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. 2015 Jan 2;290(1):601-11.
doi: 10.1074/jbc.M114.616904. Epub 2014 Nov 11.

Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin

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Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin

Ylan Nguyen et al. J Biol Chem. .

Abstract

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.

Keywords: Adhesin; Bacterial Adhesion; Crystal Structure; Pilin; Pseudomonas aeruginosa (P. aeruginosa); Pseudopilin; Twitching Motility; Type II Secretion System (T2SS); Type IV Pili.

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Figures

FIGURE 1.
FIGURE 1.
Gene organization of T4P and T2S minor components. The organization of pilin, minor pilin, pseudopilin, and minor pseudopilin genes in type IV pili and type II secretion systems of model organisms P. aeruginosa, E. coli, and Neisseria spp. Equivalent genes are similarly colored. Major subunit genes are shown in white. P. aeruginosa adhesin and anti-retraction factor PilY1 (yellow) is encoded in the minor pilin operon, whereas its Neisseria equivalents, PilC1 and PilC2, are not. Neisseria-specific minor pilin genes pilV and comP (gray) are not clustered with other minor pilin genes. P. aeruginosa gene pilY2, encoded between pilY1 and pilE, may be a pseudogene. Line breaks (//) denote noncontiguous genomic organization.
FIGURE 2.
FIGURE 2.
Incorporation of minor pilins into pili. Pili were sheared from the surface of minor pilin-pilT mutants and fractions probed by Western blot for incorporation of the minor pilins using anti-FimU (1/1000), anti-PilV (1/1000), anti-PilW (1/1000), anti-PilX (1/500), anti-PilY1 (1/1000), and anti-PilE (1/1000) antibodies. Proteins were detected at their expected masses except for PilY1, which had an apparent mass of ∼80 kDa.
FIGURE 3.
FIGURE 3.
Interaction of FimU with major pilin and minor pilins. Direct protein-protein interactions of FimU with the major pilin and other minor pilins were probed using a bacterial adenylate cyclase two-hybrid assay. FimU was N-terminally tagged with the T18 fragment and the major pilin, and other minor pilins were N-terminally tagged with the T25 fragment. A, E. coli BTH101 recombinants were grown on LB agar + X-gal and McConkey + maltose, producing blue or red colonies, respectively, if there is an interaction. B, β-galactosidase activity of the same strains was measured as described under “Experimental Procedures.” The leucine zipper constructs T18-zip and T25-zip were used as positive controls.
FIGURE 4.
FIGURE 4.
Crystal structure of FimUΔ1–28. The x-ray crystal structure of native FimUΔ1–28 was solved to 1.4 Å (top left panel). For comparison, the full-length structure of P. aeruginosa PilA (PDB code 1OQW) is shown in the right panel. The N-terminal α-helix is colored cyan, the αβ-loop is magenta, and the disulfide-bonded loop is blue. The intervening β-strands are colored gray. A topology diagram of FimU (produced using the TopDraw algorithm) depicting the organization of the structural elements is shown in the bottom left panel.
FIGURE 5.
FIGURE 5.
Structural comparison of FimU and T2S pseudopilin GspH. A, comparison of FimUΔ1–28 (left panel) with the x-ray crystal structure (middle panel) and NMR solution structure (right panel) of T2S pseudopilin GspH (EpsH) reveals similar architecture. The first four β-strands (five for V. cholerae EpsH) are colored in purple. The remaining four are colored in yellow. B, chain A of FimU (green) and EpsH (cyan) align with a root mean square deviation of 2.2 Å.
FIGURE 6.
FIGURE 6.
Pilus assembly in the absence of minor components. A, pilus assembly in the absence of minor components was probed by shearing surface proteins from a pilT mutant lacking the minor pilin fimUpilVpilWpilXpilY1pilE and minor pseudopilin xcpUVWX operons compared with single operon mutants and complementation with either operon and analyzing by SDS-PAGE. The flagellin band was used a loading control. The sheared surface protein samples were probed for the incorporation of minor pseudopilin XcpW into the pilus by Western blot analysis with an XcpW specific antibody (1/1000). B, cells recovered following shearing of surface proteins were lysed, separated by SDS-PAGE, and probed for levels of PilA, PilW, and XcpW using PilA (1/5000), PilW (1/1000), and XcpW (1/1000) specific antibodies. C, mutants made on minor pilin or minor pseudopilin retraction-deficient backgrounds were used to identify the minimum number of minor components required for pilus assembly by shearing the surface proteins from the cell and analyzing the surface pilus fractions by SDS-PAGE. The flagellin band was used a loading control.
FIGURE 7.
FIGURE 7.
Model of pilus assembly. PilVWX and PilY1 interact, creating an interface that is bound by PilE. FimU interacts directly with PilA and connects it with the PilVWXY1E subcomplex through interactions with PilV and PilE.
FIGURE 8.
FIGURE 8.
Comparison of loops in FimU, PilHNm, and EpsH. Comparison of truncated V. cholerae T2S pseudopilin EpsH (left panel) with P. aeruginosa FimU (center panel) and a Phyre2-generated model of N. meningitidis PilH (right panel) showing differences in the β1-β2 (colored green) and β4-β5 loops (colored blue).

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