Secretion and functional display of fusion proteins through the curli biogenesis pathway

Abstract

SummaryCurli are functional amyloids expressed as fibres on the surface of Enterobacteriaceae. Contrary to the protein misfolding events associated with pathogenic amyloidosis, curli are the result of a dedicated biosynthetic pathway. A specialized transporter in the outer membrane, CsgG, operates in conjunction with the two accessory proteins CsgE and CsgF to secrete curlin subunits to the extracellular surface, where they nucleate into cross-beta strand fibres. Here we investigate the substrate tolerance of the CsgG transporter and the capability of heterologous sequences to be built into curli fibres. Non-native polypeptides ranging up to at least 260 residues were exported when fused to the curli subunit CsgA. Secretion efficiency depended on the folding properties of the passenger sequences, with substrates exceeding an approximately 2 nm transverse diameter blocking passage through the transport channel. Secretion of smaller passengers was compatible with prior DsbAmediated disulphide bridge formation in the fusion partner, indicating that CsgG is capable of translocating non-linear polypeptide stretches. Using fusions we further demonstrate the exported or secreted heterologous passenger proteins can attain their native, active fold, establishing curli biogenesis pathway as a platform for the secretion and surface display of small heterologous proteins.

Structure of the nonameric bacterial amyloid secretion channel

“…Importantly, the three aromatic residues, Phe-63, Tyr-66, and Tyr-71, located at the eyelet region are crucial for recognizing curli subunits and stabilizing the nonameric channel. Because the 22 N-terminal residues of the mature CsgA curli subunit (as well as the homologous CsgB) are sufficient for targeting CsgG (18,19), we propose that critical information in this peptide fragment of CsgA and CsgB may be recognized by the eyelet filter of CsgG. (F71A, F71G, F71V, F71S, F71T, F71P, F71D, and F71E) interfere with curli production.…”

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

“…Substrate passage through the CsgG transporter is itself not sequence specific 16,23 . For clarity, a polyalanine chain was used for modelling the putative interactions of a passing polypeptide chain.…”

“…However, such proton gradients are not present at the outer membrane, requiring an alternative driving force. Whereas at elevated concentrations CsgG facilitates a non-selective diffusive leakage of periplasmic polypeptides, secretion is specific for CsgA under native conditions and requires the periplasmic factor CsgE 16,23 . In the presence of excess CsgE, purified CsgG forms a more slowly migrating species on native PAGE (Fig.…”

“…Their unique secretion and assembly properties are also rapidly gaining interest for (bio)technological application 23,28,29 . Our structural characterization and biochemical study of two key secretion components provide a tentative model of an iterative mechanism for the membrane translocation of unfolded protein substrates in the absence of a hydrolysable energy source, a membrane potential or an ion gradient (Fig.…”

Programmable biofilm-based materials from engineered curli nanofibres

“…In addition, although analogous extracellular functional amyloids are produced by many bacteria, the curli system is currently the best studied and is native to the canonical model bacterium E. coli, making it an attractive starting platform for the development of engineered materials. Finally, recent findings have shown that the curli system can be used to efficiently export natively unfolded polypeptides and was capable of producing a functional camelid antibody fragment, suggesting that the curli system can be used in a broad and modular way for the display of various functional peptides throughout the E. coli biofilm ECM 12,13 .…”