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Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20741-6. doi: 10.1073/pnas.1320528110. Epub 2013 Dec 2.

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis.

Author information

1
Department of Molecular Microbiology and Center for Women's Infectious Disease Research, Washington University School of Medicine, Saint Louis, MO 63110.

Abstract

Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.

KEYWORDS:

bacterial pathogenesis; macromolecular assembly; outer membrane usher

PMID:
24297893
PMCID:
PMC3870681
DOI:
10.1073/pnas.1320528110
[Indexed for MEDLINE]
Free PMC Article

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