The E. coli CsgB nucleator of curli assembles to -sheet oligomers that alter the CsgA fibrillization mechanism

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(17):6502-7. DOI: 10.1073/pnas.1204161109
Source: PubMed

ABSTRACT Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli, Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower and appears to proceed through large amorphous aggregates before forming filaments. Substoichiometric concentrations of CsgB induce a change in the mechanism of CsgA aggregation from that of forming amorphous aggregates to that of structured intermediates similar to those of CsgB alone. Oligomeric CsgB accelerated the aggregation of CsgA, in contrast to monomeric CsgB, which had no effect. The structured β-strand oligomers formed by CsgB serve as nucleators for CsgA aggregation. These results provide insights into the formation of curli in vivo, especially the nucleator function of CsgB.

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    • "(for review, see [2]). Unlike human amyloids, curli fibers are deliberately assembled by dedicated bacterial machinery [3]–[6]. The curli fiber biogenesis requires both structural (CsgA and CsgB) and non-structural (CsgD, CsgE, CsgF, and CsgG) components encoded by genes on two divergent operons [4], [5], [7], [8]. "
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    ABSTRACT: Urinary tract infections are the most common cause of E. coli bloodstream infections (BSI) but the mechanism of bloodstream invasion is poorly understood. Some clinical isolates have been observed to shield themselves with extracellular amyloid fibers called curli at physiologic temperature. We hypothesize that curli fiber assembly at 37°C promotes bacteremic progression by urinary E. coli strains. Curli expression by cultured E. coli isolates from bacteriuric patients in the presence and absence of bacteremia were compared using Western blotting following amyloid fiber disruption with hexafluoroisopropanol. At 37°C, urinary isolates from bacteremic patients were more likely to express curli than those from non-bacteremic patients [16/22 (73%) vs. 7/21 (33%); p = 0.01]. No significant difference in curli expression was observed at 30°C [86% (19/22) vs. 76% (16/21); p = 0.5]. Isolates were clonally diverse between patients, indicating that this phenotype is distributed across multiple lineages. Most same-patient urine and blood isolates were highly related, consistent with direct invasion of urinary bacteria into the bloodstream. 37°C curli expression was associated with bacteremic progression of urinary E. coli isolates in this population. These findings suggest new future diagnostic and virulence-targeting therapeutic approaches.
    PLoS ONE 01/2014; 9(1):e86009. DOI:10.1371/journal.pone.0086009 · 3.23 Impact Factor
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    • "Amyloid seeding effect is a common phenomenon in the amyloidogenic proteins. It has been used to generate homogeneous fibrils for structural characterization and amplification [46] or to eliminate the nucleation step to form amyloid oligomers [47], [48]. Recently, oligomer cross-seeding among different amyloid proteins, Aβ and tau, have also been shown [49]. "
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    ABSTRACT: Amyloid fibrils of α-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. α-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study α-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, >80°C, α-synuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that α-synuclein was fully reversible and the NMR studies showed no observable structural changes of α-synuclein after 95°C treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing α-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of α-synuclein also occurred. The reduction of pre-existing oligomers of α-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to α-synuclein fibrillization. Our results facilitate the understanding of α-synuclein fibrillization.
    PLoS ONE 01/2013; 8(1):e53487. DOI:10.1371/journal.pone.0053487 · 3.23 Impact Factor
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    ABSTRACT: Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB- mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB- mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.
    Journal of Biological Chemistry 08/2012; 287(42):35092-103. DOI:10.1074/jbc.M112.383737 · 4.57 Impact Factor
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