Abstract
The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, CsgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. These observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.
Highlights
The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface
Despite the inherent risk of cytoxicity, many organisms have evolved mechanisms to utilise the amyloid fold. These ‘functional amyloids’ exist in all domains of life, and operate in a host of diverse roles[4]. Several bacteria utilise these fibres as a major component of their biofilm matrix, and have evolved distinct apparatus to co-ordinate secretion[5,6,7], The first identified of such biofilm functional amyloids, and the best studied, is that of Escherichia coli[8,9], The amyloid produced by this system, termed curli fibres, has been identified in a wide range of Enterobacteriaceae, with homologous systems identified in many Gram negative phyla[10,11], Whilst providing bacteria with a structurally re-enforced scaffold, curli has been implicated in pathogenic infection[12], triggering host auto-immune responses[13], and even accelerating neurodegeneration[14]
To abate fibril formation and mimic the periplasmic state of CsgA, the curli inhibitory chaperone CsgC was added to samples at a 1:40 ratio. 1H-15N HSQC spectra were recorded in the presence and absence of CsgC, revealing no prominent chemical shift perturbations, consistent with previous work revealing the transient nature of the CsgC-CsgA encounter[35] (Supplementary Figure S1)
Summary
The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. CsgF forms a tight complex with the extracellular face of CsgG, where it co-ordinates templating of curli fibres via an interaction with the nucleator CsgB27–29, The amyloid fold of CsgA within the curli fibre has been extensively characterized, using data from a wide range of biophysical techniques[30,31,32], Complementing experimental work on the fibrous state of CsgA, a model of CsgA nucleation and assembly has been proposed using ThT-binding studies[33], and H/D exchange experiments have identified the most important repeats for driving amyloid formation[34] Whilst these have provided valuable insights into fibre assembly, structural analysis of the pre-amyloid state of CsgA present within the perisplam has been more difficult. Subsequent interaction with of the CsgE ring with the base of the CsgG pore drives encapsulation of CsgA and entropic release through the pore
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