The protein BpsB is a poly-β-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica

Abstract

Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni(2+) and Co(2+). The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms.

Keywords: Bordetella; Bps; Exopolysaccharide Biosynthesis; PNAG; biofilm; carbohydrate esterase; carbohydrate processing; enzyme structure; microbiology; structural biology.

FIGURE 1.

Schematic representation of BpsB and subcellular localization. A, domain organization and comparison of BpsB from B. bronchiseptica, PgaB from E. coli, and IcaB from S. epidermidis, from N to C terminus. Residue boundaries are listed on the top of the diagram with the predicted domains labeled. The periplasmic signal sequence is abbreviated as SS and regions with no predicted regular secondary structure are depicted as thick black lines. B, localization of BpsB. Fractions containing cytoplasmic and periplasmic proteins (lane 1), inner membrane proteins (lane 2), and outer membrane proteins (lane 3) were separated via SDS-PAGE followed by Western blotting and detection with either anti-FLAG antibodies, anti-BcfA antibodies (outer membrane protein control), or anti-BvgA antibodies (soluble protein control). Purified T7-tagged BcfA (56) or untagged BvgA (63) proteins were used as positive controls (lane 4).

FIGURE 2.

BpsB(35–307)_MMS adopts a (β/α)₇ fold with an electronegative active site. A, cartoon representation of BpsB(35–307)_MMS with the β-strands (blue) and α-helices (red) of the (β/α)₇ barrel labeled β1–7 and α1–7, respectively. The CE4 “capping” helix is colored purple. Additional ordered secondary structure elements are colored green; loops are colored light gray; the Ni²⁺ ion is a teal sphere, and the N and C termini are labeled accordingly. B, electrostatic surface representation of BpsB(35–307)_MMS shown in the same orientation as A and rotated 90° to the right show an electronegative face with loop L1 occluding the active site. Quantitative electrostatics are colored from red (−5 kT/e) to blue (+5 kT/e).

FIGURE 3.

Structural comparison of BpsB(35–307)_MMS and PgaB(43–309). A, superposition of BpsB(35–307)_MMS (blue) and PgaB(43–309) (orange) shows the differences in the canonical (β/α)₇ fold. Loop L1 (magenta) in BpsB(35–307)_MMS lies directly across the active site, and α8 is only present in PgaB. B, surface representation of BpsB(35–307)_MMS (blue) and PgaB(43–309) (orange) reveals distinct differences in loop L1 (magenta). BpsB contains a secondary groove (yellow) that leads into the active site, which is not present in PgaB because Arg-66 plugs the groove (green). C, active site comparison reveals minor differences between BpsB and PgaB. Residue numbers refer to BpsB/PgaB. The nickel ion throughout the figure is colored teal and shown as a sphere.

FIGURE 4.

Metal-dependent activity of BpsB. A, active site metal analysis of BpsB (blue sticks) shows octahedral coordination of a nickel ion (teal sphere) with Asp-114, His-184, His-189, waters W1 and W2 (red spheres), and a thiocyanate ion (green stick). The |F_o − F_c| density omit map is shown as gray mesh contoured at 3.0σ. B, fluorescamine assay of BpsB shows metal-dependent activity on β-1,6-(GlcNAc)₅ with preference for Co²⁺ and Ni²⁺. Bars represent triplicate experiments with standard deviation.

FIGURE 5.

Length-dependent activity and mutagenesis analysis of BpsB. A, fluorescamine assays of BpsB show an increase in activity with increasing PNAG oligomer length for BpsB(27–701) (white bars) but a loss of length dependence for BpsB(27–311) (gray bars). B, conserved active site residues are required for activity but not the C-terminal domain. Bars represent triplicate experiments with standard deviation.

FIGURE 6.

Detection of Bps by immunoblot and ELISA. A, exopolysaccharides were extracted from overnight-grown cultures of B. bronchiseptica strains using EDTA and proteinase K treatment. Extracted samples were spotted onto nitrocellulose membranes and probed with goat antibody raised against S. aureus deacetylated PNAG conjugated to diphtheria toxoid (8). B, B. bronchiseptica strains or PBS were added to an ELISA plate to measure the amount of Bps present on the surface of bacterial cells. The absorbed human serum at indicated dilutions was used to probe for Bps, followed by detection using goat anti-human IgG conjugated to horseradish peroxidase. Error bars are representative of the standard deviation. *** indicates a p value of < 0.0001 between Δbps and other strains, and # denotes a p value of <0.05 between WT and ΔbpsB strains. Samples were run in duplicate and are representative of one of two independent experiments.

FIGURE 7.

Localization of the Bps exopolysaccharide. Electron microscopy analysis for the presence of Bps on the surface of B. bronchiseptica was carried out by adsorbing 1 × 10⁶ bacteria onto carbon-coated gold grids in a humidified chamber for 1 h, and then exposed to 10% heat-inactivated Bps-enriched human serum for 1 h after blocking with 2% BSA in PBS. The bound antibodies were detected with 6 nm gold- labeled anti-human polyclonal antibodies at a dilution of 1:10 in the blocking buffer using negative staining with 2% phosphotungstic acid, pH 6.6, and analyzed with a Tecnai transmission electron microscope. Arrows indicate the presence of gold-labeled Bps, which is present on the surface of the WT and ΔbpsB strains.

FIGURE 8.

BpsB is required for biofilm formation. A, quantification of B. bronchiseptica biofilm formation on glass slides. Glass slides were removed at designated time points, and the adhered bacteria (CFUs) were enumerated by plating on BG agar plates. Bar represent triplicate experiments with standard deviation. WT (black bars), Bvg⁻ strain (light gray bars), Δbps strain (dark gray bars), ΔbpsB strain (white bars), complementation vector alone (hatched bars), and ΔbpsB strain complemented with bpsB on a plasmid (ruled bars). Asterisks designate a p value of <0.05 (Student's t test). B, S.E. images of B. bronchiseptica biofilms after 96 h. WT and ΔbpsB mutant strains were grown statically on vertically submerged coverslips. Bar, 10 μm.