PelA and PelB proteins form a modification and secretion complex essential for Pel polysaccharide-dependent biofilm formation in Pseudomonas aeruginosa

Abstract

The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); X-ray crystallography; biofilm; polysaccharide; protein-protein interaction.

Figure 1.

PelA interacts with PelB in P. aeruginosa. A, addition of a C-terminal hexahistidine tag (C-His₆) to PelA does not affect surface attachment (top panel), biofilm formation (middle panel), or protein levels (bottom) compared with wild type. NSB refers to a nonspecific band used as a loading control. Error bars represent the standard error of the means of two independent experiments performed in triplicate. The arrow indicates location of the pellicle. B, Western blot analysis of solubilized membranes (input (In)) and elutions (E) from nickel-affinity pulldowns from untagged PelA (wild type) or His-tagged PelA (C-His₆) strains. C, table summarizing spectral counts seen for each of the Pel proteins. The values represent an average of two biological replicates. PelE and PelG were not detected in either experiment. D, subcellular fractionation of the cytoplasmic (C), inner membrane (IM), periplasmic (P), and outer membrane (OM) components of PAO1 ΔwspF Δpsl P_BADpel strain (parental) and associated deletion and complementation strains as indicated. Fractions were probed using protein-specific antibodies with PilP and PilQ serving as inner and outer membrane controls, respectively. Molecular mass markers are indicated in kDa with the exception of PilQ, which is detected in the stacking gel.

Figure 2.

PelB is a TPR-containing protein. A ribbon representation of the structure of the PelB TPR, residues 319–436, with transparent surface representation (gray) is shown. N and C indicate the N and C termini, respectively. Individual TPRs are indicated by color. Only R9 and R10 are complete (cyan and indigo, respectively). The purple, light blue, and pink helices are incomplete TPRs.

Figure 3.

A discrete region of PelB is required for the interaction with PelA. A, schematic depicting the domain organization of PelB with the 19 TPR motifs indicated. The residue numbers for the boundaries of the domains are indicated above. SS, signal sequence. B, subcellular fractionation of the periplasmic (P) and outer membrane (OM) fractions of PAO1 ΔwspF Δpsl P_BADpel ΔpelB + pelB and the associated TPR deletion strains (R8–R19). Alkaline phosphatase (AP) and PilF serve as periplasmic and outer membrane controls, respectively. C, top panel, surface attachment determined using the crystal violet assay. Error bars indicate the standard error of the means of two independent experiments performed in triplicate. Statistical significance was calculated using one-way analysis of variance with Bonferroni correction. *, p < 0.01; **, p < 0.0001; ns, not significant. Bottom panel, standing biofilm assay. The black arrow indicates the location of the biofilm. D, Western blot analysis of the individual PelB TPR deletion strains (R8–R19) demonstrates that protein levels do not differ significantly compared with wild type. PilF serves as a loading control.

Figure 4.

Full-length PelA is required for the interaction with PelB in vitro. A, gel filtration traces of PelA(Δ46) (green), PelB(351–588) (red), and the complex (blue) all overlaid on one chromatogram. B, Coomassie-stained SDS-polyacrylamide gel corresponding to each individual peak as outlined in color. Elution fractions are indicated at the top. C, gel filtration traces of PelA(47–303) (green), PelB(351–588) (red), and the two added together (blue) all overlaid on one chromatogram. D, Coomassie stained SDS-polyacrylamide gel corresponding to each individual peak as outlined in color. Elution fractions are indicated at the top. Molecular mass standards are indicated by arrows: F, ferritin; C, conalbumin; CA, carbonic anhydrase; R, ribonuclease A; A, aprotinin with molecular masses of 440, 75, 29, 13.7, and 6.5 kDa, respectively.

Figure 5.

Interaction with PelB modulates PelA enzyme activity. A, dose-response curves examining the disruption of PAO1 ΔwspF Δpsl P_BADpel biofilm biomass in the presence of PelA, PelA + PelB, or PelB. Error bars represent the standard error of the means of three independent trials performed in triplicate. B, specific activity of PelA, PelA + PelB, or PelB hydrolysis of p-nitrophenyl acetate. Statistical significance was calculated using one-way analysis of variance with Bonferroni correction. Error bars represent the standard error of the means of four independent trials performed in triplicate. **, p < 0.001.

Figure 6.

A model of the PEL modification and secretion complex. From left to right, exopolysaccharide secretion (EPS) in PEL, PNAG, alginate, and cellulose. PelB is a two-domain protein with an outer membrane porin and a periplasmic TPR domain connected by a ∼120-amino acid linker. A discrete region of PelB is essential for the interaction with PelA. In comparison, TPR scaffolds in PNAG and alginate also mediate protein interactions with modification proteins PgaB and AlgX, respectively. BcsZ is a periplasmic glycoside hydrolase, which has not yet been shown to interact with BcsC.