Fig 1.
PgaB exhibits glycoside hydrolase activity.
Schematic representation of the (A) PgaB constructs used in this study and (B) PNAG and dPNAG composition. (C) Reducing sugar assay with 2 mg/ml dPNAG purified from S. aureus with 2 μM PgaB variants and DspB over 24 h and PgaBBb hydrolase activity against dPNAG over 2 h. Error bars represent the standard error (S.E.) from two independent experiments performed in duplicate. (D) Biofilm disruption assay performed with 1.3 μM enzyme and the indicated strains. ****P ≤ 0.0001, **P ≤ 0.01, NS: no significant difference. Statistical significance was evaluated using two-way analysis of variance and Tukey’s multiple comparison test. (E) Dose response curves examining S. carnosus, S. epidermidis, and E. coli biofilm disruption. In panels D/E the error bars represent the S.E. and n = 3. In all graphs, Bb-DAGH is coloured blue, Bb-GH red, and the D474N variant brown.
Fig 2.
Bb-GH is active in fetal bovine serum and potentiates killing by gentamicin.
(A) Dose response curves investigating the effect of fetal bovine serum on Bb-GH and DspB activity during disruption of S. epidermidis SE801 and E. coli K-12 biofilms. Error bars represent S.E., n = 3. (B) Enumeration of S. epidermidis SE801 after biofilm treatment with Bb-GH or DspB and 500 μg/ml gentamicin for 20 h. (C) Enumeration of E. coli K-12 after biofilm treatment with Bb-GH or DspB and 50 μg/ml gentamicin for 4 h. For (B) and (C) the mean was calculated from three independent experiments, error bars represent S.E.M. Statistical significance was calculated using one-way analysis of variance and Tukey’s multiple comparison test. ****P ≤ 0.0001, **P ≤ 0.01, *P ≤ 0.05, NS: no significant difference.
Fig 3.
Bb-DAGH degrades in situ produced PNAG.
(A) Schematic of the in situ PNAG digestion assay. PNAG is produced in situ using PgaCD containing E. coli membranes. PNAG production and enzymatic cleavage is verified by mass-spectrometry after purification of the hydrolyzed sample. (B) MALDI-TOF MS profiles of product released by PgaBBb variants. The profile of the Bb-DAGH treated sample is enlarged to show details in the detected dPNAG/PNAG cleavage products. Profiles of samples containing no detectable dPNAG/PNAG components are displayed in the bottom row. Symbols represent identified structures less than 1% of relative intensity, ‡: fully N-acetylated structures, †: mono-deacetylated structures, *: di-deacetylated structures.
Table 1.
Effect of treating in situ-produced PNAG with PgaB.
Fig 4.
Structural analysis of Bb-DAGH treated PNAG reveals a consensus recognition motif.
(A) MALDI-TOF MS/MS profile of the m/z ion = 2031.71, a mono-deacetylated PNAG 10-mer. Oligosaccharides were reduced by NaBH4 allowing the reducing terminus to be defined. The asterisk indicates the reducing end of the molecule. (B) Graphical representation of the dPNAG 10-mer using the same key as depicted in Fig 1B. Sugar units are numbered relative to the cleavage site at the reducing end.
Fig 5.
The (β/α)8 TIM-barrel of Bb-GH has a long and deep groove with a highly conserved central pocket.
(A) Cartoon representation of the (β/α)8 barrel with each β/α segment shown in a different color. (B) Electrostatic surface representation in the same orientation as panel A. The central pocket forming the deepest region of the groove is highlighted in green. (C) Surface representation with residues coloured based on conservation level (yellow: insufficient data). (D) Surface representation in the same colour coding and orientation as panel A. Slice 1 and 2 display different sections of the groove as indicated by the vertical lines in left panel and viewed from along the groove from left to right.
Table 2.
Summary of data collection and refinement statistics.
Fig 6.
Residue D474 aligns with the catalytic aspartate from structurally similar GH families.
(A) Sequence comparison of regions containing catalytic residues. Residues in bold form the consensus motif for each GH family. Residues in bold italics are the catalytic residues. D474 and the structurally aligned aspartates are highlighted in red. Superposition of Bb-GH with (B) DspB (PDB 1YHT) [42], (C) AMCase (PDB 2YBU) [81], and (D) α-amylase (PDB 7TAA) [82]. Bb-GH β-strands are shown in the same color scheme as Fig 5A, all other enzymes are shown in grey.
Fig 7.
Mutagenesis suggests D474 is involved in catalysis and other charged residues crucial for dPNAG binding.
(A) Reducing sugar assay with 2 mg/ml dPNAG purified from S. aureus with 2 μM PgaB constructs over 24 h. Error bars, S.E. from two independent experiments performed in duplicate. (B) E. coli biofilm disruption assay. Error bars represent the S.E. with n = 3. (C) EC50 values from E. coli biofilm disruption assay. ND: EC50 not determined as > 50% biomass remained or no plateau was reached after treatment with 5 μM enzyme for 2 h. Error bars show 95% confidence interval, and the same color scheme in A is used. (D) Transparent surface representation of the Bb-GH binding groove with mutated amino acids shown in stick representation. Note that D364 is fully buried and only visible due to the transparent representation.
Fig 8.
Sequence and structural comparison of Ec-GH and Bb-GH reveal differences in active site accessibility.
(A) Sequence alignment between Bb-GH and Ec-GH showing identical and similar residues shaded and boxed in black, respectively. Secondary structure elements of Bb-GH are shown above the sequence alignment with the canonical (β/α)8 elements labeled. Residues forming the active site pocket and analyzed by mutagenesis are highlighted with the same color scheme as in Fig 7, with loops 3 and 7 highlighted by green boxes. The sequence alignment figure was generated using ESPript 3.0 [83]. (B) Structural superposition of Bb-GH and Ec-GH shown in cartoon representation. (C) Surface representation of Ec-GH and Bb-GH. In (B) and (C) loops 3 and 7 are highlighted in red and green, respectively.
Fig 9.
Proposed recognition of dPNAG required for polymer cleavage by Bb-GH.
(A) Surface representation of Bb-GH with residues coloured based on conservation level (yellow: insufficient data). Shown in stick representation are GlcN (PDB 4P7N), GlcNAc (PDB ID 4P7Q), and (GlcNAc)4 (PDB ID 4P7R) that have been co-crystallized with Ec-GH and were modeled into the Bb-GH structure. (B) Proposed cleavage mechanism based on mutagenesis and mass spectrometry analysis. Crucial surfaces are highlighted in red, orange, and pink. Positions -4 to +5 denote expected binding sites of GlcN/GlcNAc units relative to the cleavage site. Positions +2 to +5 were taken directly from (GlcNAc) 4, position -1 coincides approximately with a GlcN monomer bound to PgaBEc (PDB 4P7N) and position -3 with a GlcNAc monomer bound to PgaBEc (PDB 4P7Q) [24].