Isolation, Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

doi:10.1074/jbc.m113.453605

Cited by 60 publications

(108 citation statements)

References 44 publications

(54 reference statements)

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“…The amyloid‐like fibers in the biofilms of our model organism for biofilm formation, the soil bacterium Bacillus subtilis , are also composed of two components. The protein TasA is the major component of the fibers and the auxiliary protein, TapA, present at about 1/100 molar ratio relative to TasA . TapA has a few roles in the formation of amyloid‐like fibers: it anchors TasA fibers to the cell surface and acts as a nucleating agent in the process of TasA fiber growth; hence it has been given the name “TasA assembling/anchoring protein” .…”

Section: Figurementioning

confidence: 99%

The Bacterial Extracellular Matrix Protein TapA Is a Two‐Domain Partially Disordered Protein

et al. 2018

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Biofilms are aggregates of microbial cells that form on surfaces and at interfaces, and are encased in an extracellular matrix. In biofilms made by the soil bacterium Bacillus subtilis, the protein TapA mediates the assembly of the functional amyloid protein TasA into extracellular fibers, and it anchors these fibers to the cell surface. We used circular dichroism and NMR spectroscopy to show that, unlike the structured TasA, TapA is disordered. In addition, TapA is composed of two weakly interacting domains: a disordered C‐terminal domain and a more structured N‐terminal domain. These two domains also exhibited different structural changes in response to changes in external conditions, such as increased temperatures and the presence of lipid vesicles. Although the two TapA domains weakly interacted in solution, their cooperative interaction with lipid vesicles prevented disruption of the vesicles. These findings therefore suggest that the two‐domain composition of TapA is important in its interaction with single or multiple partners in the extracellular matrix in biofilms.

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Section: Figurementioning

confidence: 99%

The Bacterial Extracellular Matrix Protein TapA Is a Two‐Domain Partially Disordered Protein

et al. 2018

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“…ThT fluorescence is red shifted when ThT is bound to proteins having ␤-sheet structures, and therefore, it is used as an indicator for the formation of amyloid-like structures. Our purified matrix protein preparations from wild-type cells contain oligomers that are composed of TasA and TapA at a ratio of about 100:1 (28,29). When these matrix protein preparations are treated for 10 min with 10% formic acid, evaporated to dryness, and resuspended in 20 mM Tris, 50 mM NaCl, pH 7 buffer, they polymerize into fibers, which bind ThT.…”

Section: Tapa Enhances Polymerization Of Tasa Into Fibers In Vitromentioning

confidence: 99%

“…In vitro, preparations containing 100:1 TasA-TapA mixtures can be stimulated to form fibers in two ways. In solution, fiber formation is observed after a lowering of the pH by the addition of formic acid (29). Fiber formation can be also be triggered by placing the proteins on hydrophobic (but not hydrophilic) surfaces (29).…”

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confidence: 99%

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

et al. 2014

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bBacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.

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“…The list of bacterial proteins that form functional amyloids is growing and includes the curli fibers of Escherichia coli and Salmonella sp. [8], the phenol-soluble modulins (PSMs) and the Bap protein from Staphylococcus aureus [9,10], FapC from Pseudomonas aeruginosa [11] and TasA from Bacillus subtilis [12,13].…”

Section: Introductionmentioning

confidence: 99%

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c

et al. 2017

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Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1-and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

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Isolation, Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

Cited by 60 publications

References 44 publications

The Bacterial Extracellular Matrix Protein TapA Is a Two‐Domain Partially Disordered Protein

The Bacterial Extracellular Matrix Protein TapA Is a Two‐Domain Partially Disordered Protein

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c

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