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Addressing Intracellular Amyloidosis in Bacteria with RepA-WH1, a Prion-Like Protein

  • Laura Molina-García
  • Fátima Gasset-Rosa
  • María Moreno-del Álamo
  • Susana Moreno-Díaz de la Espina
  • Rafael Giraldo
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1779)

Abstract

Bacteria are the simplest cellular model in which amyloidosis has been addressed. It is well documented that bacterial consortia (biofilms) assemble their extracellular matrix on an amyloid scaffold, yet very few intracellular amyloids are known in bacteria. Here, we describe the methods we have resorted to characterize in Escherichia coli cells the amyloidogenesis, propagation, and dynamics of the RepA-WH1 prionoid. This prion-like protein, a manifold domain from the plasmid replication protein RepA, itself capable of assembling a functional amyloid, causes when expressed in E. coli a synthetic amyloid proteinopathy, the first model for an amyloid disease with a purely bacterial origin. These protocols are useful to study other intracellular amyloids in bacteria.

Key words

Bacteria Intracellular amyloid Prionoid/Prion-like Templated fibrillation Dot/Western-blot SDD-AGE Time-lapse microscopy Microfluidics Immuno-electron microscopy 

Notes

Acknowledgments

Contributions of other past and present members of our laboratory to the development of the techniques relevant to this chapter are deeply acknowledged. We thank Ariel Lindner and his group (CRI, Descartes University/INSERM, Paris) for training F.G.R. and L.M.G. on microfluidics. This research has been supported by a grant from Spanish AEI/EU-FEDER (BIO2015-68730-R).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Laura Molina-García
    • 1
    • 2
  • Fátima Gasset-Rosa
    • 1
    • 3
  • María Moreno-del Álamo
    • 1
    • 4
  • Susana Moreno-Díaz de la Espina
    • 1
  • Rafael Giraldo
    • 1
  1. 1.Department of Cellular and Molecular BiologyCentro de Investigaciones Biológicas (CSIC)MadridSpain
  2. 2.Department of Cell and Developmental BiologyUniversity College LondonLondonUK
  3. 3.Department of Neurosciences, Ludwig Institute for Cancer ResearchUniversity of California in San DiegoLa JollaUSA
  4. 4.Department of Microbial BiotechnologyNational Centre for Biotechnology (CSIC)MadridSpain

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