Modification and Translocation of the Biofilm Exopolysaccharide Poly-β(1,6)-N-acetyl-D-glucosamine

Bacteria embedded within a biofilm are more resistant to antibiotics and host defenses than those in a planktonic state. Extracellular polysaccharides are a main component of biofilms and have been shown to play an important role in bacterial aggregation, surface attachment, and serve as a protective barrier against antibiotics and host defenses. A key exopolysaccharide required for biofilm formation by a number of pathogenic bacteria is poly-beta(1,6)-N-acetyl-D-glucosamine (PNAG). The modification of PNAG through deacetylation and/or hydrolysis by PgaB, BpsB, and IcaB is required for biofilm formation by Escherichia coli, Bordetella bronchiseptica, and Staphylococcus epidermidis, respectively. E. coli PgaB is a two-domain outer membrane lipoprotein that continually associates with PNAG throughout periplasmic translocation. Studies described herein suggest that PgaB binds and deacetylates PNAG along the cleft between the N- and C-terminal domains in a processive-like mechanism. The deacetylated PNAG polymer then wraps around PgaB to the C-terminal domain where it binds in a screw-like fashion, where it may get cleaved before being handed off to the outer membrane porin PgaA for export. BpsB is similar in structure and function to PgaB, but exhibits three mechanistic differences. First, BpsB does not require the C-terminal domain for deacetylation. Second, BpsB is not required for the export of PNAG, but is required for biofilm formation and its complex three-dimensional architecture in B. bronchiseptica. Third, the C-terminal domain displays significantly higher dPNAG hydrolase activity than PgaB, and we have identified key residues required for catalysis. The production, export, and modification machinery for PNAG biosynthesis differs in Gram-positive bacteria. Structural, functional, and mutagenesis data presented in this body of work for IcaB reveals a possible mechanism for its association with the membrane, the basis for deacetylation in Gram-positive bacteria by a single domain protein, and suggests that catalysis occurs through an altered enzymatic mechanism to known family 4 carbohydrate esterases. The structural and mechanistic data presented herein furthers our understanding of the critical modifications that occur to PNAG during biosynthesis. This work will be instrumental in developing strategies to prevent or remove PNAG-dependent biofilms for the treatment of biofilm-related infections.Ph.D.2017-06-30 00:00:0