Neuraminidase Activity in Mixed Culture Supernatant Fluids of Human Oral Bacteria.

Abstract

In view of the finding (Thonard and Blustein, J. Dent. Res., in press, 1965) that sialic acid is present in human gingival mucosa, it was decided to investigate the possibility that members of the human oral microflora might elaborate an enzyme able to attack the sialic acid-containing compounds thought to be present in the gingiva. The present report concerns the finding that neuraminidase is produced by members of the human oral microbial flora. Samples of subgingival detritis were collected with a Gracy Curette from young adults (20 to 40 years old) presenting clinical evidence of isolated areas of chronic marginal gingivitis. The scrapings from each patient were inoculated into separate screw-capped tubes containing 12 ml of Todd-Hewitt broth, and were incubated aerobically and anaerobically in 13rewer jars for 48 hr at 37 C. They were then centrifuged at room temperature for 15 min, and each supernatant fluid was divided into two 5.0-ml portions. One portion was immersed in a boiling-water bath for 30 min to inactivate any enzymes present, and served as a paired control for each sample. To detect the liberation of sialic acid from mucoprotein under the action of a neuraminidase in the cultures, an adaptation of the method employed by Rosenberg, Binnie, and Chargaff (J. Amer. Chem. Soc. 82:4113, 1960) was employed. To all culture supernatant fluids, 4 mg of powdered crystalline bovine orosomucoid (obtained from Pentex Inc., Kankakee, Ill.) and a crystal of thymol were added, and each tube was incubated at 37 C in a water bath for 2 hr. Data supplied by the manufacturer on the carbohydrate composition of the bovine orosomucoid were as follows: 14 to 15% hexose, 11 to 12% hexosamine, and 12.5 to 14.5% sialic acid. To demonstrate that this substrate was susceptible to the action of known neuraminidases, 0.1 ml (10 units) of Vibrio cholerae neuraminidase (obtained from General Biochemical Corp., Chagrin Falls, Ohio) was added to tubes containing Todd-Hewitt broth and orosomucoid and treated as described above. After 2 hr of incubation with substrate, neutralization was accomplished with NaOH, and 0.5-ml samples were assayed for the presence of free sialic acid in the form of N-aeetylneuraminic acid (NANA; Aminoff, Biochem. J. 81:384, 1961). As the Todd-Hewitt broth provided a significant background color with the Aminoff technique, a standard curve for NANA was lprepared with Todd-Hewitt broth as the solvent. Under these conditions, it was found that only in concentrations in excess of 10 Mg of NANA was it possible to obtain sensitive optical-density measurements. It was therefore decided to add 10 ,g of NANA to all tubes immediately before assaying for any additional enzyme-released NANA. A total of 30 gingival samples were testedl as described for the presence of neuraminidase under both aerobic and anaerobic conditions; representative data for the aerobic cultures are prlesented in Table 1. The amount of NANA released from the bovine mueol)rotein by the mixed culture supernatant flui(ds ranged from 1 to 20 Ag

1 Figure or Table

Cite this paper

@article{Thonard1965NeuraminidaseAI, title={Neuraminidase Activity in Mixed Culture Supernatant Fluids of Human Oral Bacteria.}, author={J. C. Thonard and C M Hefflin and Avital Steinberg}, journal={Journal of bacteriology}, year={1965}, volume={89}, pages={924-5} }