Microcin Amyloid Fibrils A Are Reservoir of Toxic Oligomeric Species

Mohammad Shahnawaz,Claudio Soto

doi:10.1074/jbc.m111.282533

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.

Highlights

Toxic forms of Microcin E492 (Mcc) are produced mainly in the exponential phase and comparatively less toxic in the stationary phase [5, 10]
In a previous study we demonstrated that the biological activity of the bacterial toxin microcin E492 is modulated in vivo by the formation of amyloid fibrils in the bacterial culture [13]
In this study we show that Mcc fibrils polymerized in vitro and in vivo harbor biologically active oligomeric species that can be released from the fibrils upon changing the environmental conditions

Summary

Introduction

Toxic forms of Mcc are produced mainly in the exponential phase and comparatively less toxic in the stationary phase [5, 10]. Fibrils (400 ␮g/ml) were disaggregated upon dilutions (1-, 5,- and 10-fold) in 50 mM PIPES-NaOH, pH 6.5, containing 500 mM NaCl. After 2 h of incubation with the new condition, samples were either used as such for turbidity assay, electron microscopy, and cytotoxicity studies, or samples were centrifuged at 16,500 ϫ g for 10 min, and supernatants were used for dot blot analysis and protein estimation by a BCA protein assay kit.

Results

Conclusion