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. 2013;8(7):1381-90.
doi: 10.1038/nprot.2013.081. Epub 2013 Jun 20.

A bacterial export system for generating extracellular amyloid aggregates

Viknesh Sivanathan  1 , Ann Hochschild
Affiliations

A bacterial export system for generating extracellular amyloid aggregates

Viknesh Sivanathan et al. Nat Protoc. 2013.

Abstract

Here we describe a protocol for the generation of amyloid aggregates of target amyloidogenic proteins using a bacteria-based system called curli-dependent amyloid generator (C-DAG). C-DAG relies on the natural ability of Escherichia coli cells to elaborate surface-associated amyloid fibers known as curli. An N-terminal signal sequence directs the secretion of the major curli subunit CsgA. The transfer of this signal sequence to the N terminus of heterologous amyloidogenic proteins similarly directs their export to the cell surface, where they assemble as amyloid fibrils. Notably, protein secretion through the curli export pathway facilitates acquisition of the amyloid fold specifically for proteins that have an inherent amyloid-forming propensity. Thus, C-DAG provides a cell-based alternative to widely used in vitro assays for studying amyloid aggregation, and it circumvents the need for protein purification. In particular, C-DAG provides a simple method for identifying amyloidogenic proteins and for distinguishing between amyloidogenic and non-amyloidogenic variants of a particular protein. Once the appropriate vectors have been constructed, results can be obtained within 1 week.

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Figures

Figure 1
Figure 1
Flow chart showing experimental stages for implementation of C-DAG. Applications that rely on the same method for protein production are grouped by color, with the Procedure Steps and timing associated with each application indicated.
Figure 2
Figure 2
pExport plasmid map with relevant features. In one example, the pExport plasmid, pVS72, contains Sup35 NM as the gene of interest. All restriction sites shown are unique.
Figure 3
Figure 3
Results for Sup35 NM and Sup35 M when using C-DAG and tested in various assays.

  1. When grown on solid medium supplemented with Congo Red, E. coli cells producing CsgAss-NM form colonies that stain red, whereas cells producing CsgAss-M form pale colonies (reprinted, with permission, from Sivanathan & Hochschild, 2012).

  2. E. coli cells secreting CsgAss-NM produce fibrillar aggregates that can be visualized by transmission electron microscopy, whereas cells secreting CsgAss-M do not produce fibrillar aggregates.

  3. The fibrillar aggregates generated by cells secreting CsgAss-NM are immunolabeled by an anti-Sup35 antibody. No fibrillar aggregates are detected for cells secreting CsgAss-M (reprinted, with permission from Sivanathan & Hochschild, 2012).

  4. E. coli cells secreting CsgAss-NM produce material that manifests apple-green birefringence when viewed by bright-field microscopy between crossed polarizers, whereas cells secreting CsgAss-M do not. Cell samples are taken from colonies formed on solid medium supplemented with Congo Red.

  5. SDS-resistant aggregates are detected using the filter retention assay for samples of cells secreting CsgAss-NM, but not for cell samples secreting CsgAss-M.

Figure 4
Figure 4
SDS-resistant aggregates detected for a dilution series of a sample prepared from cells secreting CsgAss-NM. The first four dilutions clogged the wells within the dot blot manifold to which they were applied. A two-fold reduction in signal intensity corresponding to the two-fold dilution of sample is seen only for the last three dilutions.
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References

    1. Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR. Diversity, biogenesis and function of microbial amyloids. Trends Microbiol. 2011;20:66–73. - PMC - PubMed
    1. Chapman MR, Robinson LS, Pinkner JS, Roth R, Heuser J, Hammar M, et al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science. 2002;295:851–855. - PMC - PubMed
    1. Robinson LS, Ashman EM, Hultgren SJ, Chapman MR. Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein. Mol. Microbiol. 2006;59:870–881. - PMC - PubMed
    1. Nenninger AA, Robinson LS, Hammer ND, Epstein EA, Badtke MP, Hultgren SJ, Chapman MR. CsgE is a curli secretion specificity factor that prevents amyloid fibre aggregation. 2011;81:486–199. - PMC - PubMed
    1. Sivanathan V, Hochschild A. Generating extracellular amyloid aggregates using E. coli cells. Genes Dev. 2012;26:2659–2667. - PMC - PubMed

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