Controlling Bacterial Amyloid Formation and the Influence of Curli Subunits on Pathogenic Alpha-synuclein Aggregation

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

Escherichia coli (E.coli) and other enteric bacteria are a major cause of human diseases. Biofilm formation contributes greatly to bacterial persistence and antimicrobial resistance in the host. A striking aspect of biofilms is the “division of labor” that drives unique subpopulation development. Many Enterobacteriaceae, such as E. coli, produce functional amyloid fibers called curli as a major proteinaceous component of their biofilm extracellular matrix. It is now clear that functional amyloids are widespread, with examples found throughout cellular life. The curli system in E. coli provides a rich and high throughput genetic and biochemical toolbox for the study of amyloid formation. The work has contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane-located CsgG. CsgB is anchored to the extracellular surface of the bacterium via the action of CsgF, whose ability to phase separate is tightly linked to its function in curli biogenesis. Soluble CsgA is targeted to the CsgG secretion channel by CsgE, then secreted. CsgA subunits that inappropriately polymerize in the periplasm are inhibited from amyloid accumulation via CsgE and CsgC. The exquisitely-controlled curli biogenesis system ensures that E. coli is not exposed to the potentially cytotoxic outcomes of amyloid formation. Work here will also explore how E. coli cells differentiate into transcriptionally distinct populations during biofilm development. Uncontrolled or inappropriate amyloid formation are the genesis of several neurodegenerative diseases, including Parkinson’s. Colonization of mice with curli amyloid producing bacteria results in alpha-synuclein amyloid formation and Parkinson’s-like symptoms. Furthermore, purified CsgA protein can accelerate alpha- synuclein amyloid formation in vitro. Therefore, it is imperative to learn how E. coli controls curli amyloid formation and how CsgA can accelerate alpha-synuclein amyloid formation. Interestingly, CsgC from E. coli can inhibit CsgA and alpha-synuclein amyloid formation. Knowledge gained from the following experiments will have implications for microbial pathogenesis, general protein folding, and amyloid biogenesis, thus paving the way for new therapies that rationally target these critical biological processes. To better understand curli biogenesis and develop therapeutics that target curli amyloid formation or function, the dynamic mechanism curli amyloid secretion/assembly will be determined using single molecule super-resolution microscopy and other biophysical techniques, that will reveal the location, order, and timing of the assembly of the large curli protein export machinery (Aim 1). Population development in amyloid-dependent biofilms will be studied. In Aim 2 CsgC protein chaperone and 2-pyridone molecules will be studied for their CsgA and alpha-synuclein polymerization inhibitory activities. Together the successful completion of these aims will give an overall understanding of the mechanism of amyloid inhibitory activity and the interactions between bacterial amyloid formation, biofilm development and neurodegenerative disease.