Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid

Abstract

The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.

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

“…Interestingly, similarities between the response of mammalian mitochondria to amyloid-promoted neurodegeneration and how E. coli reacts to RepA-WH1 amyloidosis were previously proposed on the basis of systems analyses carried out in the latter host (Molina-García et al, 2017). The results presented here reinforce this view and make sense in light of the common ancestry of Gram-negative bacteria and mitochondria, validating RepA-WH1 as a minimal, autonomous proxy to sketch core mitochondrial routes involved in human amyloidoses.…”

“…RepA-WH1 mutant variants were fused to the monomeric fluorescent protein mCherry in a constitutive expression plasmid (Figure S1A). These fusions, hereafter referred as WH1(WT/A31V/ΔN37)-mCherry for simplification, were the same that had been previously validated regarding their aggregation potential and toxicity in E. coli (Fernández-Tresguerres et al, 2010; Gasset-Rosa et al, 2014; Molina-Garcia et al, 2014; Molina-Garcia et al, 2017). While WH1(WT)-mCherry is soluble in the bacterial cytosol and non-cytotoxic, the hyper-amyloidogenic (A31V)-mCherry variant aggregates and is highly cytotoxic.…”

“…In a previous study, we characterized the systems-wide response of E. coli to WH1(A31V)-mCherry expression to identify pathways potentially affected by the bacterial amyloidosis (Molina-García et al, 2017). To assess cellular responses to the toxic WH1(A31V) amyloids, we performed parallel proteomic studies on three groups of samples: i) N2a cells transiently expressing WH1(WT)-mCherry or mCherry, collected after 48 h of incubation with WH1(A31V) fibres (see Figure 3; Supplemental Dataset 1); ii) THN-rtTA/SH-SY5Y cells expressing WH1(WT)-TagGFP2 or TagGFP2, harvested after 5 days of induction with Dox in co-culture with N2a cells expressing WH1(A31V)-mCherry (see Figure 5; Supplemental Dataset 2); and iii) control naïve N2a or THN-rtTA/SH-SY5Y cells incubated, respectively, with WH1(A31V) fibres or WH1(A31V)-mCherry donor cells.…”

“…Co-expression of soluble and aggregation-prone RepA-WH1 variants in E. coli demonstrated that the A31V variant can template its conformation on the parental WT protein (Molina-García and Giraldo, 2014). Systems analyses (Molina-García et al, 2017), together with in vitro reconstruction in cytomimetic lipid vesicles (Fernández et al, 2016b; Fernández and Giraldo, 2018), suggest that RepA-WH1(A31V) oligomers target the internal bacterial membrane, hampering proton motive force and thus ATP synthesis and transport through membranes, and enhance oxidative stress. In parallel, protein factors mounting the defence against stress and envelope damage co-aggregate with RepA-WH1(A31V) amyloids (Molina-García et al, 2017).…”

“…Systems analyses (Molina-García et al, 2017), together with in vitro reconstruction in cytomimetic lipid vesicles (Fernández et al, 2016b; Fernández and Giraldo, 2018), suggest that RepA-WH1(A31V) oligomers target the internal bacterial membrane, hampering proton motive force and thus ATP synthesis and transport through membranes, and enhance oxidative stress. In parallel, protein factors mounting the defence against stress and envelope damage co-aggregate with RepA-WH1(A31V) amyloids (Molina-García et al, 2017). Altogether, bacteria viability is severely compromised by RepA-WH1 amyloidosis, in a way resembling some of the central mitochondrial routes found in human amyloidoses (Haelterman et al, 2014; Norambuena et al, 2018; Mathys et al, 2019; Wang et al, 2019).…”