| Summary: | The nasopharynx is covered by mucin. Mucin is a glycoprotein and the carbohydrate moieties are potential fermentable substrates for Streptococcus pneumoniae. Previous in vitro studies showed that S. pneumoniae can grow on mucin. This study was undertaken to investigate how S. pneumoniae degrades the mucin carbohydrates to mono and disaccharides for subsequent fermentation. In silico search of pneumococcal genome identified fourteen putative glycosidases. Pneumococcal mutants of each were made and tested for growth in defined medium with mucin as the only source of carbon. Of these, two genes SPD0065 and SPD0247 were chosen for further study. Consequently two novel glycosidases were described, β-galactosidase (BgaC), encoded by gene SPD0065 and a 6-phospho-β-glucosidase (BglA) encoded by gene SPD0247. The knocked-out mutants SPD0065M and SPD0247M could not grow in Sicard’s defined medium supplemented with mucin and exhibited decreased enzymatic activity when compared to the wild type D39. Since gene SPD0562 had been previously identified as encoding a β-galactosidase (BgaA), the relative contribution of BgaA and BgaC to total β-galactosidase activity was investigated by introducing mutations in these genes individually and together. Mutation in the individual genes resulted in significant decrease in the enzymatic activity but the double mutation did not totally abolished activity. BgaC had specificity for galactose (β1,3)-N-acetylgalactosamine. Furthermore, BgaC released galactose from desialylated fetuin. The expression of SPD0065 and SPD0247 in S. pneumoniae grown in mucin containing medium or harvested from tissues from infected animals was significantly up-regulated compared to growth in glucose containing medium. When the mutants were tested in vivo, it was noted that SPD0065M had attenuated growth in the nasopharynx and SPD0247M in the lungs. In this study was demonstrated that BgaC has a role in the sequential deglycosylation of host glycoproteins and that BglA is involved in the further degradation of mucin-derived sugars.
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