Oligomeric lipoprotein PelC guides Pel polysaccharide export across the outer membrane of Pseudomonas aeruginosa

Abstract

Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal β-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.

Keywords: PEL; Pseudomonas aeruginosa; X-ray crystallography; biofilms; exopolysaccharides.

Fig. S1.

Phylogenetic distribution of PelC proteins among bacterial taxa. Circular Bayesian inference dendrogram of 126 bacterial PelC proteins. Genus and species are colored by class (see graphical legend). The E. coli LpoB outer membrane lipoprotein (WP_000164439.1) was selected as an outgroup (shown in black). In total, 80% of basal nodes had posterior probability (PP) > 0.80, and more than 90% of distal nodes had higher (0.80–1.0) PP values.

Fig. 1.

Overall structure of PelC. (A) Topology model of the PelC structure. The loops that face the center of the PelC_Pp pore are indicated in red. (B) Cartoon representation of the tetrameric arrangement of PelC_Gm in the asymmetric unit, with one protomer colored by secondary structure for clarity. (C) Cartoon representation of the dodecameric arrangement of PelC_Pp found in the asymmetric unit. In A–C, N and C represent the N and C termini, respectively. (D and E) Surface representations of the convex and protomer surfaces of PelC_Pp colored by residue conservation. Conservation, as defined by the ConSurf server, is displayed from magenta (conserved) to cyan (variable).

Fig. S2.

Structural similarity of PelC_Gm and PelC_Pp protomers. Alignment of PelC_Pp (magenta) and PelC_Gm (cyan) structures with the secondary structural elements labeled. The black arrows give the overall dimensions of the proteins. N and C denote the location of the N and C termini, respectively. The rmsd between structures is 0.881 Å over 105 C^α.

Fig. S3.

PelC adopts an α/β fold that multimerizes in the asymmetric unit. (A) PelC_Gm crystallizes with four molecules in the asymmetric unit. Density was observed for the vector-encoded thrombin cleavage site (SGLVPRGSHM) found between the N-terminal His₆ tag and the start of the PelC_Gm sequence. This region (blue) forms a β-strand and mediates a number of antiparallel intermolecular contacts between PelC molecules. The sheets formed by these dimers form a number of polar contacts with the sandwiched pair. (B) The selenomethionyl derivative PelC_Pp^L103M crystals exhibit the symmetry of space group C2 with 12 molecules per asymmetric unit. (C) The native protein crystallized in space group P6 with the four molecules (highlighted in gray) in the asymmetric unit. The symmetry of the hexagonal space group results in the formation of a dodecameric ring structure.

Fig. S4.

Structural alignment of PelC_Pp with structurally related proteins. Chain A of PelC_Pp is shown in cyan in both panels. (A) FlgT (PDB ID code 3W1E. Z score 12.6, rmsd 2.2 Å over 118 C^α). (B) CsgG (PDB ID code 4UV3; Z score 12.3, rmsd 2.9 Å over 113 C^α). N and C denote the location of the N and C termini, respectively.

Fig. 2.

PelC_Pa^DSB forms a multimer in vivo. (A) Cartoon representations depicting the model of PelC_Pa and residues targeted for mutagenesis; each cysteine from a single protomer (light blue) was designed to interact with the corresponding cysteine partner from the neighboring molecule (red). (B) Western blot analysis using PelC-specific antibodies of whole-cell lysates of the indicated P. aeruginosa strains in the presence (+) or absence (−) of reducing agent (DTT).

Fig. S5.

Equilibrium analytical ultracentrifugation analysis of PelC orthologs. (A–C) Radial distributions of the absorbance in the centrifuge cell at equilibrium at 18,000 rpm at 4 °C using a Beckman Optima XL-A analytical ultracentrifuge using an An-60 Ti rotor for PelC_Pa at 0.13 mg/mL (A), 0.18 mg/mL PelC_Gm (B), and 0.17 mg/mL PelC_Pp (C)_. (A–C, Upper) Residuals for each fit.

Fig. S6.

PelC^DSB forms a single oligomeric species and retains biofilm-forming capacity. (A) Microtiter dish biofilm (Upper) and standing pellicle (Lower) assays demonstrate that the insertion of cysteine residues into PelC_Pa does not affect pellicle formation or surface attachment. Error bars represent the SEMs of three independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction; **P < 0.0001. (B) Uncropped blots of a 16% Tris-glycine gel blotted for PelC using protein-specific antibodies (Left). A band is observed at 35 kDa in the absence of DTT; however, this band is also present in the pelC deletion strain. (Right) Uncropped blot of a 10% Tris-glycine gel blotted for PelC. This blot allowed resolution of the higher molecular-weight markers, and demonstrates that PelC_DSB migrates above the 250-kDa marker.

Fig. S7.

PelC^DSB analyzed by negative-stain EM. (A) Raw negative-stain EM image of PelC^DSB_. (Scale bar: 100 Å.) (B) Representative class averages of PelC^DSB. (Scale bar: 100 Å.)

Fig. 3.

PelC localization and analysis of membrane proximal residues. (A) The susceptibility of PelC to proteinase K (PK) treatment in whole cells (intact) and lysed cells (lysed) was observed using PelC-specific antibodies. PilF is an inner leaflet OM lipoprotein. (+) and (−) indicate the presence or absence of PK. (B) Surface representation of PelC_Pp with charge distribution of the convex surface ranging from −12 kT/e (red) to +12 kT/e (blue). (C) Cartoon representation of PelC_Pp with the aromatic tryptophan belt displayed in stick representation (red). (D) Cartoon representation of the PelC_Pa model with conserved residues targeted for mutation shown as sticks (red). (E, Top) Surface attachment determined using the microtiter dish biofilm assay; error bars indicate the SEM of 10 independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction; **P < 0.0001 compared with the wild-type strain. (E, Middle) Standing biofilm assay; the black arrow indicates the location of the biofilms. (E, Bottom) Western blot analysis of whole-cell lysates of the indicated P. aeruginosa strains using PelB- and PelC-specific antibodies.

Fig. S8.

Mutations to PelC membrane proximal residues in PA14. Microtiter dish biofilm (Upper) and colony morphology (Lower) of wild-type PA14, a PA14 ΔpelC strain and the PelC chromosomal point mutants C19S, E121A, W149A, and R151A. Error bars represent the SEMs of 10 independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction, where **P < 0.0001, comparing mutants to wild-type PA14.

Fig. S9.

Mutation to W149 in PelC^DSB is not sufficient to disrupt biofilm formation. Microtiter dish biofilm assay (Top), standing biofilm assay (Middle), and Western blot (Bottom) of PAO1 ΔwspF Δpsl P_BADpel ΔpelC + pelC (WT) and the indicated mutant strains. Error bars represent the SEMs of 10 independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction, where **P < 0.0001. n.s., not significant.

Fig. 4.

A negatively charged PelC pore is required for biofilm formation. (A) Surface representation of PelC_Pp with charge distribution of the concave surface ranging from −12 kT/e (red) to +12 kT/e (blue). (B) Cartoon representation of the PelC_Pa model with charged residues targeted for mutation shown as sticks (red). (C, Top) Surface attachment determined using the microtiter dish biofilm assay; error bars indicate the SEM from 10 independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction; *P < 0.01, **P < 0.0001 compared with the wild-type strain. (C, Middle) Standing biofilm assay; the black arrow indicates the location of the biofilm. (C, Bottom) Western blot analysis of whole-cell lysates of the indicated P. aeruginosa strains using PelC-specific antibodies. NSB, nonspecific band.

Fig. S10.

Mutation to D119K in PelC^DSB does not abrogate oligomer formation. Microtiter dish biofilm assay (Top), standing biofilm assay (Middle), and Western blot (Bottom) of PAO1 ΔwspF Δpsl P_BADpel ΔpelC + pelC (WT) and the indicated mutant strains. Error bars represent the SEMs of 10 independent trials performed in triplicate. Statistical significance was evaluated using one-way ANOVA with Bonferroni correction, where **P < 0.0001. n.s., not significant.

Fig. 5.

PelB is an outer membrane protein with a predicted β-barrel porin domain. (A) Subcellular fractionation of the cytoplasmic (C), inner membrane (IM), periplasmic (P), and outer membrane (OM) fractions of PAO1 ΔwspF Δpsl P_BADpel. PilP and PilQ serve as inner and outer membrane controls, respectively. (B) Western blot analysis of the gel mobility assay in PAO1 ΔwspF Δpsl P_BADpel using PelB-specific antibodies. Samples of cell lysate were either untreated (25 °C) or heat treated (80 °C). The molecular weight is indicated on the left.

Fig. 6.

Model of the Pel polysaccharide outer membrane secretion complex. (A) PEL becomes positively charged following deacetylation by PelA. The polymer is drawn toward the electronegatively charged concave surface of PelC, guiding PEL toward the exit channel formed by PelB. The region between the β-barrel and the TPR domains of PelB is proposed to thread through the PelC pore. Figure is not to scale. (B) Cartoon representations of PelC_Pp (Top, blue) modeled with 16-stranded β-barrel PgaA (purple; PDB ID code 4Y25) and CsgG (Bottom) (PDB ID code 4UV3). CsgG forms a 36-stranded transmembrane β-barrel (purple) from the β-strands of nine protomers. The inner diameter of each β-barrel is indicated at the top, and the overall diameter is marked below. The diameter of the PelC_Pp aromatic ring is also indicated.