Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates.

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Amyloids are proteinaceous aggregates, the deposition of which is associated with neurodegenerative diseases, such as Alzheimer's disease. In vitro protein aggregation requires high protein concentration, which is generally far from physiological concentration. Here, we utilize the interfacial properties of liquid crystals (LCs) to monitor the membrane-induced aggregation of a bacterial functional amyloid, curli, at nanomolar concentration. The binding event triggers an orientational transition of the LC, which is accompanied by the appearance of dynamic spatial patterns enabling sensitive detection of lipopolysaccharide (LPS)-mediated protein aggregation. Quantification of LC response shows a sigmoidal time profile, typical of a protein fibrillation assay. Curli is composed of two subunits (CsgA and CsgB) and is expressed on the outer membrane of Gramnegative bacteria containing LPSs endotoxin. CsgA forms the major subunit of curli, which is nucleated by the membranetethered minor subunit CsgB. Using an array of complementary tools, such as polarizing optical microscopy, fluorescence, and atomic force microscopy imaging, we found that the patterned orientation of the LC in response to the binding of curli subunits with LPS corresponds to amyloid fibril formation. Furthermore, using the curli amyloid system, we have successfully demonstrated that membrane-decorated interfaces of LC can be used to study heterotypic cross-seeding in amyloidogenesis. We believe that the LC-based system can be used as a probe to monitor mechanistic details of lipid-induced protein aggregation in the low-concentration regime.

The prion determinant of a yeast prion protein, Sup35NM, assembles into β-rich amyloid fibrils that switch the nonprion [psi] state to the prion [PSI] state of yeast. Previous studies showed that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4 and 37 °C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI] phenotype, whereas Sc37 shows a weak phenotype. To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took advantage of the nonoccurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed a conformational mobility map to be constructed. Nearly all of the residue positions demonstrated higher local flexibility in Sc4 amyloid, which exhibits a strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain than in the central region. The M-domain is partially exposed and exhibits a higher amplitude of local mobility, indicating a lower degree of chain packing in the amyloid state, as well as a higher mobility in the Sc4 state compared to the Sc37 state. The altered local conformational dynamics in these two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance patterns of strong and weak prion strains.

Curli is a biofilm-forming amyloid that is expressed on the surface of Gram-negative enteric bacteria such as Escherichia coli and Salmonella spp. Curli is primarily composed of the major structural subunit, CsgA, and interacts with a wide range of human proteins that contribute to bacterial virulence. The adsorption of curli onto the contact-phase proteins and fibrinogen results in a hypocoagulatory state. Using an array of biochemical and biophysical tools, we elucidated the molecular mechanism of interaction between human fibrinogen and CsgA. Our results revealed that a substoichiometric concentration of fibrinogen delays the onset of CsgA aggregation by inhibiting the early events of CsgA assembly. The presence of fibrinogen prevents the maturation of CsgA into fibrils and maintains the soluble state of CsgA. We also demonstrate that fibrinogen interacts more effectively with the disordered conformational state of CsgA than with the ordered β-rich state. Our study suggested that fibrinogen is an anti-curli protein and that the interplay of CsgA and fibrinogen might be a host defense mechanism against curli biogenesis, biofilm formation, bacterial colonization, and infection.