The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

Hammer, Neal D.; Schmidt, Jens C.; Chapman, Matthew R.

doi:10.1073/pnas.0703310104

Cited by 239 publications

(297 citation statements)

References 51 publications

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“…A nucleation-competent CsgB truncation mutant (called CsgB trunc ) was purified as previously described. 7 CsgB trunc has been demonstrated to efficiently seed the polymerization of wild-type CsgA. 7 Unlike CsgA seeds, CsgB trunc seeds did not efficiently promote the polymerization of CsgA slowgo under the same condition shown in Figure 7(c).…”

Section: Ala Substitutions Of Q49 N54 Q139 and N144 Results In The Dmentioning

confidence: 84%

“…5 CsgB has also been demonstrated to have amyloid-forming properties and apparently serves as a template for CsgA polymerization. 7 We previously showed that, like many other amyloids, preformed CsgA fibers could seed soluble CsgA polymerization in vitro. 8 Therefore, we proposed that the growing CsgA fiber on the cell surface could serves as a template promoting soluble CsgA polymerization in a process akin to seeding.…”

Section: Introductionmentioning

confidence: 99%

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Sequence Determinants of Bacterial Amyloid Formation

Wang

Chapman

2008

Journal of Molecular Biology

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108

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SummaryAmyloids are proteinaceous fibers commonly associated with neurodegenerative diseases and prionbased encephalopathies. Many different polypeptides can form amyloid fibers, leading to the suggestion that amyloid is a primitive main-chain-dominated structure. A growing body of evidence suggests that amino acid side chains dramatically influence amyloid formation. The specific role fulfilled by side chains in amyloid formation, especially in vivo, remains poorly understood. Here, we determined the role of internally conserved polar and aromatic residues in promoting amyloidogenesis of the functional amyloid protein, CsgA. CsgA is the major protein component of curli fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In vivo CsgA polymerization into an amyloid fiber requires the CsgB nucleator protein. The CsgA amyloid core region is composed of five repeating units, defined by regularly spaced Ser, Gln and Asn residues. The results of a comprehensive alanine scan mutagenesis screen showed that Gln and Asn residues at positions 49, 54, 139 and 144 were critical for curli assembly. Alanine substitution of Q49 or N144 impeded the ability of CsgA to respond to CsgB-mediated heteronucleation, and the ability of CsgA to self-polymerize in vitro. However, CsgA proteins harboring these mutations were still seeded by preformed wild-type CsgA fibers in vitro. This suggests that CsgA-fibril-mediated seeding and CsgBmediated heteronucleation have distinguishable mechanisms. Remarkably, Gln residues at positions 49 and 139 could not be replaced by Asn residues without interfering with curli assembly, suggesting that the side chain requirements were especially stringent at these positions. This analysis demonstrates that bacterial amyloid formation is driven by specific side chain contacts and provides a clear illustration of the essential roles of specific side chains in promoting amyloid formation.

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Section: Ala Substitutions Of Q49 N54 Q139 and N144 Results In The Dmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Sequence Determinants of Bacterial Amyloid Formation

Wang

Chapman

2008

Journal of Molecular Biology

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108

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“…For curli assembly, amyloid nucleation is proposed to be facilitated by the CsgB protein, which adopts a β-sheet-rich secondary structure and propagates its β-sheet-rich amyloid fold onto CsgA molecules (25). This cross-nucleation mechanism separates nucleation and fiber elongation and controls curli assembly to occur at correct time and location (25). In addition, wild-type nucleation is also dependent of CsgF, an outer membranelocalized chaperone-like protein (10).…”

Section: Discussionmentioning

confidence: 99%

“…During the polymerization of disease-associated amyloids, the rate-limiting step is the formation of metastable protein oligomers or nuclei (18,24). For curli assembly, amyloid nucleation is proposed to be facilitated by the CsgB protein, which adopts a β-sheet-rich secondary structure and propagates its β-sheet-rich amyloid fold onto CsgA molecules (25). This cross-nucleation mechanism separates nucleation and fiber elongation and controls curli assembly to occur at correct time and location (25).…”

Section: Discussionmentioning

confidence: 99%

Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Wang

Zhou

Ren

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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117

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Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1-R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as "gatekeepers" to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.A myloids are ordered proteinaceous fibers commonly associated with mammalian neurodegenerative diseases and prion-based encephalopathies (1). Amyloid fibers have distinct biochemical and biophysical properties, such as remarkable resistance to chemical and thermal denaturation, and specific tinctorial properties when bound to Congo red and thioflavin T (ThT) (1). The molecular basis of neurodegenerative disease development induced by amyloid propagation remains elusive, partially because of the seemingly erratic and uncontrolled nature of amyloidogenesis. An emerging focus of amyloid biosynthesis has shown that amyloids can also be an integral part of physiology found in different organisms including bacteria, fungi, and mammals (2, 3). How nature coordinates functional amyloid propagation and reduces the associated cytotoxicity is poorly understood.Curli, a bacterially produced functional amyloid, is an important component of the extracellular matrix and is involved in bacterial community behaviors (4). Because of the amyloid properties of curli fibers (5, 6), the colonies of curli-producing Escherichia coli stain red when grown on Congo red indicator plates, which provides a convenient assay to monitor curli assembly in vivo (7). In E. coli, at least six proteins are dedicated to directing efficient curli formation. Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface-tethered nucleator CsgB, which initiates CsgA polymerization (8). CsgD is a transcriptional activator for the csgBA operon (4). CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is prop...

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“…CsgA aggregation shows a distinct lag, as detected by thioflavin T (ThT) fluorescence changes, followed by a rapid increase in fluorescence to a final plateau value (22). A C-terminal truncate of CsgB, in which the C-terminal 19 amino acids have been removed (CsgB trunc ), can also assemble into fibers in vitro that bind to the amyloid-specific dyes Congo red and ThT (23). However, CsgB trunc is less efficient than full-length CsgB in mediating CsgA nucleation in vivo (23).…”

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confidence: 99%

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

Crick

Pinkner

Ford

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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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The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

Cited by 239 publications

References 51 publications

Sequence Determinants of Bacterial Amyloid Formation

Sequence Determinants of Bacterial Amyloid Formation

Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

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

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