SynBio and the Boundaries between Functional and Pathogenic RepA-WH1 Bacterial Amyloids

Abstract

Amyloids are protein polymers that were initially linked to human diseases. Across the whole Tree of Life, many disease-unrelated proteins are now emerging for which amyloids represent distinct functional states. Most bacterial amyloids described are extracellular, contributing to biofilm formation. However, only a few have been found in the bacterial cytosol. This paper reviews from the perspective of synthetic biology (SynBio) our understanding of the subtle line that separates functional from pathogenic and transmissible amyloids (prions). In particular, it is focused on RepA-WH1, a functional albeit unconventional natural amyloidogenic protein domain that participates in controlling DNA replication of bacterial plasmids. SynBio approaches, including protein engineering and the design of allosteric effectors such as diverse ligands and an optogenetic module, have enabled the generation in RepA-WH1 of an intracellular cytotoxic prion-like agent in bacteria. The synthetic RepA-WH1 prion has the potential to develop into novel antimicrobials.

Conversion of the OmpF Porin into a Device to Gather Amyloids on the E. coli Outer Membrane

“…This gene was cloned into a plasmid vector including the low-copy number replicon RK2, an IPTG-inducible P tac promoter, and its repressor ( lacI q ), thus becoming decoupled from the natural regulation of ompF expression, through OmpR and MicF, in response to variations in the environmental osmotic conditions . This parental ompF construct was used as the platform to explore the insertion of a DNA sequence encoding the amyloidogenic hydrophobic stretch from the model bacterial prion-like protein RepA-WH1, , into the sequences coding for the eight extracellular loops in the β-barrel porin. In our design, the first and the last (the eighth) of the extracellular loops in OmpF were discarded because such hydrophobic insertion could interfere with the correct BAM-mediated assembly of the porin, which involves first the binding to the C-terminal strand and last the closure of the barrel by its antiparallel antiparallel bonding with the N-terminal strand .…”

“…A second round was performed with the peptide linked to glass slides coated with an anti-fouling, low cell adhesion polymer functionalized with NHS groups, but nearly no bacteria attached, probably due to poor accessibility of the peptide within the matrix to the extracellular loops in the porin. Thus, a third strategy was successfully tested on the same NHS-activated slides (Figure a), but relying on the immobilization of RepA-WH1­(A31V)-mCherry, the protein including the amyloidogenic peptide fused to a red fluorescent construct, extensively characterized on its ability to assemble cytotoxic intracellular, prion-like amyloid aggregates. ,,, For this otherwise soluble protein, amyloidogenesis is triggered by the binding of a number of ligands, while recent evidence shows that at acidic pH, the conformational flexibility of both the α1 and α5 helices and the amyloidogenic loop is enhanced, which might promote aggregation by decreasing the stability of the fold. As a control, the isolated mCherry tag was similarly fixed to the same kind of the surface.…”

Effects of gut bacteria and their amyloids on mental health and neurodegeneration in Parkinson’s disease

“…The unusual robustness of this extracellular "functional" bacterial amyloids (FuBA) (capable of withstanding chemical denaturants and SDS) has been employed by bacteria (under tight spatio-temporal-control) for biofilmstrengthening, cell-cell communications, cell-wall construction, and for bacterial antagonism. Although the misfolded human pathological amyloid proteins are intracellular-leading to cell lysis on fibrillation, most of the bacterial functional amyloids are extracellular and rarely reported for intracellular functions [3,11].…”

“…The term "functional amyloid" has been coined to differentiate these biologically useful proteins from their toxic siblings [1,2]. Although amyloid proteins were first discovered for their link to human diseases, they now represent among a list of proteins with unique functional states which are disease-unrelated in the tree of life [3].…”

“…In silico studies on the proteins reduce the need for expensive laboratory work and enhance the rate of research and discovery [4,5]. Interests in the works on the gut-brain axis during the initial part of the new millennium have been reduced due to the lack of long-term longitudinal research [3]. In the present review, we have analyzed different types of bacterial "functional amyloids" with respect to α-synuclein of PD and their interaction with each other and in comparison to the known medication molecules.…”