INTERVENING IN FUNCTIONAL AMYLOID ASSEMBLY/DISASSEMBLY TO OVERCOME BIOFILM-DRIVEN MICROBIAL RESISTANCE

Introduction: Functional amyloids, such as the major subunit CsgA of curli fibers produced by the Escherichia coli, exemplify beneficial amyloidogenesis essential for biofilm formation and microbial survival. These extracellular β-sheet-rich fibrils reinforce biofilm structure, increasing resistance to antimicrobials and immune defenses. Modulating the molecular processes of CsgA assembly and disassembly is vital to developing novel anti-biofilm strategies addressing antimicrobial resistance (AMR). Objectives: This study aims to (1) identify small molecules that modulate CsgA amyloidogenesis; (2) quantitatively analyze their effects on fibrillation kinetics; and (3) elucidate compound-induced structural changes to guide rational drug design. Methods: Recombinant CsgA was expressed in E. coli BL21 using a pET-28 vector without affinity tags and purified under denaturing conditions. Amyloid formation kinetics were monitored by Thioflavin T (ThT) fluorescence. Circular dichroism (CD) spectroscopy tracked secondary structure transitions, confirming β-sheet enrichment during fibril formation. Results and Discussion: Preliminary results show reproducible ThT kinetics typical of CsgA amyloid assembly. CD spectra indicate progressive β-sheet increase aligning with aggregation phases. These findings provide a solid foundation for screening amyloid modulators and investigating their mechanistic impact on fibril architecture. Conclusion: This integrative biophysical approach advances understanding of functional amyloid assembly and offers promising routes for targeted disruption of biofilms to combat AMR. Funding: Supported by doctoral scholarships and grants from São Paulo Research Foundation (FAPESP; Proc. 2022/06006-0) and CAPES