Secretion of curli fibre subunits is mediated by the outer membrane‐localized CsgG protein

102

Various strains of bacteria are able to produce a unique class of functional amyloids termed curli, which are critical for biofilm formation, host cell adhesion, and colonization of inert surfaces. Curli are secreted via the type VIII bacterial secretion system, and they share biochemical and structural characteristics with amyloid fibers that have been implicated in deleterious disease in humans. Here, we report the crystal structure of Escherichia coli CsgG, which is an essential lipoprotein component of the type VIII secretion system and which forms a secretion channel in the bacterial outer membrane for transporting curli subunits. CsgG forms a crown-shaped, symmetric nonameric channel that spans the outer membrane via a 36-strand β-barrel, with each subunit contributing four β-strands. This nonameric complex contains a central channel with a pore located at the middle. The eyelet of the pore is ∼12 Å in diameter and is lined with three stacked nine-residue rings consisting of Tyr-66, Asn-70, or Phe-71. Our structure-based functional studies suggest that Tyr-66 and Phe-71 residues function as gatekeepers for the selective secretion of curli subunits. Our study describes in detail, to our knowledge, the first core structure of the type VIII bacterial secretion machinery. Importantly, our structural analysis suggests that the curli subunits are secreted via CsgG across the bacterial outer membrane in an unfolded form.CsgG | curli | amyloids | outer membrane protein | biofilm

mentioning

confidence: 99%

Structure of the nonameric bacterial amyloid secretion channel

Cao

Zhao

Kou

et al. 2014

102

“…CsgD controls expression of the csgBAC operon as well as the biosynthesis of cellulose, the primary polysaccharide component of the biofilm extracellular matrix (33)(34)(35). CsgE and CsgF are also encoded by the operon and have long been considered accessory proteins that function in concert with CsgG, the outer membrane pore responsible for the translocation of CsgA and CsgB to the outer membrane space (1,2,(36)(37)(38). CsgG forms a symmetric, nonameric, ungated, and nonselective protein secretion channel, as revealed recently (37,39).…”

mentioning

confidence: 99%

“…Deletion of csgE results in decreased stability and secretion of CsgA, CsgB, and CsgF in vivo (1,36,38). CsgE inhibits CsgA fibrillation at a ratio of 1:1 in vitro (1) and also…”

mentioning

confidence: 99%

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

Krezel

Cusumano

Pinkner

et al. 2016

Self Cite

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.biofilm formation | intrinsically disordered protein | aggregation | protein-protein interaction | CsgG

“…CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is proposed to be the curli secretion apparatus that directs the secretion of CsgA, CsgB, and CsgF across the outer membrane (9,10). CsgE and CsgF interact with CsgG at the outer membrane (9).…”

mentioning

confidence: 99%

“…CsgG is proposed to be the curli secretion apparatus that directs the secretion of CsgA, CsgB, and CsgF across the outer membrane (9,10). CsgE and CsgF interact with CsgG at the outer membrane (9). CsgF is required for efficient CsgB-mediated nucleation, and CsgE is critical for CsgA, CsgB, and CsgF stability (6, 9, 10).…”

mentioning

confidence: 99%

Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Wang

Zhou

Ren

et al. 2009

Self Cite

115

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1-R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as "gatekeepers" to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.A myloids are ordered proteinaceous fibers commonly associated with mammalian neurodegenerative diseases and prion-based encephalopathies (1). Amyloid fibers have distinct biochemical and biophysical properties, such as remarkable resistance to chemical and thermal denaturation, and specific tinctorial properties when bound to Congo red and thioflavin T (ThT) (1). The molecular basis of neurodegenerative disease development induced by amyloid propagation remains elusive, partially because of the seemingly erratic and uncontrolled nature of amyloidogenesis. An emerging focus of amyloid biosynthesis has shown that amyloids can also be an integral part of physiology found in different organisms including bacteria, fungi, and mammals (2, 3). How nature coordinates functional amyloid propagation and reduces the associated cytotoxicity is poorly understood.Curli, a bacterially produced functional amyloid, is an important component of the extracellular matrix and is involved in bacterial community behaviors (4). Because of the amyloid properties of curli fibers (5, 6), the colonies of curli-producing Escherichia coli stain red when grown on Congo red indicator plates, which provides a convenient assay to monitor curli assembly in vivo (7). In E. coli, at least six proteins are dedicated to directing efficient curli formation. Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface-tethered nucleator CsgB, which initiates CsgA polymerization (8). CsgD is a transcriptional activator for the csgBA operon (4). CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is prop...