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.
Highlights
Amyloids are stable and relatively simple, albeit polymorphic, structures in which peptide stretches from a given protein assemble as fibrillar β-sheet polymers of indefinite length (Riek and Eisenberg, 2016)
Expression of WH1(A31V)-mCh in the E. coli K-12 MDS42 strain resulted, when bacteria were observed at the microscope (Figure 1A), in a significant proportion of ‘ghost’ cells
We measured the concentration of ATP in cell lysates from bulk E. coli cultures grown aerobically in rich medium, by measuring the in vitro activity of the ATP-dependent firefly luciferase: a progressive reduction in luminiscence was observed upon the expression of the prionoid (Figure 1B, left)
Summary
Amyloids are stable and relatively simple, albeit polymorphic, structures in which peptide stretches from a given protein assemble as fibrillar β-sheet polymers of indefinite length (Riek and Eisenberg, 2016). The aggregation of proteins as amyloids is at the basis of many neurodegenerative and systemic human diseases (Eisenberg and Jucker, 2012). A Bacterial Shortcut to Amyloidosis proposed routes for amyloid cytotoxicity, including the targeting of cell membranes (Butterfield and Lashuel, 2010), co-aggregation of essential cell factors (Olzscha et al, 2011; Hosp et al, 2015), interference with intracellular traffic (Woerner et al, 2016) or overloading the protein quality triage machinery, including chaperones, the proteosome and autophagy (Hipp et al, 2014). Later proteomic studies revealed a major presence of mitochondrial factors co-aggregated with designed β-amyloid proteins (Olzscha et al, 2011). Since mitochondria have bacterial endosymbiotic ancestry (Gray, 2012), it makes sense to explore if these routes for amyloid toxicity can be reconstructed and untangled in bacteria
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