D-104. A Cell Associated Pneumococcal Gycosidase Modifies O-linked Glycoconjugates
Streptococcus pneumoniae is an important human pathogen responsible for more than a millions deaths per year worldwide. Increasing evidence suggests that the ability of S. pneumoniae to manipulate sugars contributes to colonization of the human airway. We have previously demonstrated that S. pneumoniae can modify N-linked glycans and that this may contribute to pneumococcal colonization in a number of ways including growth, affecting function of host defense molecules, and adherence of the bacteria. It is not known, however, if S. pneumoniae can modify O-linked glycans, like those which decorate mucin and are common in the airway. It has previously been reported that S. pneumoniae expresses a secreted O-glycosidase that cleaves Galactose 1-3 linked to N-acetylgalactosamine; however, the gene encoding this activity and the contribution of this enzyme to pneumococcal pathogenesis are as yet unknown. Using a colorimetric assay, we determined that O-glycosidase enzyme activity is not secreted but is cell associated in a sortase-dependent manner. Analysis of the predicted sortase-dependent surface associated proteins in the pneumococcal genome resulted in identification of a candidate gene. An unmarked non-polar mutant, 1121Δogly, was generated and lacked O-glycosidase activity against the colorimetric substrate compared to the parental strain. Furthermore, we demonstrated using lectin blots that 1121Δogly lacks that ability of the parental strain to deglycosylate the O-linked glycans of model glycoproteins and that this ability can be complemented by the addition of purified enzyme. Experiments are underway to determine the contribution of this enzyme to pneumococcal colonization and disease specifically growth on and movement through mucin. In conclusion, we have identified an O-glycosidase gene and demonstrated that a mutant at this locus is unable to deglycosylate O-linked glycans.