RepA-WH1 prionoid: Clues from bacteria on factors governing phase transitions in amyloidogenesis

Abstract

In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the 'winged-helix' N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a 'prionoid'. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI(+)] prion and RepA-WH1 generates [REP-PSI(+)], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.

Keywords: Hsp70 chaperone; RepA-WH1; amyloid polymorphism; amyloid proteinopathy; bacterial prionoid; phase transitions.

Figure 1

(See next page). Overview of RepA-WH1 amyloidogenesis, remarking hierarchical assembly in vitro (A) and phase transitions in vivo (B,C). (A) Stable dimers of RepA-WH1 (dWH1) undergo a structural transformation upon transient, low affinity binding to dsDNA, thus resulting in metastable, aggregation-prone monomers (mWH1*). The core of the WH domain is colored cyan, whereas segments showing significant conformational changes are in blue. The amyloidogenic peptide L₂₆VLCAVSLI₃₄ is depicted in red, with the side-chain of the hyper-amyloidogenic mutant residue A31V shown as spheres. Binding of dsDNA (yellow) to dWH1 disrupts the dimerization interface, thus generating partially unfolded mWH1* monomers which assemble as helical amyloid tubular filaments. Binding of RepA-WH1 to dsDNA, and thus amyloidogenesis, can be competed by molecules of S4-indigo (spheres), whereas the conformation specific antibody B3h7 (magenta) inhibits the assembly of RepA-WH1 oligomers into filaments. Filaments further associate laterally and coil to generate the mature amyloid fibers (background EM). (B) Electron micrograph showing an ultra-thin section through an E. coli cell incubated with the B3h7 antibody (arrows/dots: gold-conjugated secondary anti-mouse antibody), which reveals the preferential location of pre-amyloidogenic RepA-WH1 aggregates at the nucleoid (N; green dashed line). A fluidized C-type aggregate hydrogel is also outlined (cyan dotted line). (C) E. coli cells growing in microfluidic channels and expressing the prionoid. RepA-WH1 amyloid aggregates show two distinct appearances, i.e. single comet-like (C) aggregates behaving as a fluidized hydrogel that readily splits on cell division; or multiple globular (G) cytotoxic aggregates with the compactness characteristic of typical amyloid plaques. While the phase transition from the C to the G aggregates occurs spontaneously in vivo, the reverse uphill transition is promoted by a single cell factor: the Hsp70 chaperone DnaK.