Cysteine Metabolism Regulates Pellicle Biofilm Development by Uropathogenic Escherichia coli and the In Vitro Characterization of CsgA Amyloid Formation

Abstract

Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections. These bacteria live in complex heterogenous communities called biofilms. UPEC biofilms are encapsulated in a matrix composed of proteins, exopolysaccharides, and DNA. These biofilms are responsible for a wide variety of functions including protecting the bacteria against physical and chemical stresses. The UPEC strain UTI89 produces biofilms under different environmental conditions in the lab. In static cultures UTI89 forms pellicles at the liquid-air interface and it forms wrinkled colony biofilms on solid agar plates. The major proteinaceous component of these biofilms is a functional amyloid structure called curli. This work focuses on the development of curli-dependent biofilms and developing methods of characterizing the interactions of curli with disease associated proteins. In the first project, I established that pellicle biofilm formation can be regulated metabolically. I found that aged cultures of UPEC produced a potent pellicle biofilm inhibitor. Therefore, bacteria-free conditioned media from aged cultures of UTI89 impeded UTI89’s own pellicle formation. UTI89 grown in bacteria-free conditioned media from an aged UTI89 culture exhibited deficient pellicle formation. The antibiofilm effect was dependent on cysteine metabolism. UPEC mutants in the cysteine biosynthesis pathway were able to form pellicles in conditioned media. Interestingly, cysteine metabolism mutants, including a cysE deletion strain, were able to make curli amyloids in conditioned media. The addition of exogenous cysteine restored the antibiofilm activity. In a second project, I established a total internal reflection fluorescence microscopy assay to visualize bacterial amyloid formation in real time. The ability of curli to self-assemble into fibril structures with nucleation dependent kinetics is a process which has primarily been investigated using bulk assays. How this fibril formation is initiated and how amyloids of one type can template the formation of amyloids of another type are questions which have been poorly studied to date. I developed a method that allows the measurement of amyloid assembly in curli and allows us to investigate the interactions between the functional amyloid curli and disease associated proteins such as alpha-synuclein at the single molecule level. This work elucidates mechanisms of biofilm regulation as well as the biophysical role that components of the biofilm play in triggering other diseases and therefore, on overall health.