Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

Abstract

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...