INVESTIGATION OF POLY-N-ACETYL GLUCOSAMINE BIOSYNTHESIS INITIATION AND SPATIAL DISTRIBUTION IN BACTERIAL BIOFILMS THROUGH MICROFLUIDIC FLUORESCENT MICROSCOPY

Abstract

Bacterial cells surface is rich in diverse polysaccharides that encapsulate the cellsinvolved in crucial roles such as regulating cell permeability, mediating surrounding interactions, and offering protection. Key classes of bacterial surface polysaccharides include teichoic acids (TAs), lipopolysaccharides (LPS), capsular polysaccharides (CPS), and exopolysaccharides (EPS). These surface polysaccharides are predominately synthesized by three conserved pathways: the Wzx/Wzy-dependent pathway, the ATP-binding cassette (ABC) transporter- dependent pathway, and the synthase-dependent pathway. The initiation of polysaccharide biosynthesis in the Wzx/Wzy-dependent and ABC transporter pathways is well documented to employ a lipid initiator substrate. However, the initiations of synthase-dependent systems have yet to be eluted, with speculation of undergoing a “self-initiating” mechanism by employing a free monosaccharide as the first acceptor molecule. Here, we investigate the initiation of poly-β-D-(1→6)-N-acetyl-glucosamine (PNAG) in Gram-negative bacteria through synthase-dependent pathway via PgaCD complex. PgaCD is a transmembrane heterodimer within the glycosyl transferase (GT) 2 family, catalyzing the transfer of GlcNAc residues from activated sugar nucleotides onto an acceptor substrate. We begin by isolating active PgaCD complex and measuring its activity in vitro (Chapter 2). First, we employ an expression system that successfully incorporates both proteins within the same membrane bilayer and demonstrated positive enzymatic activity through the hydrolysis of UDP from its cognate substrate UDP-GlcNAc (2.2.1). Additional 13C NMR analysis of in vitro product confirmed the polymerization of PNAG and revealed chemical shifts characteristic of unsaturated lipids (2.2.4). Localization experiments of the de novo PNAG demonstrated membrane association mechanism after polymerization suggesting possible interactions with the membrane lipids (3.2.1). Furthermore, high concentrations of free GlcNAc residues had no effect on enzymatic activity of PgaCD, ruling out its role as a possible initiator substrate (3.2.2). Taken together, the evidence suggests that the PgaCD complex may employ a primer-dependent initiation strategy analogous to other surface polysaccharide biosynthetic systems. Stratification of biofilms could impact how EPS is spatially distributed throughout; thus, it is imperative to study biofilms in situ, without disrupting their architecture. By utilizing a microfluidic device with an integrated cell seeding zone, it allowed for simultaneous cultivation, labeling, and dispersal monitoring of bacterial biofilms (4.2.1). Through using this platform, we compared PNAG distributions in biofilms produced by different bacteria (4.2.3). Dispersal by PNAG hydrolysis using DspB wt revealed alternative cohesion profiles between the two species, showcasing the versatility of the microfluidic platform for simultaneous cultivation, imaging, and targeted dispersal of diverse bacterial biofilms.