Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells

Abstract

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways. IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

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

“…Therefore, horizontal transmissibility of RepA-WH1 was recently probed in cultured mammalian cells (Fig. 1F) (76). First, the same RepA-WH1 variants that had been fused to a fluorescent protein and assayed in bacteria (see above) were expressed in the cytosol of murine cells: while those including the hyperamyloidogenic A31V mutation formed multiple cytotoxic amyloid aggregates, the wild-type (wt) protein remained soluble and the cells were viable.…”

“…First, the same RepA-WH1 variants that had been fused to a fluorescent protein and assayed in bacteria (see above) were expressed in the cytosol of murine cells: while those including the hyperamyloidogenic A31V mutation formed multiple cytotoxic amyloid aggregates, the wild-type (wt) protein remained soluble and the cells were viable. Then these viable cells were used as receptors in an experiment in which in vitro-assembled and fluorescence-labeled RepA-WH1 amyloid fibers were added to the culture (76). In an alternative approach, the murine cells experiencing the RepA-WH1(A31V) amyloidosis were cocultured, as donors of amyloid aggregates, with human cells stably expressing RepA-WH1(wt) fused to a distinct fluorescent protein.…”

“…In an alternative approach, the murine cells experiencing the RepA-WH1(A31V) amyloidosis were cocultured, as donors of amyloid aggregates, with human cells stably expressing RepA-WH1(wt) fused to a distinct fluorescent protein. In both cases, RepA-WH1(A31V) amyloid particles were readily captured by the receptor cells, but unlike CsgA on sequence-unrelated mammalian proteins (36,37,77), intracellular aggregation occurred only in recipient cells expressing soluble RepA-WH1(wt), which could be molded into amyloid upon binding the internalized aggregates (76). It is interesting that vascin, a synthetic peptide from a nonamyloidogenic human protein (endothelial growth factor receptor 2 [VEGFR2]), like RepA-WH1, specifically seeds the aggregation of soluble full-length VEGFR2 in the recipient cells but not of any other intracellular protein (78).…”

“…It is interesting that vascin, a synthetic peptide from a nonamyloidogenic human protein (endothelial growth factor receptor 2 [VEGFR2]), like RepA-WH1, specifically seeds the aggregation of soluble full-length VEGFR2 in the recipient cells but not of any other intracellular protein (78). Finally, the proteomes of receptor cells that had been incubated with the in vitro-assembled or the in vivo-secreted RepA-WH1(A31V) amyloids were analyzed: the respiratory complexes in mitochondria, and also the protein trafficking and quality triage pathways, showed up in the cells expressing RepA-WH1(wt) but not in control cells expressing the fluorescent protein tags (76). These findings are in good agreement with the results on the toxicity exerted by RepA-WH1 in bacteria (72).…”

Protein aggregates carry non-genetic memory in bacteria after stresses

“…All these actions in bacteria assist to molecular chaperones and proteases that refold or degrade un-and misfolded proteins in order to maintain proteostasis [18,19]. Chaperones are involved in the disaggregation of aggregated proteins, and their number is directly related to the quality of the disaggregation [19][20][21]. In particular, it is indicated for the chaperone Hsp104, which carries out the fragmentation of protein aggregates [21].…”