4rchs oral Biol. Vol. 24, pp. 63 lo 66. Pergamon Press Ltd. 1979. Printed in

STRUCTURE SYNTHESIZED

Britain.

OF EXTRACELLULAR POLYSACCHARIDES FROM SUCROSE BY NEISSERZA ISOLATED FROM HUMAN DENTAL PLAQUE D.

BIRKHED, K.-G.

ROSELL*

and G. H.

BOWDEN

Department of Cariology, University of Lund, School of Dentistry, S-214 21 Malmii, Department of Organic Chemistry, University of Stockholm, Arrhenius Laboratory, S-104 05 Stockholm, Sweden, and Dental School, The London Hospital Medical College, London, England

Summary-Five strains of Neisseria, resembling N. mucosn and N. &&XXI, isolated from human dental plaque, synthesized extracellular polysaccharides from sucrose. Methylation analysis and characterization of the cleavage products by gas-liquid chromatography-mass spectrometry were the methods used. The polysaccharide material, isolated from a culture broth by high-speed centrifugation, was characterized as an a-glucan with predominantly (l-4) linkages, although l&17 per cent were (1 +3) linkages and 5-9 per cent branches linked to the 6-position. A modified Smith degradation suggested that the material consists of 2 polysaccharides, an amylopectin-type (major component) and an a-(1 + 3)-linked glucan. The mean molecular weight of methylated material was 45,000-65,000. INTRODUCTION ‘Ihe

place of bacterial extracellular polysaccharides in the aetiology of dental diseases has been reviewed many times (Newbrun, 1967; Fitzgerald, 1968; Fitzgerald and Jordan, 1968; Gibbons, 1968; Keyes, 1968; Guggenheim, 1970; Critchley, 1971). When dental plaque extracts react with sucrose they form glucans linked in the a-(1 + 6) position with branches linked a-(1 --* 3) to the main chains as well as p-(2+ 6)linked fructans with branches in the l-position (Birkhed and Rosell, 1975). Hotz, Guggenheim and Schmid (1972) found that the water-insoluble matrix of dental plaque contains glucans with predominantly a-(1 --) 3) linkages in the main chain. Extracellular polysaccharides are produced from sucrose by various oral microorganisms, e.g. glucans and fructans by Streptococcus mutans, glucans by Streptococcus sanguis and fructans by Streptococcus salivarius and Actinomyces viscosus (Ceska et al., 1972; Rosell and Birkhed, 1974; Ehrlich et al., 1975; Birkhed, Rose11 and Granath, 1979). The possible role of microbial extracellular polysaccharides in the nutrition of dental plaques and oral microorganisms has been studied (Wood, 1967; van Houte and Jensen, 1968; DaCosta and Gibbons, 1968; Staat, Gawronski and Schachtele, 1973; Dewar and Walker, 1975). Certain polysaccharides, i.e. a levan-like fructan produced by Strep. salivarius strain RI and an amylopectin-like glucan from Neisseria perjZnvu strain UODS Nl, are utilized more readily by streptococci and lactobacilli than is sucrose (Parker and Creamer, 1971). However, the characterization of extracellular amylopectin-like glucans synthesized from sucrose. by oral microorganisms is still unclear. Our aim is to describe the chemical structure of

polysaccharides synthesized from sucrose by strains of Neisseria mucosa and Neisseria subflava isolated from human dental plaque. MATERIAL AND METHODS

Bacterial strains

Five Neisseria isolates, strains A630, A636, A182, 5975 and M1107, from human dental plaque (Bowden, Hardie and Slack, 1975) were studied. They were examined by the tests described by Pike ef al. (1962), except that growth in the presence of NaCl and bile was tested on solid media. The electrophoretic mobilities of glucose&phosphate dehydrogenase and glutamate dehydrogenase produced by these microorganisms were measured (Holten, 1973). The 5 strains were saccharolytic, pH 5.2-5.6 from glucose, produced polysaccharide material from sucrose, grew in the presence of 2 but not 5 per cent bile and did not grow in 2 or 5 per cent NaCl. The mobilities of glucose-dphosphate dehydrogenase and glutamate dehydrogenase from the strains were the same as the mobilities of these enzymes from Neisseria pharyngis siccus (NCTC 4591). 17 types of strains were similarly tested and strains Neisseria pharyngis jlavus (NCTC 4590), Neisseria subjlava (ATCC 19243) and Neisseriu denitrijcuns (NCTC 10295) fell into the saccharolytic group. Type strains of Neisseria perflaw and Neisseria mucosa were unavailable. Consideration of the taxonomic characters (Bergey, 1974) suggested that the 5 strains could be classified as N. mucosa (strains A636, A630 and 5957) and N. subJava (strains Al82 and M1107). However, the differential charao teristics are few and differentiation depends on the ability to reduce nitrate. Preparation of polysaccharides

*Present address: Division of Biological Sciences, National Research Council of Canada, Ottawa KIA OR6,

Cultures were grown in the medium described by Pike et al. (1962) supplemented with 1 per cent (w/v)

Canada. 63

64

D. Birkhed. K.-G. Rose11and G. H. Bowden

sucrose and 1 litre of the same medium was inoculated with 5 ml of a 24-h culture. Incubation was performed at 37°C in air up to 3 days until the broth formed a dense white turbidity. After centrifugation at 3000 g for 20 min at 4°C the cells were discarded. The supernatant containing soluble polysaccharides was re-centrifuged at 30,000 g for 20 min at 4°C and the centrifugate dialysed against distilled water at 4°C for 48 h and lyophilized. A higher concentration of sucrose, 5 per cent, was tried but little insoluble polysaccharide material, measured by eye, was formed. General methods

Samples were concentrated under reduced pressure at temperatures not exceeding 40°C. For gas-liquid chromatography (g.1.c.k a Perkin-Elmer 990 instrument, with a flame-ionization detector was used. Separations were made with 190 x 0.15-cm glass columns containing 3 per cent OV-225 on Gas Chrom Q (10&120 mesh) at 170°C (partially-methylated alditol acetates) or at 190°C (alditol acetates) and with OV-225 S.C.O.T. columns (Soft x 20in.) at 190°C. For quantitative evaluation of the g.l.c., a Hewlett-Packard 3370B integrator was .used. For mass spectrometry (ms.), the mixture of alditol acetates, dissolved in chloroform, was injected into a Perkin-Elmer 270 combined gas chromatograph-mass spectrometer. The mass spectra were recorded at an ionization potential of 70eV, an ionization current of 80pA and an ion source temperature of 80°C. Optical rotations were measured using a lO-cm microcell in a Perkin-Elmer 141 instrument. Sugar analysis 2mg polysaccharide material and D-arabinose (as internal standard) were hydrolysed in 3 ml 0.25 M H2S04 at 100°C for 14 h. The hydrolysate was neutralized with barium carbonate, filtered and 20mg sodium borohydride added to S ml clear solution. After 2 h at room temperature, Dowex 50 (H+) resin was added in excess and then removed by filtration, Boric acid was removed by repeated distillations with methanol. The residue was acetylated by treatment with acetic anhydride-pyridine, 1: 1, (v/v; 1 ml), for 15 min at 100°C and the resulting alditol acetates analysed by g.l.c,-m.s. (Sawardeker, Sloneker and Jeanes, 1965; Chizov, Golovkina and Wt.&on, 1966). Methylation analysis

1Omg polysaccharide material was dissolved in 2 ml methyl sulphoxide in a serum flask sealed with a rubber cap. Nitrogen was flushed through the bottle and 2 M methylsulphinylmethanide in 2 ml methylsulphoxide added. The gelatinous solution was agitated in an ultrasonic bath (40 kc/s) for 30 min and left at room temperature overnight. ‘2 ml methyl iodide was added drop by drop with external cooling and the resulting turbid solution was agitated in the ultrasonic bath for 30min, giving a clear solution. Excess methyl iodide was removed by distillation and the product (12 mg) recovered by dialysis and evaporation. The material was applied to a 25 x 2-cm Sephadex LH-20 column using chloroform-acetone (2: 1) as the irrigant. The eluate was monitored polarimetrically. The product (10mg) was eluted with the

void volume and 2 mg were treated with 1 ml 90 per cent formic acid at 100°C for 2 h, concentrated to dryness and hydrolysed with 3 ml 0.25 M H#O, at 100°C for 16 h. The resulting sugars were converted to alditol acetates by reduction with sodium borodeuteride followed by acetylation and analysed by g.l.c-m.s. (BjSrndal et al., 1970; Lindberg, 1972). Smith degradation of polysaccharide material from N. mucosa strain A 636 40mg polysaccharide material was oxidized in 200ml 0.1 M sodium metaperiodate in the dark and aliquot portions withdrawn for determination of reagent consumed (Aspinall and Ferrier, 1957). The reaction was complete after 30 h and excess periodate destroyed by addition of 5 ml ethanediol. The solution was dialysed for 24 h and the per&late-oxidized polysaccharide treated with 500mg sodium borohydride overnight. Dowex 50 (H+) resin with methanol was added and evaporated to dryness repeatedly. The resulting polyalcohol was methylated and the derivative analysed as above. The majority of the methylated derivative was concentrated by heating in acetic acid-water (1: 1, 5 ml) at 100°C for 1 h and the residue dissolved in dioxaneethanol (3: 1, 10 ml), 30 mg sodium borohydride added and the solution kept overnight. Dowex 50 (H’) resin was added, the solution filtered and the filtrate repeatedly concentrated with methanol. The residual polysaccharide was further alkylated with trideuteriomethyl iodide. The methylated product was, however, isolated by the partition between chloroform and water and not by dialysis. The chloroform phase was washed with water (4 x 20 ml) and evaporated to dryness. The modified methylated giucan was converted to partially-methylated alditol acetates and analysed by g.l.c.-m.s.

Gel chromatographic determination of mol. wt distribution 5 mg methylated polysaccharide were analysed for the mol. wt distribution by gel chromatography (Granath and Kvist, 1967) on Sephadex LH-60 gel, packed and run in chloroform. The column (SR25/40, Pharmacia Fine Chemicals, Uppsala, Sweden) was previously calibrated using a series of methylated dextran fractions of known mol. wt. The flow, about 13 g/h, was monitored by a peristaltic pump provided with Viton-tubing. 5-g fractions were collected by a time-programmed collector. After a completed run, the fractions were evaporated to dryness and the carbohydrate content analysed manually by measuring the colour developed at 495 nm with the anthrone reagent. The carbohydrate concentrations of the fractions were evaluated against a standard curve and converted to the corresponding mol. wt distribution by inserting the calibration and the primary data of the run into a computer program elaborated for mol. wt-distribution analysis. RESULTS The lyophilized material isolated from the Neisseria strains contained, on acid hydrolysis, ~ghtcose in approx 100 per cent yield. The optical rotation of the polysaccharide material from the 5 strains were

65

Polysaccharides of Neisseria Table 1. Methyl ethers from the hydrolysate of methylated polysaccharides from N. mucosa (strains A636, A630 and 5957) and N. subfiaua (strains Al82 and Ml107) Mole (per cent) Sugars*

Tt

A630

A636

A182

Ml107

5957

2,3,4,6-GlucoseS 2,4,6_Glucose$ 2,3,6-Glucose$ 2,3-GlucoseS

1.00 1.82 2.32 4.50

6 10 77 7

7 17 69 7

5 12 78 5

5 12 78 5

9 13 69 9

* 2,3.4,6-glucose = 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-~-glucose, etc. t Retention times of the corresponding alditol acetates relative to 1,5-di-Oacetyl-2,3,4,6-tetra-O-methyl-o-ghtcitol on an OV-225 column. 3 Deuterium in the l-position.

[x]:& = +150” + 5 (approx 0.5, 1 M KOH), which indicate that most of the sugar units are a-linked. Methylation analysis (Table 1) showed that the polysaccharide material contained mainly (1 +4)-linked cent), glucopyranose residues (69-78 per (l--+3)-linked glucose units (l&17 per cent) as well as a small proportion of branching (5-9 per cent). The modified Smith degradation implies that the polysaccharides consist of two components, one arnylopectin-type polymer (major part) and one a-(1 --+ 3) glucan. The average mol. wt after methylation of the polysaccharides was between 45,Ot%65,000 daltons. DISCUSSION

N. perJava isolated from the human throat produces a constitutive enzyme synthesizing a high mol. wt. glycogen-amylopectin-type a-glucan from sucrose (Hehre and Hamilton, 1948; Hehre, 1949; Okada and Hehre, 1974). This enzyme, amylosucrase (EC. 2.4.1.4), converts the glucose moiety from sucrose to a polymer, by transglycosylation, with the liberation of free fructose (Okada and Helire, 1974). There is no evidence that UDP-glucose or ADP-glucose helps form the glycogen-amylopectin product. The highlypurified amylosucrase behaves like the crude enzyme in synthesizing the polysaccharide material (Okada and Hehre, 1974) indicating that the crude enzyme preparations from the Neisseria strains used in our study are suitable as purified enzymes for detailed studies on the polysaccharides formed from the sucrose However, the two sucrase activities responsible for the cc-(1-+ 4)- and x-(1 -+ 3)-linked glucans could possibly be separated on a preparative scale. The sugar analyses, methylation analysis (Table 1) and the optical rotations, all imply that the material from all the strains consists mainly of an (I--+ J)-linked r-glucan, which agrees with Hehre (1949) and Barker, Bourne and Stacey (1950). The presence of some (1 -+ 3hlinked residues as well as branching in the 6-position makes it probable that the material consists of two polysaccharides, one amylopectin-type polymer and one cc-(1- 3Flinked glucan. As the cells and thereby water-insoluble polysaccharides sticking to the cells were discarded, it is likely that this dramatically changes the proportion

of the two glucans in favour of the (l-+3)-linked material. To investigate further the structure of the polysaccharides, material from strain A636 was subjected to a Smith degradation (Goldstein et al., 1965) in a modified procedure (Lindberg et al., 1973). The material was oxidized with sodium metaperiodate and resulting aldehyde groups reduced with sodium borohydride to alcohol groups and the resulting polymeric material methylated. Analysis of this material showed that, unlike the original methylation analysis, all 2,3,4,6-tetra-O-methyl-, 2,3,6-tri-o-methyland 2,3-di-O-methyl-n-glucose were destroyed and only 2,4,6-tri-O-methyl-D-glucose was present, showing that oxidation was completed. The modified methylated material was then treated with dilute acid to remove acyclic acetals. The partially-degraded methylated material was reduced and remethylated using trideuteriomethyl iodide as alkylating agent. The material was hydrolysed and the component sugars analysed in the usual way by conversion into partially-methylated alditol acetates. The analysis showed 2,4,6-tri-O-methyl-D-glucose as the main component and no 2,3,4,6-tetra-O-methyl-nglucose with CD,-labelling in the 3-position could be detected. This supports the hypothesis that the polysaccharide material from N. muqsa strain A 636, and consequently all polysaccharides, consists of two components, one amylopectin-type polymer (major part) and one cc-(1+ 3)-linked glucan. The polysaccharides studied here were isolated from broth cultures by use of high-speed centrifugation and the polysaccharide material was not soluble in water after purification. The mol. wt deterrninations were therefore performed after methylation and must be interpreted cautiously due to possible degradation during the ultrasonic treatment. Ritz (1970) suggested that Neisseria are important in the development of human dental plaque. Little is known about the characteristics of Neisseria from plaque, although Pike et al. (1962) described 85 strains from human saliva. However, Bowden, Hardie and Dunklin (1975) reported that Neisseria (60 strains) from dental plaque fell into two major groups, saccharolytic, polysaccharide-producing (38 strains), and asaccharolytic, non-polysaccharide-producing (15 strains). Obviously Neisseria represent only a small

66

D. Birkhed, K.-G. Rose11 and G. H. Bowden

part of the plaque flora (Bowden, Hardie and Slack, 1975), of which 50 per cent or more may produce extracellular polysaccharides from sucrose. a-(1 -+ 4)linked glucans have not been found either in plaque hydrolysates (Hotz, Guggenheim and Schmid, 1972) or in plaque extracts incubated with sucrose (Birkhed and RoseIl, 1975). This indicates that the enzyme synthesizing (1 -+ 4)-linked glucans from sucrose is low in activity in human dental plaque, or that the activity of other enzymes such as salivary a-amylase, hydrolysing this type of polysaccharide, is high.

Acknowledgements-The skilled technical assistance of Miss Birthe Abrahamsson is acknowledged. We are indebted to Dr. K. Granath for estimation of the molecular weights. This work was supported by grants from the Swedish Medical Research Council (B76-03X-02522-08), from H. J. Stiftelse, S. Sigurd and Elsa G. Minne and K. and Alice W. Stiftelse.

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