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. 2016 Apr 21;165(3):690-703.
doi: 10.1016/j.cell.2016.03.016. Epub 2016 Apr 7.

A Distinct Type of Pilus from the Human Microbiome

Qingping Xu  1 , Mikio Shoji  2 , Satoshi Shibata  2 , Mariko Naito  2 , Keiko Sato  2 , Marc-André Elsliger  3 , Joanna C Grant  4 , Herbert L Axelrod  1 , Hsiu-Ju Chiu  1 , Carol L Farr  4 , Lukasz Jaroszewski  5 , Mark W Knuth  4 , Ashley M Deacon  1 , Adam Godzik  5 , Scott A Lesley  6 , Michael A Curtis  7 , Koji Nakayama  8 , Ian A Wilson  9
Affiliations

A Distinct Type of Pilus from the Human Microbiome

Qingping Xu et al. Cell. .

Abstract

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

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Figures

Figure 1
Figure 1. Operons, sequence motifs, crystal structures and clustering of the FimA pilin superfamily
(A) Operons encoding the major and minor pili in P. gingivalis strain 33277, compared to an analogous typical pilin encoding operon from the gut microbiome, exemplified by P. distasonis. (P. distasonis. structures determined in this study are colored red). (B) Sequential maturation of pilins via proteolytic and covalent modifications during export from the cytoplasm to the outer membrane. Conserved residues are marked on the top, while secondary structures are labeled at the bottom. Orange box: the lipoprotein signal peptide; asterisk: lipidated site. (C) Pilin structures determined and their possible roles (see Table S1). (D) Crystal structure of FimA4. The Arg/Lys specific cleavage site between A1 and B1 is marked by a scissor symbol. The conserved β-sheet core is colored in green, the A1 strand in blue, inserts in cyan, and the A1′ and A2′ appendage in red. (E) A topological diagram of conserved secondary structures in FimA4 colored coded as in D. (F) Crystal structures of DUF3988 members with the A1′ strand in “open” (BfrFim1I and BthFim1A) conformations, in comparison with BfrFim1L that is closely related to BfrFim1I, but containing a “closed” A1′ strand. Color coded as in D. (G) Clustering of crystal structures into three families that form the FimA superfamily and sequence features below of each family.
Figure 2
Figure 2. Structure of FimA highlighting the A1 and A1′ regions and pilus-like assemblies in the crystal lattices
(A) Grooves for binding strands A1 (blue) and A1′ (red) align with one another and form a continuous groove, as shown here using FimA4 as an example. (B) Arrangement of the two amphipathic strands A1, A1′, and the A1-B1 loop containing the cleavage site. Hydrophobic residues on the buried face of both strands are shown as sticks. The structure is colored by a B-value gradient from blue (low B-value) to red (high B-value). (C) FimA4 packs in head-to-tail columns of subunits in the crystal. The A1 and A1′ regions are colored in blue and red. (D-E) Close-up view of the interface between two subunits in one FimA4 (D) or BdiFim1A (E) column. Distances between a point near the C-terminus of G2 and a point near N-terminus of A1 of the adjacent pilin are shown as green dashed lines. (F) An example of head-to-head crystallographic dimers formed by domain swapping of the C-terminal A1′ strand (red).
Figure 3
Figure 3. The C-terminal region of FimA is critical for polymerization
(A) Alignment of the C-terminal sequences for the A2′ strands of P. gingivalis FimA genotypes (FimA1-5), FimB (strain W83), Mfa1, and Mfa2. The sequence numbering of FimA is based on the mature pilin, whereas other proteins are numbered based on the prepilin forms. (B) FimA mutants and their sequence features compared to pre- and mature FimA. PreFimA, prepilin with N-terminal signal peptide. Mature FimA, mature FimA pilin. FimA W328A, mutant of mature FimA. FimA327, mature FimA truncated after residue 327 (deletion of Trp328 and A2′). (C) Immunoblot of FimA C-terminal mutants. (D) EM micrographs of WT and fimA mutants. (E) Functional mapping of adhesion and immune epitopes onto a homology model of FimA. (F) Sequence comparison between the A1, A1′, and A2′ strands of the FimA pilin. Conserved residues that are predicted to be buried in the grooves are colored yellow. Surface residues mutated to cysteines are marked by red circles. (G) Formation of disulfide bonds between cysteine pairs introduced on the A1′ strand and NTD, respectively. Lane 1: reducing reagent β ME, 2: no β ME or H2O2, 3, 4: oxidizing reagent H2O2 in two concentrations. (H) A model of the A1′ strand replacing the A1 strand in the NTD. The Cβ-Cβ distances (Å) of Cys-Cys pairs are shown. The cross-linked Cys-Cys pairs observed are marked by green dots. (I) Formation of cross-links between the CTD and A2′ strand. (J) A model of the A2′ strand replacing the A1′ strand in the CTD.
Figure 4
Figure 4. Structure and function of anchor pilins
(A) Structures of anchor pilins (BthFim3B as example) differ significantly from structural pilins (FimA4 as example) in regions important for polymerization. (B) The C-terminus of BovFim4B adopts an “open” conformation. The sequence of the appendage with the A1′ and A2′ strands and additional C-terminal disordered region is shown in red. (C) Sequence features of anchor pilins of the minor and major pili, Mfa2 and FimB (see Figure 1B for caption). (D) Immunoblot of cell lysates of P. gingivalis strains with anti-FimA, anti-Mfa1 and anti-Mfa2 antibodies. Precursor and mature forms of pilins are marked by red and blue arrows, respectively. In lanes 3 and 4, the bands correspond to previously identified precursor forms (Kadowaki et al., 1998; Shoji et al., 2004). (E) Immunoblot with anti-Mfa2 antibody (left) and autoradiography of the SDS–PAGE gel (right) of the protein sample of P. gingivalis grown in the presence of C14-palmitic acid and immunoprecipitated with anti-Mfa2 antibody. Labeled Mfa2 are marked by red arrows (nonspecific bands marked by an asterisk). Lane 1: fimA-null, 2: fimA-null-mfa2-null, 3: fimA-null-mfa2-null complemented with mfa2+, 4: fimA-null-mfa2-null complemented with the mfa2[C29A] mutant. (F) Fractionation analysis. Lane 1: whole cell lysate, 2: cytoplasm and periplasm, 3: total membrane fraction, 4: inner membrane (soluble fraction by Triton X-100), 5: OM (insoluble fraction by Triton X-100). (G) Dot blot analysis with anti-Mfa2 antibody. Lanes as in (E).
Figure 5
Figure 5. The C-terminus of Mfa2 is essential for its incorporation into the Mfa1 pili
(A) fimA-null mutants constructed to study the impact of truncating the C-terminus of Mfa2 on the length of the Mfa1 pili. 1: fimA-null of P. gingivalis 33277, 2: mfa2-null of the mutant 1, 3: Mutant 2 complemented with mfa2+ on a plasmid, 4: Mutant 2 complemented with mfa2[C29A] on a plasmid, and 5-7: Mutant 2 complemented C-terminal truncated of mfa2 (residues 1-303, 1-314 or 1-321). (B) Expression of Mfa2 and Mfa1 in mutants 1-7 detected by antibodies. (C) Cell surface presence of Mfa2 analyzed by dot blots. Cell surface protein HBP35 was used as a control. (D) Average length (nm) of the Mfa1 pili in mutants 1-7. Error bars represent the standard deviation. * P<0.001. (E) Representative EM images of mutants 1-7 (pili are marked by arrows). (F) Mfa1 and Mfa2 interactions in mutants analyzed by western blots (Mfa2-C29A does not interact with Mfa1 since it cannot reach the OM). Asterisk, non-specific bands.
Figure 6
Figure 6. Representative structures of tip pilins
(A) Crystal structure of the tip pilin Mfa4. The unbiased experimental electron density map (solvent modified) of the A1-B1 loop and the C-terminus are shown (contoured at 1 σ, orange). (B) The structure of BovFim1C consists of a prototypical pilus assembly domain (gray/blue/red) connected to a C-terminal CTLD (cyan). (C) Surface representation of BovFim1C color coded as in B with inserts in green. (D) Structural comparison of the CTLD domain of BovFim1C with another bacterial CTLD. The common core regions are colored in violet/gold/cyan.
Figure 7
Figure 7. A proposed assembly mechanism for type V pili
(A) A schematic model for pilus assembly. (B) Proposed atomic model of a FimA pilus filament. FimA subunits are colored alternately in red and cyan. (C-D) Topological diagram of one assembled subunit in a type V pilus assembly (C) compared to that of type I pilus (D). Donor strands are colored red.
See this image and copyright information in PMC

Comment in

  • A New Pillar in Pilus Assembly.
    Coyne MJ, Comstock LE. Coyne MJ, et al. Cell. 2016 Apr 21;165(3):520-1. doi: 10.1016/j.cell.2016.04.024. Cell. 2016. PMID: 27104974

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