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Amyloid aggregation of the eukaryotic translation terminator eRF3/Sup35p, the [PSI +] prion, empowers yeast ribosomes to read-through UGA stop codons. No similar functional prion, skipping a stop codon, has been found in Escherichia coli, a fact possibly due to the efficient back-up systems found in bacteria to rescue non-stop complexes. Here we report that engineering hydrophobic amyloidogenic repeats from a synthetic bacterial prion-like protein (RepA-WH1) into the E. coli releasing factor RF1 promotes its aggregation and enables ribosomes to continue with translation through a premature UAG stop codon located in a β-galactosidase reporter. To our knowledge, intended aggregation of a termination factor is a way to overcome the bacterial translation quality checkpoint that had not been reported so far. We also show the feasibility of using the amyloidogenic RF1 chimeras as a reliable, rapid and cost-effective system to screen for molecules inhibiting intracellular protein amyloidogenesis in vivo, by testing the effect on the chimeras of natural polyphenols with known anti-amyloidogenic properties. Resveratrol exhibits a clear amyloid-solubilizing effect in this assay, showing no toxicity to bacteria or interference with the enzymatic activity of β-galactosidase.
The yeast prion [PSI +], which is the amyloid-aggregated form of the Sup35p translation termination/ribosome releasing factor three (eRF3), expands the diversity of the proteome by reading through the UGA stop codon and is a widely studied basic model for amyloidogenesis and epigenetics1. In bacteria translation termination is dependent on the releasing factor 1 (RF1), which recognises the UAG and UAA stop codons, and releasing factor 2 (RF2) that recognizes UAA and UGA. Both factors are released from the ribosomal A site by the action of a GTPase (RF3, a Sup35p orthologue)2,3. The elucidation of bacterial ribosome structures in their different functional states has facilitated the characterization of the mechanism of action of RF1 and RF24,5. These proteins are structural analogues of tRNAs that occupy the ribosomal A site over the termination codons, thereby placing amino acid side-chains to recognise the stop triplet, while projecting their respective N-terminal domains towards the exterior of the ribosome (Fig. 1a). Under conditions wherein the availability of RF1/RF2 is drastically reduced, when a stop codon is reached by the ribosome there is a pause in the translation and an anti-terminator aminoacylated tRNA inefficiently replaces RF1/2, thereby allowing for stop codon read-through in a discrete fraction of the pause events. The strict quality control exerted in E. coli 6–8, either on the stability of incompletely translated messengers or on partially synthesised proteins following a stop or a pause during elongation, may be a reason why translation termination in bacteria has not yet been exploited for biotechnological purposes, such as drug screening.
Model systems of protein amyloidoses, spanning from Alzheimer’s, Parkinson’s and prion neurodegenerative diseases to diabetes, have been successfully developed on the basis of eukaryotic organisms such as yeast, worms, flies, mice and, more recently, human stem cells9. Beyond their contribution to our understanding of amyloidoses, such models have enabled screening for anti-amyloidogenic compounds10. However, issues including the time needed to get a measurable signal or phenotype and, in the case of animal models, the requirement of fulfilling the two first of ‘the three Rs’ of Bioethics (the Replacement of animals and the Reduction in the number of them used)11, still press in favour of developing alternative screening platforms for biotechnological applications. Bacteria such as Escherichia coli, in spite of their ease and robustness of handling and the subsequent savings in cost and time, have been less used as proxies to amyloid diseases due to the evolutionary distance between prokaryotes and humans. There are a few exceptions, including extracellularly secreted bacterial amyloids, such as curli/CsgA12 and related amyloids, which scaffold biofilms and have been recently engineered to survey the amyloidogenic potential of any secreted protein and to test the anti-amyloidogenic activity of natural compounds13. More recently, bacteria have been also engineered to develop an assay in which the aggregation of β-lactamase in the periplasm, and thus the resistance levels against β-lactam antibiotics, is modulated by the aggregative potential of peptides fused to the enzyme14. Regarding intracellular amyloids, straight fusions of GFP to amyloidogenic peptides have been used to get a read-out of aggregation correlated with a loss in fluorescence emission15.
In this work, we have challenged the view that, upon ribosome stalling on a stop codon, the trans-translation checkpoint cannot be easily overcome in bacteria6–8, by exploring the feasibility of modifying one of the releasing factors in E. coli (RF1) to become inactivated through amyloid aggregation. Thus, we have generated chimeras between RF1 and a hydrophobic peptide of bacterial origin: the amyloidogenic stretch in the RepA-WH1 prionoid16. This prion-like protein was chosen as a proof of concept because it is the prototype of a bacterial intracellular amyloidosis, relevant as a model for human amyloid diseases since it elicits in E. coli a vertically (mother-to-daughter cells) transmissible amyloid proteinopathy17, propagating as alternative Hsp70 chaperone-modulated conformational strains18. Furthermore, RepA-WH1 exhibits cross-seeding both in vitro and in vivo 17,19, and shares pathways of toxicity with mitochondrial routes of amyloid disease (i.e., membrane targeting and ROS generation)20,21. The amyloidogenic, hydrophobic sequence in RepA-WH1 was recently found to functionally replace the polar repeated sequences that enable the yeast prion [PSI +] to aggregate22. As a proof of concept, we also show that these chimeras constitute in bacteria a useful intracellular screening system for anti-amyloidogenic compounds, as described for analogous reporters built on [PSI +] in yeast23,24.
In yeast Sup35p, polar Q/N-rich sequences govern the aggregation of the protein as the [PSI +] prion, which enables stop-codon read-through by ribosomes1,23,24. We have recently shown that 2-4 repeats of a hydrophobic and amyloidogenic sequence (LVLCAVSLI) from the bacterial prion-like protein RepA-WH125, can functionally replace in yeast the polar Q/N oligopeptide repeats in [PSI +] resulting in a new synthetic prion, [REP-PSI +]22. To explore the possibility of generating an analogous synthetic prion in bacteria, thus enabling an epigenetic control on bacterial translation termination, we have now engineered the same amyloidogenic repeats of the RepA-WH1 sequence, in short WH1(R0-4), at the N-terminus of the E. coli RF1.
A strain carrying a thermosensitive allele of prfA 26, the gene encoding RF1, in which we had deleted the lacZ gene (MRA8-lacZ, prfA ts) was used as host. In this strain, RF1ts allows a normal termination of translation at UAG codons at 30°C, whereas at 42°C RF1ts is disabled and thus translation termination is impaired, depending on complementation by a functional prfA gene. The expression of the WH1(R0-4)-RF1 chimeras in conditions promoting amyloidogenic aggregation (42°C) would make translation termination defective, thus leading to read-through stop codons (Fig. 1b).
A first plasmid construct series allowed for IPTG-induced expression of the His6-tagged WH1(R0-4) repeats fused to RF1, enabling complementation of the host RF1ts (Fig. 2a). Two compatible reporter plasmids allowed for arabinose induction of lacZ by carrying either a UAA stop codon at its natural terminus (lacZ-WT), or an additional premature amber (UAG) mutation (lacZ-amber) generating a truncated and inactive enzyme (Fig. 2b). Such lacZ-amber reporter gives a blue or white coloration to bacteria depending, respectively, on the synthesis, or not, of the full length β-galactosidase enzyme (Fig. 2c).
In cells expressing WH1(R0-4)-RF1 and carrying the lacZ-WT reporter, when grown at the permissive temperature (30°C) on agar plates carrying the chromogenic substrate X-Gal, the characteristic blue-green colour was observed (Fig. 3a, top-left). In contrast, this blue-green colour was not observed in bacteria grown in the same conditions but carrying the lacZ-amber reporter, indicating an efficient translational termination, as expected (Fig. 3a, bottom-left). At the non-permissive temperature (42°C), there was no apparent change, neither in colour nor in growth, in cells carrying the lacZ-WT reporter (Fig. 3a, top-right), due to the end of translation determined by RF2 at the terminal UAA stop codon. However, in the case of the lacZ-amber reporter only those bacteria bearing RF1 chimeras with at least two amyloidogenic repeats, WH1(R2-4)-RF1, displayed a blue-green colour at 42°C (Fig. 3a, bottom-right). This pointed to the efficient read-through by the ribosomes of the premature amber codon in the high copy-number lacZ-amber mRNA, as a consequence of a loss of function in the WH1(R2-4)-RF1 chimeras. To test the ability of the engineered translation termination device to generate a signal amenable to high-throughput screening, bacteria were also grown in minimal liquid medium in multi-well plates. Colour phenotypes similar to those observed for the agar plates were obtained (Fig. 3b). Accordingly, β-galactosidase assays in vitro showed that at 42°C the enzymatic activity for WH1(R2-4)-RF1 in lacZ-amber backgrounds, normalized to the corresponding value for the same chimeras in combination with the lacZ-WT reporter, increased progressively with the number of amyloidogenic tandem repeats, peaking at 3h post-induction (Fig. 3c). With the lacZ-WT reporter, similar levels of β-galactosidase activity were achieved for all the chimeras, with no significant difference between the number of repeats in WH1(R2-4)-RF1 (Fig. 3c, inset).
Altogether, these results indicate that the aggregation of RFs can enhance stop codon read-through, and constitute a proof of concept on the feasibility of engineering a bacterial sensor for the amyloid aggregation of peptide stretches based on the same principle governing the [PSI +] prion in yeast.
To determine the feasibility of using the bacterial sensor in screens for anti-amyloidogenic compounds, we tested a number of distinct polyphenolic molecules known to have such activity (Fig. 4)10. We found that, at 42°C and with the lacZ-amber reporter, the sensor including the WH1(R2-4)-RF1 chimeras efficiently monitored the anti-amyloidogenic activity of resveratrol and, to a lesser extent, of epigallocathequin-3-gallate (EGCG) (Fig. 4a). These are two of the most active compounds in counteracting amyloidogenesis in a number of proteins involved in human disease10. An issue of concern in any sensor system is the possibility of having false positive results. To check if this were the case for WH1(R2-4)-RF1, the experiments with resveratrol and EGCG were repeated with the lacZ-WT reporter (Fig. 4b). Both the visual inspection of the liquid bacterial cultures (Fig. 4b, top) and β-galactosidase assays with the WH1(R3)-RF1 chimera (Fig. 4b, bottom) indicated that while resveratrol, compared to its carrier solvent DMSO, had some enhancing effect of the enzymatic activity of β-galactosidase, EGCG significantly inhibited the reporter. In addition, serial culture dilutions on agar plates showed that EGCG had some inhibitory effect on bacteria growth, because it led to a smaller size of colonies, while resveratrol did not have such effect (Fig. 4c). Therefore, the reporter system based on the WH1(R2-4)-RF1 and lacZ-amber is robust enough to screen for inhibitors of protein amyloidosis, provided that appropriate control assays on the compounds selected in the screening were performed. This must be also the case for any other reporter gene of choice (e.g., encoding luciferase or fluorescent proteins).
The effect of resveratrol on the WH1(R2-4)-RF1 chimeras was further characterized with the lacZ-amber reporter in liquid cultures in vivo (Fig. 5a). In β-galactosidase assays (Fig. 5a, bottom), as expected for a solubilizing effect of this polyphenol on the three chimeras, the levels of enzymatic activity, normalized to those measured for the untreated cells, decreased up to 50%, indicating a net increase in successful translation termination elicited by WH1(R2-4)-RF1. In parallel, we checked that resveratrol did not show any significant effect on the growth of bacteria (Fig. 5b).
To determine if the reduced stop codon read-through indeed was a consequence of decreased aggregation of the RF1 chimeras due to the action of the polyphenol, the effect of resveratrol on the WH1(R3)-RF1 chimera, which efficiently promoted amber stop codon read-through (Figs 3 and and4),4), was tested by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) (Fig. 5c). This is a technique capable to resolve large polydispersed amyloid aggregates on the basis of their resistance to solubilization by SDS27. Cell extracts from bacteria expressing WH1(R3)-RF1 in the presence of DMSO contained the chimeric protein as several oligomeric species, ranging from low to high molecular weights. However, those incubated in the presence of resveratrol showed a marked decrease in the amount of the largest aggregates. These results demonstrated that the reduction in the stop codon read-through by the ribosomes, as reported by the sensor, is a direct consequence of the ability of resveratrol to prevent the aggregation of the RF1 chimeras.
In parallel to the re-assignment of stop codons to code for non-natural amino acids28–30, this report shows that a translation termination factor (RF1) can also be engineered in bacteria to survey the propensity to amyloid aggregation of a given protein sequence. Moreover, we show here that amyloid-tagged RF1 is amenable for testing in bacteria compounds targeting intracellular amyloidosis. Future developments will imply to test chimeras of RF1 with peptide sequences involved in human amyloidoses and to explore the robustness of the assay in large-scale screenings. Once these issues have been addressed, the amyloidogenic RF1 chimeras, due to the handiness and simplicity of bacteria, may be included as a first in vivo screening assay in drug discovery programs targeting amyloid proteinopathies.
The prfA gene, encoding the bacterial translation termination factor RF1, was amplified by PCR from pELI0226. Besides a SacII site, which is necessary to clone the prfA gene, SmaI and PvuI targets were included at the 5′ end of the forward primer for the subsequent introduction of tandem repeats of the RepA-WH1(A31V) amyloidogenic peptide (L26VLCAVSLI34)22,25 (Fig. 2a). The amplified prfA was cloned into pRG-SD1, a vector carrying a suboptimal translation initiation sequence (to assure low intracellular levels of the cloned chimeras) and expressing hexahistidine fusion proteins under the control of an IPTG-inducible Ptac promoter31. The parental construction including prfA and none of the WH1 repeats was named pRG-WH1(R0)-RF1. Starting from this plasmid, the Ptac-His6-WH1(R0)-RF1 module was transferred to a plasmid with a low copy-number RK2 replicon18, to get pRK2-WH1(R0)-RF1. For the construction of the rest of the chimeras, the inserts with the tandem repeats of the amyloidogenic peptide were obtained by means of enzymatic digestion (SmaI and EcoRV) of the pUKC1620_R1-4 plasmids22. Fragments were then ligated with pRG-WH1(R0)-RF1, previously digested with SmaI, to build pRG-WH1(R1-4)-RF1. Finally, the inserts containing the R1-4, repeats fused to prfA were transferred (SmaI and BamHI digestion) to pRK2-WH1(R0)-RF1, thus generating the pRK2-WH1(R1-4)-RF1 vector series.
The lacZ gene was amplified from pMLM13232 using oligonucleotides with SpeI (forward) and SmaI (reverse) 5′ ends. Both the PCR product and the pFus vector33 were digested with those enzymes and then fragments were ligated to give the pFus-lacZ-WT plasmid, which carried the lacZ gene under the control of the arabinose-inducible ParaBAD promoter. The pFus-lacZ-amber plasmid (Fig. 2b) was generated by means of site-directed mutagenesis using Pfu Turbo (Stratagene) and specific oligonucleotides to introduce a premature amber termination codon (UAG) at the position 1545–1547 of lacZ (LacZ residue change: A515*)34.
MRA8, an E. coli K-12 strain thermosensitive for RF1 due to the R137P mutation in the prfA gene35, was selected as host strain. The choice of a prfA ts rather than a null strain, was imposed by the fact that prfA/RF1 is essential in E. coli 36. MRA8 was transformed with pKD46, a plasmid expressing the λ-red recombinase under the control of the ParaBAD promoter37. Upon expression of λ-red (0.2% arabinose), 2ml cultures were grown for 2h at 30°C and 1,100rpm (Thermo Mixer Compact, Eppendorf). Cells were harvested and prepared in sterile H2O/glycerol for electroporation (MicroPulser, BioRad) with a PCR product including a Km-parE cassette flanked by 50bp from the 5′ an 3′ ends of lacZ, to get the MRA8 lacZ::Km-parE strain. Using this strain as template, two 500-bp lacZ flanking regions were separately amplified by means of PCR. Both were ligated and then amplified, to generate a 1 kbp fragment that was used in a second recombination step in MRA8 lacZ::Km-parE, as described above. To select for the recombinant cells, platting was performed on M9 agar supplemented with 0.5% rhamnose, which induces the parE toxin38: only those cells having lost the Km-parE insert would grow under those conditions, giving place to the MRA8 lacZ strain. All constructs were sequenced (Secugen Inc., CIB-CSIC).
All the assays were performed by expressing the WH1(R0-4)-RF1 chimeras in E. coli MRA8-lacZ cells, in such a way that genome-encoded RF1 is soluble and functional at 30°C, but not at 42°C. Cultures were grown in LB, or minimal medium plus casaminoacids (M9+CAA), supplemented with 100µg/ml ampicillin and 50µg/ml kanamycin, overnight at 30°C. On the following day, they were inoculated (1/100) into fresh medium and allowed to grow at 30°C until they reached an OD600nm of 0.3.
Serial dilutions of cultures were prepared (10−1–10−5). Subsequently, 7µl drops of each dilution were added to LB plates with 100µg/ml ampicillin, 50µg/ml kanamycin, 0.02mM IPTG, 0.001% arabinose, 0.003% glucose and 40µg/ml X-Gal. Then bacteria were grown at 30°C or 42°C overnight to evaluate the read-through capacity of each chimera (i.e., colonies with a blue colouration).
M9+CAA, a colourless medium, was used. Five hundred µl of each culture (OD600nm=0.3) were added to a p24 multi-well plate (Falcon). Each well was supplemented with 0.02mM IPTG, 0.001% arabinose, 0.003% glucose and 40µg/ml X-Gal, and the plates were grown at 42°C and 300rpm (Thermo Mixer Compact, Eppendorf) for 24h. The same protocol was used in the case of the assays with inhibitors of amyloid aggregation (Sigma; 25mM stocks in DMSO): 50µM curcumin, 100µM quercetin, 100µM epigallocatechin-3-gallate (EGCG), 100µM resveratrol and 37.5µM myricetin.
Cultures at OD600nm=0.3 were distributed into eight aliquots and the expression of β-galactosidase (LacZ) and the RF1 chimeras was simultaneously induced with 0.001% arabinose (plus 0.003% glucose) and 0.02mM IPTG, respectively. Half of the samples were further grown at 30°C and the other half at 42°C. LacZ activity was evaluated in cell extracts by a colorimetric assay: the degradation of ONPG39. At least 3 independent experiments were performed for each chimera. For the assays performed in LB and with the lacZ-WT reporter, 200µl culture aliquots were diluted to a final volume of 1ml with Z buffer39, supplemented with 50µl 0.1% SDS and 100µl chloroform, and incubated at 28°C for 10min. Then ONPG (Sigma) was supplemented to 4µg/ml and the reaction was left to proceed for 5min, when it was stopped with 500µl 1M Na2CO3. For the chimeras combined with the lacZ-amber reporter, cells from 8ml of culture were processed and the reaction was carried out for 15min. In the case of the assays in M9+CAA medium, 200µl of cultures and a reaction time of 5min were selected for lacZ-WT, whereas 300µl and 15min were used with the lacZ-amber reporter. With cells recovered from multi-well plates, the assay was performed at 24h post-induction. β-galactosidase activity was quantitated according to the following formula39:
where A420 is the absorption of the ONPG degradation products; (1.75×A550) a light scattering correction factor at 420nm; and OD600, a correction for the number of cells.
The presence of SDS-resistant amyloid oligomers, an indication for protein amyloidogenesis, was evaluated by means of semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), as described18,19. Bacteria were grown at 30°C to OD600=0.3, when they were supplemented with 1mM IPTG and resveratrol (1mM), or its solvent (DMSO) as a control, and then transferred to 42°C for 3h. The high concentrations of both the inducer and the polyphenol, compared with those used in the reporter β-galactosidase assays (see above), were due to the intrinsically limited sensitivity of SDD-AGE and, consequently, to the need of assuring sufficient intracellular amounts of the aggregates and their titration by resveratrol. Then harvested bacteria were mechanically lysed using a Fast-Prep (MP Biomedicals; 1.0 mm Ø Lysing Matrix C). Thirty µl of the bacterial cell lysates were resolved by electrophoresis in a 1.5% agarose-TAE gel supplemented with 0.1% SDS. Gels were then blotted to a PVDF membrane by wet electro-transfer (Mini Trans-blot, BioRad). Anti-His monoclonal (1/500) and anti-mouse polyclonal (1/5,000) antibodies (Sigma) were used in sequential incubations. Detection was performed with the ECL Plus luminescence kit (GE Healthcare) and AGFA Curix RP2 films.
We are indebted to the members of our group for much encouragement and discussions. Thanks are due to Damián Lobato-Márquez (CIB-CSIC) for the kind gift of the pFus plasmids and for help with deletion of chromosomal lacZ. Richard Poole and Arantza Barrios (UCL) are acknowledged for the critical reading of the manuscript. This work has been financed with grants (CSD2009-00088, BIO2012-30852 and BIO2015-68730-R) from Spanish AEI and UE-FEDER. R.G. is a member of the CIB-CSIC Intramural Research Program “Macromolecular Machines for Better Life” (MACBET).
L.M.G. performed the experiments and analysed data. R.G. conceived the project, analysed data and wrote the manuscript.
A patent on the fusions between amyloidogenic sequence repeats and RF1 and their usage for the screening of anti-amyloidogenic compounds has been filed at the European Patent Office (PCT/EP2016/057543).
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