Master of Science (MSc)
Faculty of Science
Dr. Joel Weadge
Cellulose biofilms consist of a community of bacterial cells encased in a self-produced matrix of polymers (e.g. exopolysaccharides, such as cellulose) that facilitate a firm adherence to surfaces. The acetylation (addition of an acetyl group on carbohydrates) is crucial in virulence; thereby in some cases allowing opportunistic bacteria to cause harmful diseases. Pertaining to this research, the wrinkly spreader (WS) genotype of Pseudomonas fluorescens colonizes the air-liquid interface on food, water sources and human tissue to form a robust biofilm with the ability to spread across surfaces. The composition of this biofilm largely consists of bacterial cellulose polymers produced and acetylated by the proteins encoded by the wssoperon. Specific mutations of the wssFGHI genes result in the production of a non-acetylated cellulose-containing biofilm variant, which is not as adherent to surfaces. This is important as other pathogenic bacteria also contain the wss acetylation operon, such as the emergent human pathogen Achromobacter insuavis. In relation to the proposed project, the pathogens P. fluorescensand A. insuavis contain the wssI gene that is proposed to encode for an O-acetyltransferase protein, WssI. The purpose of this research was to characterize WssI from both pathogens, to understand its role in the production of acetylated cellulose and investigate its predicted role as an O-acetyltransferase protein. All research objectives were achieved including optimizing the purification of WssI, and functionally characterizing the esterase activity, optimal pH, and kinetic parameters. While acetyltransfer could not be confidently detected under conditions tested in activity assays, WssI did exhibit weak binding affinity of avicel initially detected in pulldown assays and low levels of acetyltransfer were further verified by mass spectrometry with pNP-Ac and acetyl-CoA as donors. In order to extrapolate information about the mechanism of action, the conserved active site residues were studied in a series of experiments. Consistent with other close homologs of published acetyltransferases, typical serine protease/esterase inhibitors did not lead to significant reductions in activity; however, site directed mutagenesis of the genes encoding two of the predicted catalytic triad residues (Ser and His) resulted in up to 50% inhibition of activity dependent upon acetyl donor. Lastly, WssI was tested against potential enzyme inhibitors by High Throughput Screening (HTS) assays. A small subset of the finalized list of 12 compounds successfully inhibited WssI in the micromolar range, one of which is a commercial tyrosine kinase inhibitor, implying that blockage of the ester cleavage may inhibit the overall activity of WssI. The findings of this study outline the functional properties of WssI, which have never been studied in vitro from any group in the literature. This thesis therefore contributes novel information in regard to the Wss system for the purpose of advancing biofilm research for further development of anti-acetylation or anti-biofilm agents in vivo.
Burnett, Alysha, "Functional Characterization of O-Acetyltransferase WssI: Role in Bacterial Cellulose Acetylation of Achromobacter insuavis and Pseudomonas fluorescens" (2019). Theses and Dissertations (Comprehensive). 2136.