Addressing the propagation and conformational dynamics of a model prionoid in bacteria

Trabajo presentado en PRION 2014, celebrado en Trieste (Italia) del 27 al 30 de mayo de 2014.Protein amyloids arise from the conformational conversion and templated assembly of a soluble protein into fibrillar aggregates with a crossed β-sheet backbone, leading to human neurodegenerative and systemic proteinopathies.1 Besides epigenetic determinants, such as prions in yeast, functional amyloids are assembled in bacteria as extracellular scaffolds but, until recently, no proteinopathic amyloidosis had been found in microorganisms.2 In some bacterial plasmids, RepA protein initiates DNA replication and then, by physically coupling plasmid molecules through its origin-bound WH1 domain, RepA assembles as an amyloid to inhibit further replication rounds.3,4 In vitro, RepA-WH1 can assemble into amyloid fibers upon binding to short, plasmid-specific DNA sequences.5,6 RepA-WH1 causes an amyloid proteinopathy in E. coli, hampering cell proliferation.7 RepA-WH1 amyloidosis is vertically transmissible from mother to daughter cells, but not infectious, enabling conformational templating by cross-seeding in vitro and in vivo, thus it qualifies as a prionoid.2 Through microfluidics, we have directly assessed the dynamics of the RepA-WH1 prionoid in E. coli cells.8 Bacterial lineages maintain two mutually exclusive types (strains) of RepA-WH1 amyloids: either multiple globular particles that inhibit cell division, or a single elongated aggregate, mildly detrimental to growth. DnaK, the Hsp70 chaperone in E. coli, contributes to RepA-WH1 amyloidogenesis in vivo, but the Hsp104 chaperone ClpB does not have a major effect on the vertical spread of the RepA-WH1 amyloid aggregates. We have engineered repeats of the RepA-WH1 amyloid stretch to replace the Q/N-rich oligopeptide repeats in Saccharomyces cerevisiae Sup35p/[PSI+], thus building chimeric [REP-PSI+] prions. These are functional as epigenetic determinants, and become independent on Hsp104p for propagation.