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.

In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the ‘winged-helix’ N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a ‘prionoid’. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI+] prion and RepA-WH1 generates [REP-PSI+], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.

1RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of 2 plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-2007; Torreira et al., 2015). When expressed in E. coli, the hyper-amyloidogenic domain variant RepA-1 WH1(A31V) forms particles of various sizes, distributed across the bacterial cytosol (Fernández-2 Tresguerres et al., 2010). These particles are transmissible vertically (during cell division, i.e. from 3 mother to daughter cells) as two distinct aggregate strains with remarkable appearances and phenotypes: 4 elongated and mildly cytotoxic, and compact and acutely cytotoxic, respectively (Gasset-Rosa et al., 5 2014). Co-expression of soluble and aggregation-prone RepA-WH1 variants in E. coli demonstrated that 6 the A31V variant can template its conformation on the parental WT protein (Molina-García and 7 Giraldo, 2014). Systems analyses (Molina-García et al., 2017), together with in vitro reconstruction in 8 cytomimetic lipid vesicles (Fernández et al., 2016b; Fernández and Giraldo, 2018), suggest that RepA-9 WH1(A31V) oligomers target the internal bacterial membrane, hampering proton motive force and thus 10 ATP synthesis and transport through membranes, and enhance oxidative stress. In parallel, protein 11 factors mounting the defence against stress and envelope damage co-aggregate with RepA-WH1(A31V) 12 amyloids (Molina-García et al., 2017). Altogether, bacteria viability is severely compromised by RepA-13 WH1 amyloidosis, in a way resembling some of the central mitochondrial routes found in human 14 amyloidoses (Haelterman et al., 2014; Norambuena et al., 2018; Mathys et al., 2019; Wang et al., 2019).15 However, E. coli is not suitable for addressing the issues of cell-to-cell transmissibility of protein 16 aggregates and the subsequent intracellular amyloid cross-aggregation, since this Gram-negative 17 bacterium does not uptake large protein particles due to the insurmountable obstacle of its three-layered 18 cell envelope. 19To explore the capability of the prion-like protein RepA-WH1 to propagate in a heterologous host, 20 here we exposed murine neuroblastoma cells, transiently expressing mCherry-tagged soluble RepA-21 WH1(WT), to in vitro assembled RepA-WH1(A31V) amyloid fibres. In addition, we studied the 22 intercellular induction of protein aggregation by co-culturing murine cells releasing RepA-WH1(A31V) 23 aggregates with human neuroblastoma cells stably expressing soluble RepA-WH1(WT). Confocal 24 microscopy and biochemical studies showed that the mammalian cells can take up RepA-WH1(A31V) 25 amyloid fibres, that cross-seed the cytosolic aggregation of the endogenous RepA-WH1(WT) in the 26 recipient cells. Moreover, co-culture of cells releasing the RepA-WH1(A31V) variant also induced the 27 aggregation of RepA-WH1(WT) in bystander cells, suggesting intercellular transmission of RepA-28 5 WH1(A31V) seeds. In both setups of experimental transmission, the induced RepA-WH1(WT) 1 aggregates were cytoto...