Aggregation interplay between variants of the RepA-WH1 prionoid in Escherichia coli

Area of Science:

• * Molecular biology
• * Protein misfolding and aggregation
• * Bacterial genetics

Background:

• * The N-terminal domain (WH1) of Pseudomonas plasmid RepA protein forms amyloid fibers in vitro.
• * Expression in E. coli causes intracellular amyloid proteinopathy, hindering bacterial proliferation.
• * This bacterial amyloidosis is heritable but not infectious, defining it as a synthetic prionoid.

Purpose of the Study:

• * To investigate the cross-aggregation properties of different RepA-WH1 variants.
• * To determine if distinct RepA-WH1 variants can induce aggregation of wild-type RepA-WH1 in vivo.
• * To compare the aggregation behavior of a hyperamyloidogenic variant with a variant forming inclusion bodies.

Main Methods:

• * Expression of wild-type RepA-WH1 (RepA-WH1(WT)) and mutant variants (RepA-WH1(A31V), RepA-WH1(ΔN37)) in Escherichia coli.
• * Analysis of intracellular aggregate formation and propagation.
• * Investigation of cross-aggregation between different RepA-WH1 variants in vivo.

Main Results:

• * Hyperamyloidogenic RepA-WH1(A31V) aggregates enhanced the growth of new amyloid particles from soluble RepA-WH1(WT).
• * RepA-WH1(ΔN37), forming inclusion bodies, induced aggregation of RepA-WH1(WT) only at high molar ratios.
• * This demonstrates cross-aggregation between different RepA-WH1 variants within the bacterial cytoplasm.

Conclusions:

• * Cytotoxic bacterial prionoid RepA-WH1 exhibits cross-aggregation between variants, a hallmark of eukaryotic amyloids.
• * This cross-aggregation behavior contributes to the propagation and modulation of bacterial amyloidosis.
• * The findings provide insights into the mechanisms of prionoid formation and propagation in bacteria.