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Molecular Mechanism of Curli Amyloid Formation: A Folding-Limited Nucleation Process Balancing Efficient Polymerization and Avoiding Cytotoxicity


Core Concepts
The curli amyloid fibrils of Gram-negative bacteria form via a folding-limited one-step nucleation process, which has emerged as an evolutionary balance between efficient extracellular polymerization and avoiding pre-emptive nucleation in the periplasm.
Abstract
The study investigates the molecular mechanism underlying the formation of curli amyloid fibrils, which are a major component of the extracellular matrix in Gram-negative bacteria and serve as a functional scaffold for biofilms. Key insights: NMR analysis reveals that the curli subunit CsgA exists in a partially unfolded, molten globule-like state in its pre-amyloid form, sampling local secondary structure rather than being fully disordered. Native mass spectrometry detects the presence of CsgA dimers as transient, on-pathway species during the early stages of amyloid nucleation. Cryo-EM imaging resolves minimalistic amyloid nuclei composed of two or three CsgA molecules, suggesting a folding-limited one-step nucleation process. Sequence analysis indicates that the curli strand motif is enriched in amino acids with low β-strand propensity, hinting at a folding landscape with a local minimum corresponding to a helical intermediate state. The authors propose that the folding-limited nucleation mechanism has emerged as an evolutionary strategy to balance efficient extracellular polymerization of curli fibrils while avoiding premature nucleation and potential cytotoxicity within the periplasmic space of the bacterial cell.
Stats
The radius of gyration (Rg) of the unfolded CsgA monomer is estimated to be 2.8 ± 0.8 nm, while the folded CsgA monomer has a predicted Rg of 1.65 nm. The diffusivity of CsgAslowgo is measured to be 1.37 x 10-10 m2/s, corresponding to a hydrodynamic radius (Rh) of 1.82 ± 0.03 nm. The average amide exchange rate in the curlin repeats of CsgAslowgo is 0.69 s-1, compared to a predicted random coil average of 3.0 s-1.
Quotes
"Arguably the best studied case is that of curli. Curli fibers are a major component of the extracellular matrix of Gram-negative bacteria and are expressed under biofilm forming conditions where they fulfill a scaffolding and cementing role in the extracellular milieu to reinforce the bacterial community." "What remains to complete our picture of de novo curli formation is an identification of the nucleus species." "Taken altogether, a picture emerges of folding-limited one-step nucleation that has emerged as an evolutionary balance between efficient extracellular polymerization, while steering clear of pre-emptive nucleation in the periplasm."

Deeper Inquiries

How do the structural features and folding kinetics of curli amyloids compare to those of pathological amyloids associated with human neurodegenerative diseases?

The structural features and folding kinetics of curli amyloids differ significantly from those of pathological amyloids associated with human neurodegenerative diseases. Curli amyloids, such as those formed by the CsgA protein, exhibit a β-solenoid structure composed of stacked β-strands that form a non-helical, polar protofilament. This structural arrangement is distinct from the cross-beta architecture typically observed in pathological amyloids. Additionally, the folding kinetics of curli amyloids involve a folding-limited one-step nucleation process, where the monomeric protein transitions from a partially unfolded state to a β-solenoidal structure. In contrast, pathological amyloids often involve the rapid formation of reversible, non-native oligomers that undergo a structural reconversion into amyloid fibrils, leading to cytotoxicity and disease progression.

What are the potential implications of the folding-limited nucleation mechanism for the design of synthetic functional amyloids with controlled assembly properties?

The folding-limited nucleation mechanism observed in curli amyloids has significant implications for the design of synthetic functional amyloids with controlled assembly properties. By understanding the molecular mechanism of amyloid formation in curli, researchers can potentially apply similar principles to engineer synthetic amyloids for various applications. The fine-tuning of the rate of monomer folding via modulation of secondary structure propensity, as seen in curli amyloids, could be leveraged to design synthetic amyloids with precise control over their assembly kinetics and structures. This could enable the development of functional amyloids for use in synthetic biology applications, such as in the construction of biomaterials, drug delivery systems, or biosensors, where controlled assembly properties are crucial.

Could the insights into the evolutionary tuning of curli amyloid formation be leveraged to develop novel antimicrobial strategies targeting biofilm formation in pathogenic bacteria?

The insights gained from the evolutionary tuning of curli amyloid formation could indeed be leveraged to develop novel antimicrobial strategies targeting biofilm formation in pathogenic bacteria. Understanding the molecular mechanisms that govern the formation of curli amyloids, which play a crucial role in the extracellular matrix of Gram-negative bacteria, provides a potential target for disrupting biofilm formation. By interfering with the folding and assembly of curli amyloids, it may be possible to prevent the formation of biofilms, which are key for bacterial survival and virulence. This could lead to the development of innovative antimicrobial approaches that specifically target the amyloid formation process in bacteria, offering a new avenue for combating biofilm-related infections and enhancing current antimicrobial strategies.
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