of sialic acid-mediated functions of host glycoproteins (Corfield, 1992). Indeed, in vivo studies have demonstrated that mutants of B.fragilis deficient in sialidase production exhibited a diminished ability to proliferate in the rat pouch model of infection (Godoy et al., 1993). In the present study, sialidase production by S.oralis has dual relevance to the nutrition of the organism in that it releases NeuNAc which is, per se, fermented and in addition opens the glycans of AGP to further attack by exo-glycosidases ([beta]-d-galactosidase, [alpha]-l-fucosidase, N-acetyl-[beta]-d-glucosaminidase and [alpha]-d- and [beta]-d-mannosidases), which release additional sources of fermentable carbohydrate. Inhibition of the sialidase activity of S.oralis by 2,3-dehydro-NeuNAc inhibited growth on AGP by preventing desialylation and further deglycosylation of the glycans.

Using synthetic fluorogenic substrates, sialidase, [beta]-d-galactosidase, and N-acetyl-[beta]-d-glucosaminidase were demonstrable; [alpha]-l-fucosidase and mannosidase activities were, however, not detected, and these activities were only apparent from analyses of AGP glycans during growth of S.oralis. Synthetic glycosides have on other occasions been shown to be poor substrates for glycosidases, and their failure to respond does not therefore necessarily indicate absence of that activity. Van der Hoeven and Camp (1991) demonstrated, using conventional methods of carbohydrate analysis, the production of an [alpha]-l-fucosidase activity by S.oralis when the organism was cultured in the presence of mucin but were unable to detect this glycosidic activity using the chromogenic substrate. Similarly, Kilian et al. (1989) monitored mannosidase production by all recognized species of viridans streptococci, but were unable to demonstrate this activity in S.oralis strains using a [beta]-d-linked p-nitrophenyl substrate.

In this present study we have demonstrated that during growth of S.oralis Man is cleaved from the glycans of AGP and that the released monosaccharide is utilized as a source of fermentable carbohydrate. Furthermore, the smallest detectable oligosaccharide associated with the polypeptide was the core pentasaccharide (Man3GlcNAc2). One explanation for the absence of Man-containing tetra- and tri-saccharides in the glycoprotein-containing fractions removed during the later stages of growth (e.g., when 15% of the total Man was still present) is that the pentasaccharide core undergoes rapid modification by the bacterium. This suggests the action of highly active [alpha]-d- and [beta]-d-mannosidases which quickly liberate Man, with the free monosaccharide then rapidly metabolized by the bacterium. This is, as far as we are aware, the first report of the production of mannosidase activity by S.oralis.

S.oralis released the constituent monosaccharides of AGP (NeuNAc, Gal, Fuc, GlcNAc, and Man) in a sequential manner consistent with the canonical structure of AGP and the production of the appropriate exo-glycosidic activities until the only monosaccharides remaining were the GlcNAc residues covalently linked to the asparagine residues of the polypeptide backbone. All monosaccharides released from AGP glycans during growth of S.oralis were utilized with the exception of Fuc, which was found free in culture supernatants at levels consistent with its complete cleavage from AGP. NeuNAc and Gal were detected as the free monosaccharides in culture supernatants following release from the polypeptide backbone of AGP during the growth cycle of S.oralis. This is consistent with the presence of inducible transport systems for these sugars, these systems being activated by the presence of the free monosaccharides. A specific inducible transport system for NeuNAc has been characterized in both Lactobacillus plantarum and E.coli; the latter system has the characteristics of a proton motive force-dependent permease (Rodriguez-Aparicio et al., 1987; Andreadaki et al., 1990; Martinez et al., 1995). GlcNAc and Man were liberated from AGP glycans, but remained below detectable levels in culture supernatants. In streptococci, these monosaccharides are transported via phosphenolpyruvate:sugar phosphotransferase systems (PTS); specific, inducible transport systems are present, but transfer of these sugars across the cell wall is also mediated via the constitutive glucose PTS (Liberman and Bleiweis, 1984; Homer et al., 1993). Thus, GlcNAc and Man are transported immediately following cleavage from AGP glycans and the free sugars were not detected in culture supernatants of S.oralis.

S.oralis is phylogenetically closely related to the pathogen S.pneumoniae (Bentley et al., 1991; Kawamura et al., 1995), which is a major cause of respiratory disease and meningitis. S.pneumoniae produces a range of glycosidases, including both endo- and exo-N-acetyl-[beta]-d-glucosaminidase activities, the former enzyme acting to cleave the glycosidic bond between the two GlcNAc residues within the pentasaccharide core, releasing a tetrasaccharide (Glasgow et al., 1977). Recently, Tyagarajan et al. (1996) have demonstrated the feasibility of assessing the linkage specificity of glycosidases from a number of sources using HPAEC-PAD to monitor mono- and oligosaccharide release from oligosaccharide substrates. In this study we were unable to demonstrate the presence of endo-glycosidic activities in S.oralis by monitoring throughout the growth cycle for the presence of AGP-derived oligosaccharides in culture supernatants using HPAEC-PAD.

It can be concluded from these results that S.oralis produces exo-glycosidases that sequentially remove the sugar residues from the glycans of AGP and that the released monosaccharides are then metabolized (except for Fuc). Each glycan present in AGP was extensively deglycosylated until the only monosaccharide remaining was the terminal GlcNAc linked to asparagine. This is, we believe, the first demonstration of the detailed mechanism by which pathogenic streptococci are able to utilize glycan chains of a serum glycoprotein to sustain growth. It would seem reasonable to assume that similar mechanisms will operate on other serum glycoproteins and tissues which contain complex-type N-linked glycans and suggest that the ability to utilize glycans in this way may have implications in the pathology of such infections. Furthermore, the results indicate that in the presence of attenuated host defense mechanisms S.oralis is particularly aggressive among the viridans streptococci, as it produces the array of glycosidases needed to cleave glycans in the manner described here and uses the liberated monosaccharides for growth. Until recently, the viridans streptococci were not considered major pathogens, with the exception of their association with dental caries and infective endocarditis (Bochud et al., 1994a), and routine treatment with penicillins was a standard regimen. The emergence of penicillin resistance within this group (Doern et al., 1996; Reichmann et al., 1997) is now limiting therapeutic options for such infections, and therefore an understanding of the mechanisms by which these organisms proliferate may be important in the development of future antimicrobial therapies.

Materials and methods

Materials

AGP was supplied by Bioproducts Ltd., Elstree, UK. HPAEC-grade NaOH and NaOAc were purchased from Fisons (Loughborough, Leicestershire, UK). PNGase F was obtained from New England Biolabs (Hitchin, Hertfordshire, UK). Polyacrylamide desalting columns were from Pierce and Warriner Ltd. (Chester, UK). All solvents and reagents used in the release and purification of AGP monosaccharides and glycans were of analytical grade. All other materials and substrates were purchased from Sigma, unless otherwise stated.

Bacterial strain and culture

S.oralis strain AR3, isolated from subacute infective endocarditis, was routinely cultured on Fastidious Anaerobe Agar supplemented with 5% (v/v) defibrinated horse blood and incubated in an anaerobic atmosphere comprising 10% H2, 80% N2 and 10% CO2 at 37°C for 24 or 48 h. Single colonies were removed into 20 ml volumes of Brain Heart Infusion broth and incubated until bacterial growth reached late log-phase. Minimal medium, in which streptococcal growth is dependent on the addition of a source of fermentable carbohydrate, was prepared at double strength, as described previously (Byers et al., 1996). Minimal medium was supplemented with AGP to a final concentration of 5 mg/ml in culture volumes of up to 20 ml and inoculated with a 5% (v/v) of BHI starter culture of S.oralis. Cultures were incubated anaerobically for periods of up to 100 h and aliquots removed at intervals for determination of cell growth, monosaccharide and glycan analyses and glycosidase assays. Cell growth was monitored by measuring the absorbance of 200 µl aliquots of cultures at 620 nm in a 96-well plate-reading spectrophotometer (MCC340, ICN-Flow). In order to determine the effect of the sialidase inhibitor, 2,3-dehydro-NeuNAc, on growth minimal media were supplemented with AGP or glucose at concentrations of 5 mg/ml and 10 mM, respectively, and containing 0, 10, or 100 µM 2,3-dehydro-NeuNAc. Media were dispensed into a 96-well microtiter tray in 200 µl aliquots and inoculated with BHI starter. Cultures were incubated at 37°C in a shaking, plate-reading spectrophotometer (iEMS, Labsystems, Life Sciences International, Hampshire, UK), and growth was measured over a 38 h period by monitoring A620 at hourly intervals.

Prior to further analyses, cells were pelleted from culture by centrifugation at 14,600 × g for 5 min, supernatants were decanted, and cells were washed in 50 mM sodium phosphate buffer, pH 7.5. Cells were resuspended in this same buffer and these suspensions and a portion of each culture supernatant used in the assay of glycosidase activity. The remaining culture supernatant from each incubation period was heated at 100°C for 15 min to inactivate glycosidases. Aliquots of whole culture supernatant were retained for the direct analysis of free mono- and oligosaccharides, and the remainder was stored at -20°C prior to desalting by gel filtration, SDS-PAGE and mono- and oligosaccharide analyses.

Glycosidase assays

Bacterial glycosidase activities (sialidase, N-acetyl-[beta]-d-glucosaminidase, [beta]-d-galactosidase, [alpha]-l-fucosidase, [alpha]-d-mannosidase, and [beta]-d-mannosidase) were measured at pH 7.0 using the appropriate 4-MU-linked substrates. Substrates were included in assays at a final concentration of 0.1 mM using previously described assay conditions and release of 4-MU was monitored by recording fluorescence values in a fluorimeter fitted with a 96-well plate-reading attachment (LS-3B, Perkin-Elmer) at excitation and emission wavelengths of 380 and 460 nm, respectively (Byers et al., 1996). 4-MU in assays was quantified by comparison of fluorescence values with those of standard concentrations of the authentic compound. Cell- and supernatant associated activities of individual glycosidases were related to the cell-associated protein of bacterial suspensions and are given as nanomoles 4-MU released/min/mg of cell-associated protein.

Protein estimations

Protein concentrations of whole cell suspensions of S.oralis and AGP preparations were determined using the bicinchoninic acid assay (BCA; Smith et al., 1985) using a commercially available kit. Protein determinations were carried out according to the manufacturers' instructions and bovine serum albumin over the concentration range 0-1 mg/ml was used as standard.

Preparation of culture supernatants prior to mono- and oligosaccharide analyses

Glycoprotein-containing culture supernatants (500 µl aliquots) were applied to a 5 ml polyacrylamide, desalting column (6 kDa cut-off) equilibrated in and eluted with 0.2 M ammonium bicarbonate (pH 8.0). The void and included volumes were determined by calibrating with blue dextran and NaCl, respectively. The void volume fractions which contained all residual glycoprotein (confirmed by the BCA protein assay), were pooled, lyophilized (three times) to remove ammonium bicarbonate, reconstituted in 1 ml of distilled water, and stored at -20°C prior to analyses. Fractions from the included volume of the column, containing free monosaccharides, were also retained for further analyses following lyophilization and reconstitution, as described above.

SDS-PAGE analysis of AGP and residual glycoproteins

SDS-PAGE of intact and bacterially-modified AGP was performed after the method of Laemmli (1970). In addition, AGP was treated with PNGase F according to the manufacturer's instructions and the deglycosylated protein analyzed by SDS-PAGE. Desalted glycoprotein was mixed with SDS-PAGE sample buffer containing 25% [beta]-mercaptoethanol and heated at 100°C for 5 min. Samples containing 10 µg of protein (as determined using the BCA protein assay) were applied to a 20 × 20 cm 12% homogeneous gel and resolved at a constant voltage of 100 V over 4 h. The molecular mass of the glycoprotein was determined by comparison with low- and high-molecular mass protein marker sets. The gel was stained overnight by treatment with Coomassie brilliant blue and destained in double distilled water with gentle agitation.

Release of AGP monosaccharides by acid hydrolysis

Glycoproteins (50 µg) were hydrolyzed in 4 M trifluoroacetic acid (TFA; 500 µl) at 120°C for 2 h in order to liberate neutral and amino sugars (Gal, Man, Fuc, and GlcNAc, this latter being converted to glucosamine (GlcN) as a result of the hydrolysis procedure). The hydrolysate was lyophilized (twice) and the residue, dissolved in water (200 µl), was analyzed by HPAEC. Glycoproteins (50 µg) were hydrolyzed in 40 mM TFA (200 µl) at 80°C for 1 h in order to liberate NeuNAc. The hydrolysate was then analyzed directly by HPAEC.

NeuNAc, Gal, Man, Fuc, and GlcNAc (50 µM) were subjected to acid hydrolysis under these same reaction conditions. The resulting hydrolysates were used as standards for the quantitation of AGP-derived monosaccharides by HPAEC-PAD.

Desialylation of AGP

AGP, 5 mg dissolved in 10 mM acetate buffer (pH 5.0) containing 0.01% NaN3, was treated with neuraminidase (sialidase) immobilized on agarose (Type X-A from Clostridium perfringens; Sigma) at 37°C. NeuNAc release was monitored by HPAEC-PAD and was complete after 17 h incubation. The immobilized enzyme was removed by centrifugation and the supernatant dialyzed (10 kDa molecular weight cut-off) against distilled water (3 × 1 l) to remove salts and NeuNAc. The retentate was lyophilized and subjected to hydrazinolysis to release a pool of fully desialylated AGP glycans.

Release of AGP oligosaccharides by treatment with hydrazine

Oligosaccharides of intact AGP, AGP treated with neuraminidase immobilized on agarose (incubated for 17 h at 37°C to ensure complete desialylation) or desalted preparations of the glycoprotein sampled from growth cultures were cleaved from the peptide by treatment with hydrazine using methodology originally described by Takasaki et al. (1982) and purified essentially as described by Lifeley et al. (1995). Salt-free preparations of glycoprotein (4.5 mg) were dried in a glass ampoule under vacuum over phosphoric oxide. Anhydrous hydrazine (100 µl) was added, and the ampoule was sealed and heated at 95°C for 5 h. This preparation was reconstituted in butanol/ethanol/acetic acid (8/2/1; v/v/v) and applied to a 1 ml column containing microcrystalline cellulose previously equilibrated in this same solvent. The column was washed by sequential elution with butanol/ethanol/acetic acid (8/2/1, v/v/v), butanol/ethanol/water (4/1/1, v/v/v) and methanol, this washing procedure removing unreacted hydrazine, peptide material and oligosaccharides smaller than tetrasaccharides. Oligosaccharides were re-N-acetylated in situ by treatment with methanolic acetic anhydride (10/4; v/v) for 30 min at ambient temperature prior to further sequential washing with methanol, butanol/ethanol/water (4/1/1; v/v/v), and methanol. Glycans were eluted from the column with 0.2 M NaOAc and the eluent treated with 0.1 ml of acetic anhydride for 30 min at room temperature. The acetylhydrazone derivatives of the oligosaccharides were hydrolyzed by addition of 0.1 ml of Cu(II) acetate (0.1 M in 0.1 M acetic acid) and incubation for 30 min at room temperature. Cations were removed from the preparation by passage through an ion-exchange column containing Dowex AG50 and Chelex (both in the H+ forms) and the purified oligosaccharides eluted with distilled water prior to passage through a 0.2 µm membrane to remove particulates and lyophilization. Samples were stored at -20°C until required for analysis by HPAEC-PAD and MALDI at which time they were reconstituted in water.

In the case of the glycoprotein remaining when growth was complete, the preliminary washings, in addition to the final fraction, were retained for analysis. These washings were dried at 40°C, the residue dissolved in 0.2 M NaOAc and treated sequentially with acetic anhydride (200 µl), Cu(II) acetate (0.1 M, 100 µl) and then processed in a similar manner to the final fraction (Lifely et al., 1995). The products obtained from this latter process were then analyzed for the presence of monosaccharides by HPAEC using a PA1 column. AGP was treated and analyzed similarly.

Hydrolysis of intact and residual AGP by treatment with alkaline borohydride

The supernatant from culture after 100 h of incubation was separated into residual glycoprotein and a low-molecular weight fraction by gel filtration, as described. Residual glycoprotein or low-molecular weight fraction from 4.5 mg of AGP were treated with 1 M NaOH, 1 M NaBH4 (2 ml) under reflux for 6 h. The reaction mixtures were cooled in ice and adjusted to pH 4 by the addition of acetic acid and freeze-dried. Dimethylsulfoxide (2 ml), 1-methylimidazole (200 µl), and acetic anhydride (2 ml) were then added to the residue and the mixtures kept at room temperature for 3 h. Water (5 ml) was added to destroy excess acetic anhydride, the solution was extracted with dichloromethane (3 × 10 ml), and the combined extracts were washed with water (2 ×5 ml). The organic solutions were then dried using MgSO4, filtered, and evaporated to dryness. The residues were treated overnight with methanolic sodium methoxide, pH 10. The solutions were then neutralized with Amberlite AG50 (H+ form), filtered, and evaporated to dryness. The residues in water (200 µl) were analyzed by HPAEC. AGP (1 mg) and GlcNAc, Fuc, or N,N-diacetylchitobiose (0.1 mg each) were treated and analyzed in a similar manner.

High pH anion exchange chromatography (HPAEC)

HPAEC was performed using a Dionex DX500 system fitted with gradient pumps and a PAD detector (Dionex, UK). Data were collected and analyzed using the Dionex Peaknet software. Eluents comprised 200 mM NaOH, 1 M NaOH, and 1.0 M NaOAc prepared in 18 M[Omega] water, and these were degassed by sparging with helium for 30 min prior to chromatographic analyses. An appropriate volume of each sample was injected onto the column from an AS3500 autosampler (Thermoseparations, UK) equipped with a 100 µl sample loop. All separations were performed at a flow rate of 1 ml/min at ambient temperature.

Separation of neutral and amino sugars (Fuc, GlcN, Gal, and Man) released from AGP by acid hydrolysis was performed on a Carbopac PA1 (250 × 4 mm) column (Dionex) at an isocratic concentration of 18 mM NaOH over 20 min. NeuNAc from acid hydrolysates was resolved using this same column and a linear gradient of NaOAc (20-100 mM over 30 min) in 100 mM NaOH. The detection limits for each monosaccharide (NeuNAc, Gal, GlcN, Man, and Fuc) were <0.05 µM.

Intact and residual oligosaccharides released from glycoproteins by hydrazinolysis were analyzed using a gradient of NaOAc (20-100 mM over 0-30 min and 100-300 mM over 30-60 min) in 250 mM NaOH. The elution positions of mono-, di-, tri-, and tetra-sialylated glycans of AGP were calibrated by reference to the retention times of oligosaccharides released from fetuin by treatment with hydrazine, as described by Townsend et al. (1989). The saccharide species released from residual AGP following growth of S.oralis and reduced saccharides (glucosaminitol, N,N-diacetylchitobiitol, and fucitol) were resolved on a MA1 (250 × 4 mm) column (Dionex) using isocratic elution with 80 mM NaOH over 40 min.

MALDI

For the determination of the molecular mass of glycans released from AGP by treatment with hydrazine, a MALDI mass spectrometer with pulsed extraction (Kratos Maldi 4 spectrometer, Kratos Analytical Ltd., Manchester, UK) was used in positive-ion and linear modes. Spectra were acquired following irradiation of samples with a nitrogen laser giving a 337 nm output with 3 ns pulse width and molecular ions accelerated at a potential of 20 kV. Pools of oligosaccharides released from ~20 nmol of glycoprotein were dissolved in 0.5 ml of 1% aqueous TFA, and 0.5 µl of this was mixed with an equal volume of matrix solution on the target plate. The matrix solution comprised 10 g/l 2,5-dihydroxybenzoic-acid in 0.1% aqueous TFA/acetonitrile (2/1, v/v). Bovine pancreas insulin (molecular mass 5734.6) was used as an external standard for instrument calibration.

Acknowledgments

Bio Products Limited, Elstree, UK are thanked for providing the sample of AGP. Kratos Analytical Ltd., Manchester, UK are thanked for providing access to MALDI facilities. This work was supported in part by grants from the British Heart Foundation (PG/95064), The Freemasons' 250th Anniversary Fund and the Joint Research Committee (King's College School of Medicine and Dentistry).

Abbreviations

AGP, [alpha]1-acid glycoprotein; BCA, bicinchoninic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPAEC, high pH anion-exchange chromatography; PAD, pulsed amperometric detection; NeuNAc, N-acetylneuraminic acid; ManNAc, N-acetylmannosamine; GlcNAc, N-acetylglucosamine; GlcN, glucosamine; Gal, galactose; Fuc, fucose; Man, mannose; GlcNAc-ol, N-acetylglucosaminitol; 2,3-dehydro-NeuNAc, 2,3-dehydro-N-acetylneuraminic acid; MALDI, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; 4-MU, 4-methylumbelliferone; PNGase F, peptide: N-glycosidase F.

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