Microcin Amyloid Fibrils A Are Reservoir of Toxic Oligomeric Species

Abstract

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (>8.5), low NaCl concentrations (<50 mM), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases.Bacteriocins are a group of polypeptide antibiotics excreted by different bacterial genera including Gram-negative and Gram-positive species (1-3). Once released, they restrain the growth of competing bacterial strains in a receptor-mediated manner employing several mechanisms. Mcc 2 is a low molecular weight (ϳ7.8 kDa) bacteriocin produced by Klebsiella pneumoniae RYC492 (4 -6). Antibacterial activity of Mcc appeared to be restricted to the Enterobacteriaceae species, closely related to the producer strain that directly competes with Klebsiella to occupy a spatial niche in the ecosystem (5, 7). Mcc has been shown to exert its toxic effect by forming ion channels on the cell membrane of target cells in a receptor-mediated fashion, which leads to a rapid depolarization and thus permeabilization of the cell membrane (6,8,9). Unlike other bacteriocins, toxic forms of Mcc are produced mainly in the exponential phase and comparatively less toxic in the stationary phase (5, 10). Surprisingly, neither apparent differences in the amounts of Mcc nor degradation or post-translational modifications have been shown to be responsible for the loss of activity (11). Some reports have described that these changes in Mcc toxicity may be due to the production of a microcin antagonist known as enterochelin in the stationary phase (12). More recently, the loss of Mcc toxicity in the stationary phase has been attributed to changes in the folding and polymerization of Mcc into aggregated structures similar to amyloid fibrils (13). Compelling in vitro and in vivo studies have shown that the changes on the Mcc...

Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates

“…S1). Previous studies have shown that fibrils can serve as reservoirs of toxic oligomers (38,39). Because of the relative instability of out-ofregister fibrils, we hypothesize that they are more prone to release oligomers than in-register fibrils.…”

Amyloid Structures as Biofilm Matrix Scaffolds

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