Addressing Intracellular Amyloidosis in Bacteria with RepA-WH1, a Prion-Like Protein

Abstract

Bacteria are the simplest cellular model in which amyloidosis has been addressed. It is well documented that bacterial consortia (biofilms) assemble their extracellular matrix on an amyloid scaffold, yet very few intracellular amyloids are known in bacteria. Here we describe the methods we have resorted to characterize in Escherichia coli cells the amyloidogenesis, propagation and dynamics of the RepA-WH1 prionoid. This prion-like protein, a manifold domain from the plasmid replication protein RepA, itself capable of assembling a functional amyloid, causes when expressed in E. coli a synthetic amyloid proteinopathy, the first model for an amyloid disease with a purely bacterial origin.These protocols can be useful to study other intracellular amyloids in bacteria.

Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death

“…His-SOD1 G93A-mCherry fibrilization was induced by adding SOD1 A4V aggregates produced in bacteria. Briefly, SOD1 A4V protein aggregates were induced in BL21 cells, bacteria cell pellet was collected, lysed, and centrifuged/resuspended for 5 cycles (Molina- García et al, 2018). Then a sucrose discontinuous gradient was prepared: in a 2 mL Eppendorf tube, successively was displayed 200 mL of 60 (bottom)> 50 > 40 > 30 > 20% (top) sucrose, freezing each layer at À80 C before adding the next one.…”

Optogenetic Navigation of Routes Leading to Protein Amyloidogenesis in Bacteria

“…Samples were run in duplicate: one set for Coomassie blue staining, and the other for Western blotting. Transference to PVDF membranes was carried out by semi-dry blotting, followed by blocking in TTBS plus powder milk, as described [51]. Primary antibodies were used at 1:20,000 dilution, either mouse anti-His (Sigma) or rabbit anti-mCherry (Abcam), then incubated with HRP-conjugated secondary anti-mouse/rabbit antibodies at 1:20,000 (Sigma).…”

“…Amyloidogenesis assays in vitro were carried out as described [51]. Two mL Eppendorf tubes were set-up with 50 µL of 25µM RepA-WH1(A31V) in fibril assembly buffer (0.1M Na 2 SO 4 , 4 mM MgSO 4 , 20 mM Hepes•NaOH pH 8.0, 7% PEG4000, 3% MPD).…”

“…Cells from 15 mL of the cultures were harvested and washed twice with PBS. Pellets were resuspended in 1 mL of PBS and, while 0.9 mL were centrifuged and cells stored at -70 ºC for solubility tests, bacteria from the other 0.1 mL were fixed with 4% paraformaldehyde (Sigma) [51]. Fixed cells were stained with ThS, displayed on glass slides and mounted with Fluoromount-G (SouthernBiotech) before observation in a Nikon Eclipse 90i microscope (see above), using a 100x Plan Apo oil immersion objective (NA= 1.4).…”

Structural analysis of a replication protein encoded by a plasmid isolated from a multiple sclerosis patient

“…This regulation is also critical for the replication of plasmidderived, bacteriophage-like or virus-like DNA genomes (Ruiz-Masó et al, 2015). Reps are essential for the replication of multidrug-resistant bacteria in humans (Schumacher et al, 2014) and studies have suggested that Reps have a role in transmissible amyloid proteinopathy (Molina-García et al, 2018;Giraldo et al, 2016Giraldo et al, , 2011.…”