Amyloid like fibers-formation, structure, and disruption

Biofilms are surface-aggregates of microbial cells that secret extracellular biopolymers, mainly proteins and polysaccharides that form an intercellular network, termed the extracellular matrix (ECM).The ECM is related with the increased resistance of biofilms to antibiotic activity, and even though it can serve as a target for anti-biofilm drugs, little is known about its composition and structure. In our model organism for biofilm formation, Bacillus subtilis, TasA is the major protein component of the ECM. It aggregates to form amyloid-like fibers but their mechanism of formation and structure has not yet been determined. In a systematic study of TasA aggregation, we examine the effect of protein and salt concentration, as well as pH in solution on the aggregation kinetics and on the structure of the fibers. In parallel to the kinetic and structural analysis of the aggregation process, we also study their disruption by common enzymes and surfactants. To achieve these goals, we use a range of biochemical and biophysical tools, including Atomic Force Microscopy (AFM), gel electrophoresis, wide and small angle scattering, and kinetic fluorescence measurements (using the amyloid binding dye, thioflavin T). We have found that the aggregation of TasA is indeed affected by parameters such as salt and pH. Preliminary results show that TasA fibers are structured, however unlike classical amyloid fibers they can be disrupted by enzymes and surfactants. Our study spans the aggregation mechanism of TasAin vitrounder different environmental conditions and it will be useful in the design of drugs that will inhibit biofilm formation by interaction with its extracellular amyloid-like fibers.