Interspecies Cross-seeding Between Curli Subunits and Curli-dependent Pellicle Biofilm Development by Escherichia coli.

Abstract

The Earth’s bacterial population is primarily found in heterogeneous communities called biofilms. Bacteria in biofilms are enclosed in a matrix composed of proteins, DNAs, and polysaccharides. Under laboratory conditions, enteric bacteria form two distinct types of communities: rugose colony biofilms that grow on plates and pellicles that float at the air-liquid interface in static liquid culture. Formation of both types of biofilms is dependent on a class of surface structure called curli. Curli are structurally and biochemically similar to amyloids, which are historically linked with neurodegenerative conditions. Curli are the prototypical ‘functional amyloid’, where the amyloid fold is utilized as an important part of normal cellular physiology. Functional amyloids are being rapidly discovered and occur in all walks of cellular life.

My work focuses on the interspecies interactions of curli subunits and the development of curli-dependent biofilms. Like disease-associated amyloids, curli subunits self-assemble into an amyloid with nucleation-dependent kinetics. The polymerization can be promoted by preformed curli fibers in a process known as seeding. Seeding is highly specific and cross-species seeding is typically limited. I found that curli subunits from E. coli, Salmonella typhimurium LT2, Citrobacter koseri, and a distantly related homolog from Shewanella oneidensis, were able to cross-seed in vitro, suggesting seeding specificity among curli homologs is less stringent. Cross-seeding of curli proteins was also observed in mixed colony biofilm with E. coli and S. typhimurium. Remarkably, interspecies curli assembly promoted bacterial surface attachment and facilitated pellicle formation.

Biofilm development is a dynamic process. Bacteria produce antibiofilm signals as the culture ages. I found conditioned media of uropathogenic E. coli (UPEC) contained a factor that impeded its own pellicle formation. The antibiofilm activity was dependent on cysteine/cystine metabolism, as cysteine auxotrophs were unable to produce the antibiofilm factor and exogenous cysteine or cystine restored the antibiofilm activity. Taken together, both structural and metabolic components are involved in biofilm regulation. Curli function as a structural signal to promote interspecies community formation whereas a cysteine-dependent product prevents biofilm development. This work is the first study on the seeding specificity of bacterial functional amyloids, and also sheds light on biofilm regulations.