Development of a new bead movement based computational framework shows bacterial amyloid curli reduces bead mobility in biofilms.

Biofilms exist in complex environments including the intestinal tract as a part of the gastrointestinal microbiota. The interaction of planktonic bacteria with biofilms can be influenced by material properties of the biofilm. During previous confocal studies, we observed amyloid curli-containing Salmonella Typhimurium and Escherichia coli biofilms appeared rigid. In these studies, Enterococcus faecalis, which lacks curli-like protein, showed more fluid movement. To better characterize the material properties of the biofilms, a 4D model was designed to track the movement of 1 μm glyoxylate beads in 10-20 μm thick biofilms over approximately 20 minutes using laser-scanning confocal microscopy. Software was developed to analyze the bead trajectories, the amount of time they could be followed (trajectory lifespan), the velocity of movement, the surface area covered (bounding boxes) and cellular density around each bead. Bead movement was found to be predominantly Brownian motion. Curli-containing biofilms had very little bead movement throughout the low and high-density regions of the biofilm as compared to E. faecalis and isogenic curli mutants. Curli-containing biofilms tended to have more stable bead interactions (longer trajectory lifespans) than biofilms lacking curli. In biofilms lacking curli, neither the velocity of bead movement nor the bounding box volume was strictly dependent on cell density, suggesting other material properties of the biofilms were influencing the movement of the beads and flexibility of the material. Taken together these studies present a 4D method to analyze bead movement over time in a 3D biofilm and suggest curli confers rigidity to the extracellular matrix of biofilms.Importance Mathematical models are necessary to understand how material composition of biofilms can influence their physical properties. Here we developed of 4D computational toolchain for the analysis of bead trajectories which laid the groundwork for establishing critical parameters for mathematical models of particle movement in biofilms. Using this open source trajectory analyzer, we determined that the presence of bacterial amyloid curli changes the material properties of a biofilm making the biofilm matrix rigid. This software is a powerful tool to analyze treatment- and environment-induced changes in biofilm structure and cell movement in biofilms. The open source analyzer is fully adaptable and extendable in a modular fashion using VRL-Studio to further enhance and extend its functions.