Pre-amyloid oligomers of the proteotoxic RepA-WH1 prionoid assemble at the bacterial nucleoid.

María Moreno-Del Álamo,Susana Moreno-Díaz De La Espina,Rafael Giraldo,M Elena Fernández-Tresguerres

doi:10.1038/srep14669

María Moreno-Del Álamo, Susana Moreno-Díaz De La Espina + Show 2 more

Open Access

https://doi.org/10.1038/srep14669

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Abstract

Upon binding to short specific dsDNA sequences in vitro, the N-terminal WH1 domain of the plasmid DNA replication initiator RepA assembles as amyloid fibres. These are bundles of single or double twisted tubular filaments in which distorted RepA-WH1 monomers are the building blocks. When expressed in Escherichia coli, RepA-WH1 triggers the first synthetic amyloid proteinopathy in bacteria, recapitulating some of the features of mammalian prion diseases: it is vertically transmissible, albeit non-infectious, showing up in at least two phenotypically distinct and interconvertible strains. Here we report B3h7, a monoclonal antibody specific for oligomers of RepA-WH1, but which does not recognize the mature amyloid fibres. Unlike a control polyclonal antibody generated against the soluble protein, B3h7 interferes in vitro with DNA-promoted or amyloid-seeded assembly of RepA-WH1 fibres, thus the targeted oligomers are on-pathway amyloidogenic intermediates. Immuno-electron microscopy with B3h7 on thin sections of E. coli cells expressing RepA-WH1 consistently labels the bacterial nucleoid, but not the large cytoplasmic aggregates of the protein. This observation points to the nucleoid as the place where oligomeric amyloid precursors of RepA-WH1 are generated, and suggests that, once nucleated by DNA, further growth must continue in the cytoplasm due to entropic exclusion.

Highlights

Enhanced capabilities in DNA replication[16,17]
The results reported here provide evidence on the nucleoid, the complex subcellular structure that organizes the bacterial chromosome[36], as the place where oligomeric particles of the synthetic prionoid RepA-WH1 are generated in E. coli (Fig. 4a)
This observation is in agreement with the fact that the E. coli genome contains multiple copies of the DNA sequence that was previously found to be maximally efficient in promoting the amyloidogenesis of RepA-WH118,21

Summary

Introduction

Enhanced capabilities in DNA replication[16,17]. We found that transient binding to short (11 bp) specific plasmid-derived dsDNA sequences in vitro modulated the assembly of the protein into amyloid fibres[18]. As in the case of PrP5–11, the evidence for a role of dsDNA in RepA-WH1 amyloidogenesis in vivo is still indirect and feeble, including the enhancer effect on aggregation of the effector dsDNA sequence when cloned as multiple repeats in an expression vector[21] This is not an absolute requirement, due to the presence in the E. coli genome of several sequences closely matching that of the DNA effector[21]. Development of new tools to survey nucleic acids-dependent RepA-WH1 amyloidogenesis in vivo is compulsory Chemical probes such as Congo red, thioflavin-T and polythiophenes are widely used to screen for the amyloid state in protein assemblies[25], antibodies specific for either oligomeric or fibrillar amyloid conformations are outstanding tools to screen, characterize and obtain insight into protein amyloidogenesis[26,27,28,29]. B3h7 revealed that, inside the E. coli cells, amyloid precursors are generated at the nucleoid, a finding compatible with the previously reported ability of dsDNA sequences to promote RepA-WH1 amyloidogenesis in vitro

Methods

Results

Conclusion