Fiber Formation across the Bacterial Outer Membrane by the Chaperone/Usher Pathway

Institute of Structural Molecular Biology, University College London and Birkbeck College, Malet Street, London, WC1E 7HX, United Kingdom.
Cell (Impact Factor: 33.12). 06/2008; 133(4):640-52. DOI: 10.1016/j.cell.2008.03.033
Source: PubMed

ABSTRACT Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins. Copyright © 2015 Lieberman et al.
    mBio 03/2015; 6(2). DOI:10.1128/mBio.00322-15 · 6.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in man and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determine host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG and each feature a related but yet distinct receptor binding profile. Here the X-ray structure of FaeGad bound to lactose provides the first structural insight in the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D'-D″″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe150-Glu152 and Val166-Glu170 of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A haemagglutination based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly the crucial D'-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket amongst the FaeG serotypes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; 290(13). DOI:10.1074/jbc.M114.618595 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The formation of adhesive surface structures called pili or fimbriae ('bacterial hair') is an important contributor towards bacterial pathogenicity and persistence. To fight often chronic or recurrent bacterial infections such as urinary tract infections, it is necessary to understand the molecular mechanism of the nanomachines assembling such pili. Here, we focus on the so far best-known pilus assembly machinery: the chaperone-usher pathway producing the type 1 and P pili, and highlight the most recently acquired structural knowledge. First, we describe the subunits' structure and the molecular role of the periplasmic chaperone. Second, we focus on the outer-membrane usher structure and the catalytic mechanism of usher-mediated pilus biogenesis. Finally, we describe how the detailed understanding of the chaperone-usher pathway at a molecular level has paved the way for the design of a new generation of bacterial inhibitors called 'pilicides'. © 2015 The Author(s) Published by the Royal Society. All rights reserved.
    Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences 03/2015; 373(2036). DOI:10.1098/rsta.2013.0153 · 2.86 Impact Factor