CONTRIBUTIONS OF FUNCTIONAL BACTERIAL AMYLOIDS TO THE RELEASE OF NEUTROPHIL EXTRACELLULAR TRAPS: CURLI PROTEIN OF E. COLI

Amyloid fibrils (AF) are composed of misfolded proteins organized in a β-cross structure and, although they are the hallmark of amyloid diseases, such as Alzheimer's and Parkinson's, they can also act as functional amyloids. Different bacteria secrete proteins, such as curli protein secreted by Escherichia coli, which can form AF that helps in the structuring and resistance of the biofilm, making it more refractory to the action of different agents and making it difficult to eliminate these bacteria. Neutrophil Extracellular Traps (NET) are DNA fibers decorated with antimicrobial proteins and, while they play an important role in immune response, they have already been associated with several diseases. Our group showed for the first time that human neutrophils when incubated with pathological AF, release NET in a manner dependent on NADPH oxidase 2 (NOX2) and that elastase present in NET fragments AF into toxic species. Now, our group aims to elucidate the role of the curli protein, a functional amyloid, in the release of NET. For this purpose, bacteria were stimulated or not to produce curli and characterized by binding to Congo Red, Transmission Electron and Fluorescence Microscopy. Human and NOX2 knockout mice neutrophils were stimulated with curli+ and curli- (knockout for curli protein) bacteria and NET release, ROS production, and NOX2 dependence were evaluated by fluorescent probes. The curli+ and curli- strains, when grown in conditions not favorable to curli production and biofilm formation (37°C, agitation), induced NET independently of NOX2 but accompanied by ROS production. When curli+ and curli- strains were grown in conditions favorable to curli production and biofilm formation (28°C, stagnant), we observed by immunocytochemistry that there was less NET induction in both strains, probably because in the biofilm form there is less neutrophil-bacteria contact. We are now seeking to elucidate the contribution of curli in the induction of NET.