Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation.

Roger D Klein,Kanna Nagamatsu,Matthew R Chapman,Aaron J L Lynch,Wenjie Wang,Chao Wu,Neha Jain,Jerome S Pinkner,Carl Frieden,Scott J Hultgren,Gaya K Amarasinghe,Zachary T Cusumano,Nathaniel C Gualberto,Qin Shu

doi:10.1128/mbio.01349-18

Roger D Klein, Kanna Nagamatsu + Show 12 more

Open Access

https://doi.org/10.1128/mbio.01349-18

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Abstract

ABSTRACTCurli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.

Highlights

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA
To identify residues responsible for mediating the CsgE-CsgA interaction (Fig. 1A), we examined the nuclear magnetic resonance (NMR) chemical shift perturbation of all assigned CsgE W48A/F79A peaks in the presence and absence of CsgA
We identified 11 residues (E31, S32, D33, Y34, S45, A46, R47, W48, R71, E85, and L88) that exhibited a greater-than-2fold-higher chemical shift perturbation than average, suggesting that these residues are involved in the CsgE-CsgA interaction (Fig. 2A to C)

Summary

Introduction

No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. We use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis

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