Synthetic Bacterial Amyloids

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Rafael Giraldo

Group Leader

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Amyloids are stable protein assemblies that regulate phenotypes and enable their epigenetic inheritance. However, when they are the outcome of protein misfolding, amyloids can trigger diseases (i.e., human neurodegenerative and systemic amyloidosis). We create, through bottom-up Synthetic Biology, bio-resources based on bacterial amyloids with two major aims: i) understanding the molecular determinants of the shift between function and toxicity in natural amyloids; and ii) generating new devices based on amyloids as constructive resources in Biotechnology and Biomedicine.

 

Publications

Pantoja-Uceda D, Oroz J, Fernández C, de Alba E, Giraldo R, Laurents DV. Conformational priming of RepA-WH1 for functional amyloid conversion detected by NMR spectroscopy. Structure 2020; 28: 336-47.

Revilla-García A, Fernández C, Moreno-del Álamo M, de los Ríos V, Vorberg IM, Giraldo R. Intercellular transmission of a synthetic bacterial cytotoxic prion-like protein in mammalian cells. mBio 2020; 11: e02937-19.

Giraldo R. SynBio and the boundaries between functional and pathogenic RepA-WH1 bacterial amyloids. mSystems 2020; 5: e00553-20.

Giraldo R. Optogenetic navigation of routes leading to protein amyloidogenesis in bacteria . J Mol Biol 2019; doi: 10.1016/j.jmb.2019.01.037

 

Functional amyloids are protein assemblies that enable the epigenetic inheritance of phenotypes. However, when due to protein misfolding, amyloids can trigger diseases (i.e., human neurodegenerative and systemic amyloidosis). We create, through bottom-up Synthetic Biology, bio-resources based on bacterial amyloids with two major aims: i) understanding the molecular determinants of the shift between function and toxicity in natural amyloids; and ii) generating new constructive resources for Biotechnology and Biomedicine based on amyloids.

RepA is a protein from a bacterial plasmid whose WH1 domain undergoes conformational changes capacitating it as a transcriptional repressor, or as a DNA replication initiator or, through assembling amyloid oligomers, to hinder premature re-replication rounds. RepA-WH1 dimers become metastable monomers upon allosteric binding to plasmid-specific dsDNA sequences or acidic phospholipids, thus triggering amyloidogenesis. We engineered RepA-WH1 to become a biosafe prion-like protein (prionoid) that is transmitted from mother-to-daughter Escherichia coli cells, causing a synthetic ‘generic’ amyloid proteinopathy. RepA-WH1 aggregates propagate as two strains with distinct appearance and cytotoxicity, modulated by the Hsp70 chaperone DnaK. RepA-WH1 amyloidosis recapitulates in bacteria the hallmarks of mitochondrial routes associated with human amyloid diseases, including the formation of oligomeric pores at the internal membrane and the generation of reactive oxygen species.

We have used RepA-WH1 as a benchmark for the design of synthetic tools to probe protein amyloidogenesis, including gold nanoparticles-based sensors, screening devices exploiting amyloid-promoted overriding of translation termination, both in yeast or in bacteria, and in vitro expression devices to address amyloidosis within cytomimetic lipid vesicles. Recently, control on RepA-WH1 amyloidogenesis has also been achieved through optogenetics, i.e., the fusion of a blue light-responsive plant domain (LOV2) to the N-terminus of WH1. Expressing LOV2-WH1-mCherry in E. coli under blue light illumination leads to the assembly of oligomers that hamper bacterial growth. We are now exploring these devices as novel antimicrobials (‘optobiotics’).

Giraldo 2020 Figure

Figure legend. A ‘generic’ synthetic model of amyloidosis engineered from bacterial RepA.

(a) RepA is a dimeric transcriptional repressor that dissociates as monomers to initiate plasmid replication. Finally, through its WH1 domain assembles post-replicative, inhibitory amyloid oligomers.

(b) Fraying terminal helices in RepA-WH1 dimers prime RepA-WH1 dissociation and the assembly of the monomers as filaments, involving an amyloidogenic loop (red). Amyloidogenesis can be driven by DNA and acidic phospholipids (aPLs) or by gold nanoparticles (Au-NRs), and inhibited by S4-indigo, or by a conformation-specific antibody (B3h7). Fusion of a plant photosensor domain (LOV2) to the N-terminal helix in RepA-WH1 enables optogenetic modulation of amyloidogenesis: blue light illumination generates cytotoxic oligomers.

(c) The amyloidogenic stretch in RepA-WH1 can functionally replace prionogenic NM sequences in the yeast prion [PSI+]. The same repeats fused to E. coli RF1 enable stop codon read-through by ribosomes, counteracted by anti-amyloid compounds.

(d) RepA-WH1 is vertically inherited in E. coli as two distinct amyloid strains: cytotoxic globular (G), or harmless comet-shaped (C) particles. Hsp70 chaperone detoxifies aggregates by favoring the C strain. G oligomers make pores at the inner membrane and enhance oxidative stress.

(e) Horizontal spread of RepA-WH1 can be achieved in mammalian cells, but it is restricted by the need of its heterologous expression.

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Name
Position
Contact
Rafael Giraldo Principal investigator
Cristina Fernández Postdoctoral scientist
Leticia Lucero Predoctoral student
Sol Vendrell Predoctoral student
Silvia Marín Technical Assistant

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