Streptococcus mutans SMU.623c codes for a functional, metal-dependent polysaccharide deacetylase that modulates interactions with salivary agglutinin

Abstract

The genome sequence of the oral pathogen Streptococcus mutans predicts the presence of two putative polysaccharide deacetylases. The first, designated PgdA in this paper, shows homology to the catalytic domains of peptidoglycan deacetylases from Streptococcus pneumoniae and Listeria monocytogenes, which are both thought to be involved in the bacterial defense mechanism against human mucosal lysozyme and are part of the CAZY family 4 carbohydrate esterases. S. mutans cells in which the pgdA gene was deleted displayed a different colony texture and a slightly increased cell surface hydrophobicity and yet did not become hypersensitive to lysozyme as shown previously for S. pneumoniae. To understand this apparent lack of activity, the high-resolution X-ray structure of S. mutans PgdA was determined; it showed the typical carbohydrate esterase 4 fold, with metal bound in a His-His-Asp triad. Analysis of the protein surface showed that an extended groove lined with aromatic residues is orientated toward the active-site residues. The protein exhibited metal-dependent de-N-acetylase activity toward a hexamer of N-acetylglucosamine. No activity was observed toward shorter chitooligosaccharides or a synthetic peptidoglycan tetrasaccharide. In agreement with the lysozyme data this would suggest that S. mutans PgdA does not act on peptidoglycan but on an as-yet-unidentified polysaccharide within the bacterial cell surface. Strikingly, the pgdA-knockout strain showed a significant increase in aggregation/agglutination by salivary agglutinin, in agreement with this gene acting as a deacetylase of a cell surface glycan.

FIG. 1.

Structure-based sequence alignment of CE4 esterases. The sequences of three known de-N-acetylases, a de-O-acetylase, and PgdA_Sm from the CE4 family are shown: S. mutans PgdA, C. lindemuthianum chitin de-N-acetylase, B. subtilis PdaA, Streptomyces lividans xylan de-O-acetylase, and S. pneumoniae PgdA. The secondary structures of PgdA_Sm and PgdA_Sp are indicated above and below the alignment, respectively. The secondary structure is highlighted as red helices, and blue strands represent the CE4 esterase domain. Secondary structure not present in the canonical CE4 fold is shown in green. The five CE4 active-site motifs (MT1 to MT5, yellow) are indicated below the alignment. The metal coordinating residues are colored cyan, and the catalytic residues are colored magenta. Residues highlighted in orange show large shifts in surface-exposed loops. The alignment was performed using the Aline program (written and kindly provided by Charlie Bond, University of Western Australia, and Alexander Schüttelkopf, Dundee University).

FIG. 2.

Images of S. mutans UA159 wild type and the pgdA-knockout strain. Closeup images of a single colony of S. mutans UA159 (wild type; left) and the ΔpgdA strain (right). Bacteria were grown anaerobically on brain heart infusion agar plates at 37°C for 7 days. Images were taken with a digital Zeiss camera installed on a Zeiss stereomicroscope (Stemi SV6; Hallbergmoos, Germany) at ×32 magnification.

FIG. 3.

AI of S. mutans UA159 wild type and the pgdA-knockout strain during growth. AIs of the wild-type strain are presented as gray bars; those of the pgdA-knockout strain are presented as white bars. OD₆₀₀ values of the cultures at time points are given in squares for the wild type and triangles for the pgdA knockout. Both AI and OD₆₀₀ values shown are means of three independent samples with standard deviations.

FIG. 4.

Susceptibility of S. mutans to lysozyme. S. mutans wild type and the pgdA knockout were grown in TH broth. In the early exponential phase or at the beginning of the stationary phase (as indicated by the arrows), lysozyme (40 μg/ml) was added to the experimental cultures. (A and C) Wild-type strain; (B and D) pgdA knockout. Solid symbols indicate the control cultures without the addition of lysozyme. Open symbols indicate the cultures treated with lysozyme.

FIG. 5.

Susceptibility of S. mutans toward aggregation by agglutinin. Overnight cultures of S. mutans UA159 wild type (blank squares) and the pgdA knockout (solid diamonds) were washed with buffer and tested for adherence to increasing concentrations of SAG (A) using the fluorescence of SYTO-13 and WGA (B) using the fluorescence of the Alexa Fluor 488 conjugate, as described in Materials and Methods. The values shown are the means of three independent samples with standard deviations.

FIG. 6.

De-N-acetylase activity of S. mutans PgdA. (A) S. mutans PgdA exhibits metal-dependent de-N-acetylase activity. The de-N-acetylase activity of recombinant PgdA_Sm protein purified in the absence of EDTA. Assay mixtures containing 1 mM chitohexaose and 1 μM PgdA_Sm were preincubated for 5 min in solution with different EDTA concentrations. The addition of a 10-fold excess of CoCl₂ and ZnCl₂ was used to reactivate the protein after EDTA treatment. (B) S. mutans PgdA steady-state kinetics. PgdA_Sm (1 μM) was incubated with various concentrations of chitohexaose. The experiments were performed in triplicate, and the mean arbitrary fluorescent units (afu) were converted to the molar concentration of product using a glucosamine calibration curve under identical conditions. The reaction maintained first-order kinetics for 16 h (data not shown), and initial velocities were measured after 12 h.

FIG. 7.

Overview of the S. mutans PgdA structure. (A) Comparison of the overall structures of the S. mutans PgdA and the S. pneumoniae PgdA. α-helices are colored red and β-strands are colored blue in the CE4 esterase domain. Secondary structure elements at the termini of the proteins, outside the typical CE4 fold, are shown in green. Exposed loop regions which differ significantly due to inserts in the S. mutans PgdA structure compared to PgdA_Sp (2C1G) are shown in orange. Secondary structure elements are named in accordance with the sequence alignment in Fig. 1. (B) Stereo image of the active site of S. mutans PgdA. A phosphate ion (yellow) was observed coordinating with the zinc ion (magenta) and other ligands including a water molecule (shown in cyan) in an octahedral manner. The unbiased 1.45-Å |F_o| − |F_c|, φ_calc electron density map is shown (blue) contoured at 2.5 σ. (C) S. mutans PgdA contains an extended surface groove containing exposed aromatic residues. PgdA_Sm and PgdA_Sp (2C1G) structures are shown in surface representation. All aromatic residues are represented as sticks and colored blue. A putative intermediate of PgdA_Sp deacetylation of GlcNAc₃, as described previously (7), is shown in stick representation and colored green. This potential tetrahedral intermediate was superposed onto the PgdA_Sm structure using the PgdA_Sp coordinates to generate a model of a PgdA_Sm-chitooligosaccharide complex. Aromatic residues that line the active site or putative oligosaccharide binding site of PgdA_Sm are labeled. Surface representations of the metal binding triad and the four active-site residues are colored pink.