The differential affinity of the usher for chaperone-subunit complexes is required for assembly of complete pili - PubMed (original) (raw)

The differential affinity of the usher for chaperone-subunit complexes is required for assembly of complete pili

Qinyuan Li et al. Mol Microbiol. 2010 Apr.

Abstract

Attachment to host cells via adhesive surface structures is a prerequisite for the pathogenesis of many bacteria. Uropathogenic Escherichia coli assemble P and type 1 pili for attachment to the host urothelium. Assembly of these pili requires the conserved chaperone/usher pathway, in which a periplasmic chaperone controls the folding of pilus subunits and an outer membrane usher provides a platform for pilus assembly and secretion. The usher has differential affinity for pilus subunits, with highest affinity for the tip-localized adhesin. Here, we identify residues F21 and R652 of the P pilus usher PapC as functioning in the differential affinity of the usher. R652 is important for high-affinity binding to the adhesin whereas F21 is important for limiting affinity for the PapA major rod subunit. PapC mutants in these residues are specifically defective for pilus assembly in the presence of PapA, demonstrating that differential affinity of the usher is required for assembly of complete pili. Analysis of PapG deletion mutants demonstrated that the adhesin is not required to initiate P pilus biogenesis. Thus, the differential affinity of the usher may be critical to ensure assembly of functional pilus fibres.

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Figures

Fig. 1

Model for the assembly and secretion of P pili by the CU pathway. The Pap proteins are represented by single letters (A, PapA; H, PapH; etc.). The dimeric PapC usher is depicted with the central β-barrel domain forming a channel that spans the OM, the N- and C-terminal domains (labeled N and C, respectively) located in the periplasm, and the plug domain (labeled P) gating the channel shut. Pilus subunits cross the inner membrane via the Sec general secretory pathway and then form binary complexes with the PapD chaperone. PapDG complexes have highest affinity for the usher and initiate pilus assembly by binding to the N-terminal domain of an usher monomer. The usher catalyzes subunit incorporation into the pilus fiber and allows secretion of the fiber to the cell surface.

Fig. 2

PapC F21 is required for P pilus assembly. A. Alignment of the chaperone-subunit binding regions of the N-terminal domains of the PapC and FimD ushers. Phenylalanine residues are indicated with grey shading. Residue numbers relative to the mature N-termini of PapC and FimD are indicated above and below the alignment, respectively. B. Pilus purification. Strain AAEC185/pMJ2 (Δ_papC pap_ operon) was complemented with vector only, WT PapC, or the indicated PapC mutant. Pili were isolated from the bacterial surface by heat extraction and magnesium precipitation, and then subjected to SDS-PAGE. The PapA major rod subunit (A, upper panel) was visualized by Coomassie blue staining. Pilus tip subunits (PapG, E and F, lower panel) were detected by immunoblotting with anti-P pilus tips antiserum.

Fig. 3

PapC F21A binds to pilus subunits in vitro and in vivo. A. Overlay assay for binding of PapDG to PapC. OM fractions were isolated from strain SF100 expressing vector only, WT PapC, or the indicated PapC mutant. Duplicate samples were subjected to SDS-PAGE and either stained with Coomassie blue to show the amount of PapC present (arrow head, upper panel), or transferred to a PVDF membrane for the overlay assay. Binding of PapDG to the usher (arrow head, lower panel) was determined by immunoblotting with anti-PapDG antibody. B. Co-purification of pilus tip subunits with the usher. OM fractions were isolated from strain SF100/pPAP58 (papDJKEFG) expressing vector only, WT PapC, or the indicated PapC mutant. OM fractions were solubilized with non-denaturing detergent and PapC was purified by affinity chromatography. The peak fraction containing PapC was subjected to SDS-PAGE and either stained with Coomassie blue to show the amount of PapC present (C, upper panel) or immunoblotted with anti-P pilus tips antiserum to detect pilus tip subunits that co-purified with the usher (PapG, E and F, lower panel).

Fig. 4

Binding affinities of PapC F21A and R652A for PapDG chaperone-adhesin complexes. Fluorescently labeled PapDG was added to cuvettes at 25 nM final concentration. WT PapC (A), PapC F21A (B) or PapC R652A (C) was then titrated into the cuvettes and fluorescence spectra recorded. The graphs represent normalized changes in total fluorescence emission intensity plotted as a function of PapC concentration, where the total changes in intensity ranged from 21 to 34%. The points represent means ± standard deviation of at least three independent experiments, with one-to-three replicates per experiment. Apparent _K_d values were obtained by fitting the data to a sigmoidal curve.

Fig. 5

Binding affinities of PapC F21A and R652A for PapDA chaperone-rod subunit complexes. Fluorescently labeled PapDAnteK was added to cuvettes at 25 nM final concentration. WT PapC (A), PapC F21A (B) or PapC R652A (C) was then titrated into the cuvettes and fluorescence spectra recorded. The graphs represent normalized changes in total fluorescence emission intensity plotted as a function of PapC concentration, where the total changes in intensity ranged from 16 to 72%. The points represent means ± standard deviation of at least three independent experiments, with one-to-three replicates per experiment. Apparent _K_d values were obtained by fitting the data to a sigmoidal curve.

Fig. 6

PapG is not required for P pilus assembly. A. Pilus purification. Pili were isolated by heat extraction and magnesium precipitation from the surface of strains AAEC185/pBR322 (vector control), AAEC185/pPAP5 (WT pap gene cluster), AAEC185/pPAPΔG (Δ_papG pap_ gene cluster), ZAP594/pHGM98 (chromosomal pap gene cluster with plasmid-encoded papI regulator) and ZAP594ΔpapG/pHGM98 (chromosomal Δ_papG pap_ gene cluster with plasmid-encoded papI regulator). The pilus preparations were subjected to SDS-PAGE and the PapA major rod subunit (A, upper panel) was visualized by Coomassie blue staining. Pilus tip subunits (PapG and E, lower panel) were detected by immunoblotting with anti-P pilus tips antiserum. B. Strains AAEC185/pPAP5, AAEC185/pPAPΔG, ZAP594/pHGM98 and ZAP594ΔpapG/pHGM98 were examined by whole-bacteria, negative-stain transmission electron microscopy. Scale bars = 500 nm.

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The differential affinity of the usher for chaperone-subunit complexes is required for assembly of complete pili - PubMed (original) (raw)