Isolation and analysis of sialidase-defective mutants of streptococcus oralis

Date
1998
DOI
Authors
Nalbantoglu, Eser Rengin
Version
OA Version
Citation
Abstract
Streptococcus sanguis, Streptococcus oralis and Streptococcus mitis are among the earliest colonizers of the tooth surface during plaque formation. In addition, these species are also associated with extraoral infections including infective endocarditis. Sialidases, enzymes that cleave sialic acids from carbohydrates and glycoproteins, are believed to play a role in plaque formation and extraoral infections by oral streptococci. Previously, a sialidase gene was isolated and sequenced from S. oralis C104. The gene is homologous (>70% identity) to the nanA gene from Streptococcus pneumoniae. In this study, isogenic sialidase- defective mutants were constructed by allelic exchange. A streptococcal erythromycin resistance cassette from pBSem [superscript r] inserted into a unique Hpal site at nucleotide 1233 within the sialidase coding region. This plasmid was used for electroporation of S. oralis C104. Erythromycin-resistant colonies were isolated and tested for sialidase expression using fluorogenic substrate, 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (MUN). One sialidase-defective mutant, designated S. oralis EU68, was isolated and Southern hybridization was used to confirm the insertion of the erythromycin resistance cassette within the sialidase gene. The production of sialidase activities by S. oralis C104 was investigated using MUN. Growth in a minimal medium supplemented with glucose (Glc) resulted in suppression of sialidase activity. In contrast, S. oralis C104 exhibited higher levels of sialidase activity when the medium was supplemented with porcine gastric mucin (PGM), a model glycoprotein. S. oralis C104 and its isogenic sialidase-defective mutant, S. oralis EU68 grew little with PGM as a sole carbon source. Similarly, S. oralis C104 and EU68 showed poor growth in minimal medium supplemented with N-acetylneuraminic acid (NANA). Glucose (Glc), glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) supplemented minimal medium supported the growth of both S. oralis C104 and EU68. These data suggest that growth of bacteria does not dependent of sialidase production. S. oralis C104 grown with PGM and NANA exhibits increased levels of sialidase activities which may play a role in the virulence of this organism and explain its predominance in extra-oral diseases.
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Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1998 (Pediatric Dentistry).
Includes bibliographical references: (leaves 92-121).
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