Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone

Abstract

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time-lapse microscopy and a microfluidic set-up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA-WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain-like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet-shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA-WH1 aggregates. However, the propagation of the comet-like species is DnaK (Hsp70)-dependent. The bacterial RepA-WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.

Aggregation Interplay between Variants of the RepA-WH1 Prionoid in Escherichia coli

“…Bacteria carrying the fluorescent mCherry/YFP fusion proteins were observed, as described previously (12), with a Nikon Eclipse 90i microscope equipped with a CFI Plan APO VC ϫ100 (numerical aperture [NA], 1.40) oil immersion objective and a Hamamatsu ORCA-R 2 charge-coupled-device (CCD) camera. The following excitation (EX) and emission (EM) filters and exposure times were used: for mCherry, EX at 543/22 nm, EM at 593/40 nm, and exposure of 600 ms; for YFP, EX at 500/24 nm, EM at 542/27 nm, and exposure of 700 ms.…”

“…All experiments were carried out in the E. coli K-12 strain MDS42 recA, whose reduced genome has been depleted of mobile genetic elements (14) to avoid inactivation of the repA-WH1 gene by transposon insertion (11). Plasmids (Table 1) of the red series expressing either RepA-WH1(A31V) or RepA-WH1(⌬N37), a deletion mutant lacking its amyloidogenic peptide stretch, are derivatives of the RK2-based, low-copy-number pSEVA121 vector (http://seva.cnb.csic .es/SEVA) (15) and were described elsewhere (12), while construction of the wild-type (WT) variant was performed by PCR (Pfu DNApol) using the oligonucleotides described in Table 2. Plasmids of the yellow series were constructed by consecutive ligation of PCR-amplified fragments carrying the appropriate Ptac-repA-WH1-YFP (where YFP is yellow fluorescent protein) expression module from pWH1s (9) but including a cMycencoding tag in the 5= primer (Table 2), a downstream lacI q repressor cassette, and the p15A replicon plus the Cm resistance gene (from the pACYC184 vector).…”

“…Protein expression levels were determined by means of Western blotting, targeting with antibodies the N-terminal peptide tags His 6 (mCherry fusions) and cMyc (fusions to YFP). Cells from 25 ml of culture at an OD 600 of 2.0 were sedimented and lysed, and electrophoresis was run as described previously (12). Primary mouse antibodies (anti-His, 1:50,000; anti-cMyc, 1:10,000 [Sigma]) were incubated for 2 h, and a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) was incubated for 1 h. Chemiluminescence detection was performed with an ECL Prime kit (GE Healthcare), and band intensity analysis was carried out, with samples from three independent cultures, using Quantity One software (version 4.6.3; Bio-Rad).…”

“…Furthermore, fusions of RepA-WH1(A31V) to a fluorescent protein tag allowed tracking of the aggregation of the protein in the cytoplasm of E. coli, revealing a drastic reduction in cell proliferation upon protein aggregation (11). In addition to such toxicity, RepA-WH1(A31V) aggregates differ from conventional IBs in having a higher degree of staining with a specific amyloidotropic fluorophore and in exhibiting dynamic interconversion between distinct amyloid species, which resemble prion strains (12). The latter process is modulated by the Hsp70 chaperone DnaK that generates oligomeric RepA-WH1(A31V) particles that are readily transferred to the progeny during bacterial division (12).…”

“…In addition to such toxicity, RepA-WH1(A31V) aggregates differ from conventional IBs in having a higher degree of staining with a specific amyloidotropic fluorophore and in exhibiting dynamic interconversion between distinct amyloid species, which resemble prion strains (12). The latter process is modulated by the Hsp70 chaperone DnaK that generates oligomeric RepA-WH1(A31V) particles that are readily transferred to the progeny during bacterial division (12). Being noninfectious but vertically transmissible from mother to daughter cells, RepA-WH1 qualifies as the first entirely bacterial prionoid (13).…”