Using Biofilm Exopolysaccharide Hydrolase Enzymes as Investigative Tools to Decipher the Role of Exopolysaccharides in Biofilm Formation

Abstract

Biofilms pose a formidable challenge in healthcare settings by impeding the host immune response, blocking decontamination efforts, and inhibiting conventional antibiotic treatments. Hydrolysis of biofilm exopolysaccharides by glycoside hydrolase (GH) enzymes is an established strategy for biofilm disruption. Dispersin B (DspB) catalyzes the hydrolysis of poly-β-(1→6)-N-acetyl-D-glucosamine (PNAG), a fundamental exopolysaccharide in both Gram-positive and Gram-negative bacteria, has garnered attention as a potential therapeutic agent for biofilm dispersal. Despite its promising potential, the application of DspB is limited by its relatively low catalytic activity compared to other GH enzymes. Previous studies have shown that this reduced efficiency is primarily due to suboptimal substrate recognition and binding, indicating the need for further protein engineering. This knowledge gap highlights the necessity for an effective, high-throughput screening method to guide the engineering of improved DspB variants.In this study, we developed a high-throughput screening assay based on bacterial cell surface display to evaluate the activity of DspB (Chapter 2). This designed fusion protein construct with ice nucleation protein (InaP) and enhanced green fluorescent protein (eGFP) enabled quantification of surface displayed DspB via fluorescence while preserving enzymatic function (2.2.1). The expression and localization of the fusion protein were confirmed, and enzymatic activity assays demonstrated that the displayed DspB retained its hydrolytic function (2.2.3). A strong correlation was observed between IPTG induction levels, eGFP fluorescence intensity, and enzymatic activity, validating the system's reliability (2.2.4). To assess the applicability of the assay in a biologically relevant context, the method was further tested using Staphylococcus epidermidis biofilms. The surface-displayed DspB effectively dispersed PNAG-dependent biofilms, and results were supported by statistical analysis (2.2.5). In addition to PNAG, several bacteria produce additional biofilm exopolysaccharides, but there is little information on their interactions or spatial distribution. To address the challenge of studying heterogeneous exopolysaccharides in E. coli biofilms, we developed catalytically inactive GH enzyme-based probes that bind phosphoethanolamine (pEtN)-modified cellulose (Chapter 3-4). A colorimetric reducing-end assay was first established and optimized to quantitatively assess the catalytic activity of BcsZ, an E. coli endoglucanase enzyme, across different substrates, pH ranges, and enzyme concentrations. Site-directed mutagenesis was used to generate BcsZD243A-moxBFP and BcsZE55A,D243A-moxBFP fusion protiens, the latter of which was found to have a complete loss of catalytic activity and was selected for further studies. Fluorescence microscopy revealed that this probe effectively binds cellulose-rich regions in native biofilms formed by the E. coli strain AR3110 (chapter 4). In sum, this thesis has explored novel strategies to screen the activity of hydrolase enzymes, particularly through the development of a high-throughput screening platform targeting the Dispersin B protein. Alongside this, new investigative probes derived from bacterial hydrolase enzymes (Bacterial cellulose synthase Z) were designed to deepen our understanding of E. coli biofilm architecture. Together, these advancements not only provide powerful methodologies for studying and manipulating biofilm structures but also open up promising avenues for the disruption and control of biofilm-related bacterial infections.