RepA-WH1 prionoid: A synthetic amyloid proteinopathy in a minimalist host

The intricate complexity at the molecular and cellular levels of the processes leading to the development of amyloid proteinopathies is somehow counterbalanced by their common, universal structural basis. The later has fueled the quest for suitable model systems to study protein amyloidosis under quasi-physiological conditions in vitro and in simpler organisms in vivo. Yeast prions have provided several of such model systems, yielding invaluable insights on amyloid structure, dynamics and transmission. However, yeast prions, unlike mammalian PrP, do not elicit any proteinopathy. We have recently reported that engineering RepA-WH1, a bacterial DNA-toggled protein conformational switch (dWH1→mWH1) sharing some analogies with nucleic acid-promoted PrP^C→PrP^Sc replication, enables control on protein amyloidogenesis in vitro. Furthermore, RepA-WH1 gives way to a non-infectious, vertically-transmissible (from mother to daughter cells) amyloid proteinopathy in Escherichia coli. RepA-WH1 amyloid aggregates efficiently promote aging in bacteria, which exhibit a drastic lengthening in generation time, a limited number of division cycles and reduced fitness. The RepA-WH1 prionoid opens a direct means to untangle the general pathway(s) for protein amyloidosis in a host with reduced genome and proteome.Key words: RepA-WH1, bacterial prionoid, synthetic prionoid, amyloid proteinopathy, aging in bacteriaThe development of suitable model systems for the study of the complex neurodegenerative and systemic human diseases caused by the aggregation of proteins into amyloid cross-β assemblies has been successfully attempted in different ways.¹ From the point of view of the macromolecules involved, besides those proteins directly involved in amyloid diseases (Alzheimer''s β-amyloid and Tau, Creutzfeldt-Jakob''s PrP, Parkinson''s α-synuclein, Huntington''s huntingtin or β₂-microglobulin in dialysis-related amyloidosis), an ever increasing number of disease-unrelated proteins can be forced to unfold, and subsequently assemble, as amyloids under extreme, non-physiological physicochemical conditions. Both kinds of model proteins have been crucial to establish our current understanding of the common molecular basis for protein amyloidogenesis.¹ At the organisms side, although animal models, most notably mice, have returned invaluable information on protein amyloidosis, the complexity of the intricate regulatory and biochemical networks inherent to metazoans and their cultured cells, has hampered the outlining of a clear scenario on the mechanism(s) leading to cytotoxicity. Cytotoxicity can arise either from properties common to most amyloidogenic proteins, such as targeting of cell membranes by amyloid oligomers or co-aggregation of essential cell factors, or through pathways particular to each protein and its associated disease.² The key to such a riddle relies on comprehensive systems biology analyses, but also on resorting to experimental models with their number of potential variables (proteins and their interactions) drastically reduced, but yet showing the same (cytotoxic) response.Since the discovery of the Ure2p/URE3⁺] and Sup35p/PSI⁺] prions in yeast, these (relatively) simple eukaryotic microorganisms have been instrumental in addressing the molecular basis for amyloid conformational templating, structural polymorphism and cell-to-cell transmissibility.^3–5 However, two limitations to the applicability of yeast prions as universal models for amyloidosis are noteworthy: (1) the amyloidogenic sequence stretches in yeast prions are consistently Gln/Asn-rich, unlike most proteins involved in amyloid proteinopathies (which bear hydrophobic stretches) with the exception of the proteins involved in Huntington disease and in related ataxias; (2) even more importantly, while yeast prions are the epigenetic determinants of distinct, mildly advantageous phenotypes that improve adaptability to environmental challenges,^6,7 they are not the causative agents of a proteinopathy in yeast albeit, when overexpressed, Sup35p/PSI⁺] becomes detrimental for cell growth. Although this prion has recently been successfully propagated in Escherichia coli,⁸ it still does not behave as a proper pathogenic agent in this microorganism. Natural amyloids have also been described and characterized in bacteria such as E. coli (curli/CsgA)⁹ and Pseudomonas (FapC),¹⁰ but, invariantly, they are extracellularly secreted and functional in scaffolding cellular consortia such as biofilms. A case apart is posed by inclusion bodies, intracellular protein aggregates accumulated in bacterial cytoplasm upon heterologous expression of recombinant proteins, which exhibit some amyloid features¹¹ but with a discrete detrimental effect on cell fitness.^12,13