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 b...

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Autores principales:	Aida Revilla-García, Cristina Fernández, María Moreno-del Álamo, Vivian de los Ríos, Ina M. Vorberg, Rafael Giraldo
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	bacterial amyloidosis prion-like RepA-WH1 intercellular transmission proteotoxic cross-aggregation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a467b52c25fa4171beec31fddca98605
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spelling	oai:doaj.org-article:a467b52c25fa4171beec31fddca986052021-11-15T15:57:02ZIntercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells10.1128/mBio.02937-192150-7511https://doaj.org/article/a467b52c25fa4171beec31fddca986052020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02937-19https://doaj.org/toc/2150-7511ABSTRACT 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.Aida Revilla-GarcíaCristina FernándezMaría Moreno-del ÁlamoVivian de los RíosIna M. VorbergRafael GiraldoAmerican Society for Microbiologyarticlebacterial amyloidosisprion-likeRepA-WH1intercellular transmissionproteotoxic cross-aggregationMicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacterial amyloidosis prion-like RepA-WH1 intercellular transmission proteotoxic cross-aggregation Microbiology QR1-502
spellingShingle	bacterial amyloidosis prion-like RepA-WH1 intercellular transmission proteotoxic cross-aggregation Microbiology QR1-502 Aida Revilla-García Cristina Fernández María Moreno-del Álamo Vivian de los Ríos Ina M. Vorberg Rafael Giraldo Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
description	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.
format	article
author	Aida Revilla-García Cristina Fernández María Moreno-del Álamo Vivian de los Ríos Ina M. Vorberg Rafael Giraldo
author_facet	Aida Revilla-García Cristina Fernández María Moreno-del Álamo Vivian de los Ríos Ina M. Vorberg Rafael Giraldo
author_sort	Aida Revilla-García
title	Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
title_short	Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
title_full	Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
title_fullStr	Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
title_full_unstemmed	Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells
title_sort	intercellular transmission of a synthetic bacterial cytotoxic prion-like protein in mammalian cells
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a467b52c25fa4171beec31fddca98605
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Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells

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