1.B.48 The Curli Fiber Subunit, CsgA, Porin, CsgG (CsgG) Family

Amyloids are insoluble fibrillar protein deposits with an underlying cross-β-structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a diverse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell-cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)] (Pham et al. 2014).

Evidence has been presented showing that the CsgA curli subunit is exported across the outer membranes of enteric bacteria via the CsgG protein of 277 aas. CsgG (of the DUF3897 or COG5616 family) forms oligomeric pores in the outer membranes of enteric bacteria. (Chapman et al., 2003; Robinson et al., 2006). CsgA forms extracellular fimbriae that enable biofilm formation and promote pathogenicity. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. Taylor et al. 2011 determined the structure of CsgC and derived a structural model of the outer-membrane subunit translocator CsgG. CsgC proved to be related to the N-terminal domain of DsbD (TC#5.A.1.1.1), both in structure and oxido-reductase capability. Furthermore, CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly.

As noted above, CsgAs are amyloid fibers associated with biofilm formation, host cell adhesion and invasion. They are the major fiber subunits. CsgG forms a complex with two other curli assembly proteins, CsgE and CsgF. (Robinson et al., 2006).  Purified CsgG  assembles into an oligomeric complex with an apparent central pore.  Antibiotic sensitivity assays demonstrated that overexpression of CsgG rendered Escherichia coli susceptible to the antibiotic erythromycin. A 22-amino-acid sequence at the N-terminus of CsgA was sufficient to direct heterologous proteins to the CsgG secretion apparatus (Robinson et al. 2006; Epstein et al. 2009).

Amyloid fibers have a yield-strength comparable to steel (Smith et al., 2006) and have protease and detergent resistant-properties that have been harnessed by a variety of organisms. Extracellular amyloid, helps create a proteinaceous matrix that enables surface adhesion and colony formation. The primary structural component of the curli fibers, CsgA, is secreted from bacterial cells in a soluble form (Chapman et al., 2002; Barnhart and Chapman, 2006). CsgB nucleates fibrillization of CsgA (Fowler et al., 2007). Curli fibers are generated in a highly regulated process involving six proteins encoded by two operons. Expression of the components of curli is regulated by several environmental conditions including temperature, salt and nutrient availability (Barnhart and Chapman, 2006). It has been hypothesized that curli fibers are involved in host invasion and pathogenesis through their activation of host extracellular matrix remodeling enzymes (Barnhart and Chapman, 2006).

CsgA is actively secreted to the extracellular milieu, but CsgB is surface located. The curli export/assembly protein, CsgG, is an outer membrane lipoprotein (Loferer et al. 1997), resistant to protease digestion both in vivo and in vitro. It is possible to modulate the steady-state levels of CsgA and CsgB by varying the amounts of CsgG. Curli production and steady-state levels of CsgA and CsgB can be increased above wild-type levels by overexpression of CsgG.

Extracellular curli fimbriae enable biofilm formation and promote pathogenicity. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. The structure of the outer-membrane subunit translocator CsgG has been determined (Taylor et al. 2011) and updated (Goyal et al. 2014 (PMID# 25219853)). CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability.  

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Goyal et al. (2014) reported the X-ray structure of E. coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor, CsgE, forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å³ pre-constriction chamber. The structural, functional and electrophysiological analyses reported by Goyal et al. (2014) imply that CsgG is an ungated, non-selective, protein secretion channel that employs a diffusion-based, entropy-driven transport mechanism.

The reaction catalyzed by CsgG is:

CsgA (periplasm) → CsgA (external medium)

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