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Abstract
A prominent feature contributing to pathogenicity within the Streptococcus genus is the involvement of surface glycans, including capsular polysaccharides (CPS), cell wall associated polysaccharides and O-glycosylated proteins. In general, these carbohydrates are utilized to block immune detection, serve as decoys or aid in host colonization. In this thesis, we employed S. pneumoniae (major cause of bacterial pneumonia) and S. mutans (primary etiological agent for caries) to better understand the importance of bacterial glycosylation among Streptococci. The human host produces a poorly studied immune protein, human intelectin-1 (hIntL-1), that has been shown to bind the CPS of certain S. pneumoniae serotypes. Investigation of S. pneumoniae interactions with hIntL-1 showed that hIntL-1 causes bacterial agglutination but does not kill bacteria directly or in complement-dependent manner. Instead, binding of this lectin leads to increased bacterial attachment to host cells, a phenomenon that has previously been described for Streptococcal strains capable of forming long bacterial chains. However, in the presence of fresh human blood cells, hIntL-1 causes enhanced killing of S. pneumoniae through phagocytosis specifically by neutrophils. In this study, I demonstrated how a bacterial surface structure typically used for the evasion of host defenses, could in turn be targeted by a specialized innate immune lectin. In S. mutans, the Pgf glycosylation machinery has recently been described to contribute to the pathogenicity of the organism in various ways. Using a truncated version of the Pgf target protein Cnm, we identified its glycosylation sites that are heavily modified with HexNAc2. We further characterized the key Pgf enzyme involved in HexNAc biosynthesis, PgfE. Computational modeling, NMR and capillary electrophoreses confirmed that PgfE is an NAD+ dependent UDP-GlcNAc/UDP-GalNAc 4-epimerase. The second 4-epimerase expressed by S. mutans, GalE, is required for galactose metabolism, but not protein glycosylation. The loss of GalE leads to defects in cell division and septum formation, presumably due to the loss of glucose which is one of the main building blocks of the S. mutans cell wall polysaccharide. These results not only provided new insights into protein O-glycosylation in S. mutans, but also demonstrate the importance of a single epimerase for proper cell division.