A factor from Actinomyces viscosus T14V that specifically aggregates Streptococcus sanguis H1

J Mizuno, J O Cisar, A E Vatter, P V Fennessey, F C McIntire
1983 Infection and Immunity  
A highly specific aggregation factor for Streptococcus sanguis Hi (AFR1) was obtained by lysozyme treatment of Actinomyces viscosus T14V. At 1 [.g/ml, AFH1 aggregated a suspension of S. sanguis Hi, with which A. viscosus T14V coaggregates by a mechanism not inhibited by lactose; even at much higher levels AFH1 caused little or no aggregation of streptococci from other coaggregation groups (J. 0. Cisar et al., Infect. Immun. 24:742-752, 1979). The most active fraction of AFH1 obtained by gel
more » ... obtained by gel chromatography (near the void volume of Bio-Gel A1.Sm) reacted as a single antigen with anti-A. viscoslus T14V serum and was unrelated to the fimbrial antigens of A. viscosus T14V. Smaller molecular fractions, at high levels, inhibited aggregation of S. sanguis HI by high-molecularweight AFH1 as well as coaggregation of S. sanguis Hi with A. viscosus T14V. The AFH1 fraction with high aggregating activity was composed of approximately 53% cell wall components (alanine, glutamine, lysine, N-acetylglucosamine, and N-acetylmuramic acid), 40% polysaccharide (N-acetylgalactosamine, rhamnose, and 6-deoxytalose), and 7% protein; teichoic acid was not detected. The fraction which inhibited aggregation and coaggregation contained much less of the cell wall constituents and more of the polysaccharide than the fraction with potent aggregating activity. Aggregation was completely prevented either by treating AFH1 with 0.01 M periodate at 25°C for 4 h or by treating S. sanguis Hi with heat or pronase. A role for electrostatic forces in the aggregation was indicated by: (i) NaCl inhibition of aggregation, and (ii) a great decrease in aggregation potency as a result of chemical modification of either cationic or anionic groups of AFH1. On the other hand, NaCl reversed the aggregation only very weakly. The overall data suggest that a carbohydrate-protein interaction may be dominant in the aggregation of S. sanguis Hi by AFH1 and in the coaggregation of S. sanguis Hi with A. viscosus T14V. The importance of specific interspecies surface reactions among bacteria in the development of dental plaque was implied in the artificial plaque model of Bladen et al. (2) and was postulated by Gibbons and Nygaard (13) who demonstrated numerous examples of interbacterial aggregation when specific pairs of bacteria were mixed together (coaggregation). Subsequently, there have been several contributions which have been reviewed previously (4, 7, 21, 23, 24). Many strains of Streptococcus sanguis from human dental plaque were reported to coaggregate with a high percentage of oral strains of t Present address: Department of Preventive Dentistry, Kagoshima University Dental School, Usuki-Cho, Kagoshima 890, Japan. Actinomyces viscosus and Actinomyces naeslundii, and both genera were separated into several groups based upon their coaggregation patterns (7, 21). These coaggregations are thought to depend on the reaction of a lectin on one cell with a carbohydrate on the other. Lactose inhibits all coaggregations in which the actinomycete carries a lectin for carbohydratecontaining receptors on certain streptococci. In contrast, this sugar generally does not inhibit those coaggregations in which the lectin-like component appears on the streptococcal surface with its receptor on the actinomycete. As nothing was known about the structure of the putative carbohydrate components in coaggregations not inhibited by lactose, we chose to investigate one of them. 1204 on May 4, 2020 by guest http://iai.asm.org/ Downloaded from A. VISCOSUS T14V. S. SANGUIS HI COAGGREGATION Previous studies (7, 21) showed that S. sanguiis Hi coaggregated with A. 0iscosus T14V and with most of the other oral isolates of A. liscosiis and A. naeslindii. In these coaggregations, the presence of a lectin on S. sanguis Hi was postulated, with heat-and protease-resistant molecules, presumably carbohydrate, on the actinomycetes. In this communication we describe the isolation and the partial characterization of a very potent factor (AFHI) from A. v'iscosus T14V which specifically aggregates S. sangiuis Hi, which appears to carry a putative carbohydrate that is essential in A. v'iscosus T14V-S. sanguis HI coaggregation and which accounts for one of the different receptors on A. viscosus T14V (20). MATERIALS AND METHODS Bacteria. Strains of bacteria used in this study are listed in Table 1 . Actinornvces WVa-963 strains EIJ-8 and D47B-27B are different from either A. iiScOSi(S or A. naesliindii (Paul E. Kolenbrander, personal communication). Two streptococcal strains, PK1317 and PK1318, were mutants of S. sanguis Hi with decreased ability to coaggregate with A. liscosius T14V (20). These four strains were kindly provided by Paul E. Kolenbrander (National Institute for Dental Research, Bethesda, Md.). Organisms were grown overnight in a medium described previously (7) under an atmosphere of 85% N.-10% H.-5% CO, with shaking. Harvested cells were suspended in 50% glycerol and kept at -20°C until used. Preparation of crude aggregation factor (AFHI). To the washed cell suspension of A. i'scosus T14V (approximately 2 mg/ml, dry weight) in 5 mM potassium phosphate buffer (pH 7.0), egg white lysozyme (grade VI; Sigma Chemical Co., St. Louis, Mo.) was added at 2 mg/ml. The reaction mixture, with 0.03% NaN3 as a preservative, was incubated at 36°C for 2, 4, 6, 8, and 24 h, and the aggregating activity of the supernatant and the coaggregating activity of the cell residue were measured. Incubation for 8 h gave the best yield of S. sanguiis HI aggregating activity in the supernatant and was therefore chosen for routine preparation of AFH1. After the incubation with lysozyme, A. v4iscosus T14V cells were removed by centrifugation; the cell-free supernatant was kept at 4°C, and solid trichloroacetic acid was added to 5% (wt/vol). After 1 h at 4°C, the precipitate was removed by centrifugation; the supernatant was adjusted to pH 7 and dialyzed against distilled water. The non-dialyzable part was lyophilized. A 72-liter volume of A. i'scosus T14V culture yielded 5.2 g of crude AFH1. Chromatography of AFH1. On a column (25 mm inner diameter by 900 mm) packed with Bio-Gel A1.Sm (Bio-Rad Laboratories, Richmond, Calif.), 150 mg of crude AFHI was applied, and the column was developed with 5 mM P04-50 mM NaCI-0.1 mM CaCI. (pH 6.5) buffer at 4°C. Each 6-ml fraction was analyzed for aggregating activity and methylpentose content.
doi:10.1128/iai.40.3.1204-1213.1983 fatcat:um442mldsreklbzqhs4md5om64