Characterization of WssF; a putative acetyltransferase from Achromobacter insuavis and Pseudomonas fluorescens

Biofilms are a survival mechanism commonly employed by communities of bacteria for adherence and protection. Bacteria produce a matrix of polymers (e.g. exopolysaccharides, such as cellulose) that allow them to exert control on their local environment. In the case of cellulose biofilms, acetylation (addition of acetate on carbohydrates) is paramount for polymer integrity and in some cases virulence. For this research, the wrinkly spreader (WS) genotype of the emergent human pathogen Achromobacter insuavis facilitates infections of the eyes of contact lens wearers and the lungs of Cystic Fibrosis patients (CF). Chronic infections have created a growing concern for the protective role biofilms play in antimicrobial resistance. Cellulose biofilms are an important mechanism for other pathogenic bacteria such as Pseudomonas fluorescens, which affect produce and water sources by forming a robust cellulose-containing biofilm that enhances the ability of the bacteria to spread across surfaces. Both A. insuavis and P. fluorescens contain the wssF gene that encodes a putative O-acetyltransferase protein, in that site-specific mutagenesis of wssF resulted in non-acetylated cellulose. The purpose of this research was to characterize WssF from both species, to understand its role in the production of acetylated cellulose and investigate its role as a putative O-acetyltransferase protein. This research progressed successfully through three primary objectives; expression and purification of WssF, functional characterization of acetyltransferase activity and structural characterization of the WssF protein. Esterase activity, the first step in acetyl transfer, was successfully characterized through a kinetic workup, which found KM(2.88 – 7.18 mM), kcat (0.24 – 0.34 s-1) and, Vmax (47 – 83 s-1*M-1) values comparable to homologous acetyltransferases. Following this, acetyl transfer was confirmed through the use of Thin Layer Chromatography (TLC) and Mass Spectrometry, both mono and diacetylated cellopentaose was produced in the presence of unmutated WssF. Site Directed Mutagenesis (SDM) of the predicted catalytic triad residues (Ser, Asp and, His) resulted in up to 85% inhibition of esterase activity and at least 95% of acetyl transfer thereby confirming their role in catalysis. Successful functional characterization of WssF has allowed for screening of potential enzyme inhibitors by High Throughput Screening (HTS) assays. A set of 18 compounds has been selected for further study as potential WssF acetyltransferase inhibitors. Finally, structural characterization of WssF has been advanced, with optimal conditions for crystallization having been determined and (~2Å) data sets collected but solving a structural model of WssF is ongoing. The findings of this research successfully outline the functional and structural characteristics of WssF, which are the first for a cellulose acetyltransferase involved in biofilm formation. This thesis further develops our understanding of biofilm modification, a vital step in combating antimicrobial resistance