title: Pre-amyloid oligomers of the proteotoxic RepA-WH1 prionoid assemble at the bacterial nucleoid.
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.
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas - CSIC, Madrid E28040, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Moreno-Del Álamo
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas - CSIC, Madrid E28040, Spain.
authors.forename: Susana Moreno-Díaz
authors.initials: SM
authors.lastname: de la Espina
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas - CSIC, Madrid E28040, Spain.
authors.forename: M Elena
authors.initials: ME
authors.lastname: Fernández-Tresguerres
authors.affiliation: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas - CSIC, Madrid E28040, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Giraldo
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid; D000818:Animals; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D011817:Rabbits; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000682:Amyloid; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases