A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis

Sarah L Rouse,Hagan Bayley,Daniel Otzen,Sebastian Lambert,Uma Mackie,R Marc L Morgan,Jamie-Lee Berry,Per H Nielsen,William J Hawthorne,Wiebke Ewert,Sandra A Ionescu,Carol V Robinson,Stephen Hare,Fisentzos Stylianou,Morten Dueholm,Dror S Chorev,Florian-Alexander Herbst,Stephen Matthews

doi:10.1038/s41467-017-00361-6

Abstract

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.

Highlights

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix
We used a combination of biophysical techniques including native mass spectrometry (MS), singlechannel current recordings and circular dichroism in order to elucidate the role of the periplasmic N-terminal domain of full-length FapF (FapF1–406) and derive a structural model for the translocation complex
Removing the N-terminal domain comprising a 39-residue helical region followed by a 42-residue disordered linker and adding an OmpA signal sequence and a hexahistidine tag to allow purification directly from the E. coli outer membrane (OM) led to larger, well-diffracting and reproducible crystals for FapFβ (Fig. 1a)

Summary

Introduction

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. Bacterial amyloid fibres are a major protein component of biofilms, and play important functional roles in bacterial persistence either in animal hosts or on surfaces[2,3,4,5,6]. We used a combination of biophysical techniques including native mass spectrometry (MS), singlechannel current recordings and circular dichroism in order to elucidate the role of the periplasmic N-terminal domain of full-length FapF (FapF1–406) and derive a structural model for the translocation complex. Based on this new insight we designed several mutants to probe the functional determinants of FapC secretion. Our findings contribute to a growing understanding of how bacteria can safely handle amyloidogenic polypeptides and provides the inspiration for new approaches in the control of bacterial biofilms

Methods

Results

Conclusion