Abstract
Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.
Highlights
Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract
Ushers contain five domains: a central 24-stranded ß-barrel domain that inserts into the outer membrane (OM) to form the secretion channel, an internal plug domain that forms the channel gate, a periplasmic N-terminal domain (NTD) that functions in subunit recruitment, and two tandem periplasmic C-terminal domains (CTD1 and CTD2) that function in pilus assembly and secretion[15,16,17,18]
To generate a three-subunit intermediate, we replaced the PapK N-terminal extension (NTE) with the NTE from PapE (PapKE-NTE) and deleted papE, resulting in a ΔE P pilus tip fiber composed of PapK bound to PapF bound to PapG (Supplementary Fig. 1b)
Summary
Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili promote bacterial adherence to the globoseries of glycolipids in the kidney, leading to pyelonephritis, whereas type 1 pili mediate binding to mannosylated proteins in the bladder, leading to cystitis[4,5] Given their central roles in initiating and sustaining infection, there is intense interest in understanding the molecular mechanisms of pilus assembly and function. Periplasmic chaperone–subunit complexes are recruited to the OM usher (PapC for P pili, FimD for type 1 pili) for subunit assembly into pili and secretion of the pilus fiber to the cell surface (Fig. 1b). P pilus tip fibers, composed of the PapK, E and F subunits, and the PapG adhesin, are longer and more flexible compared to type 1 pili, and the variable incorporation of PapE subunits (~5–10 copies per pilus) creates structural heterogeneity (Supplementary Fig. 1b). Structures of in vivo P pilus assembly intermediates are needed to broaden understanding of CU pilus biogenesis and allow for the rational design of therapeutics that disrupt pilus biogenesis
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