NamZ1 and NamZ2 from the Oral Pathogen Tannerella forsythia Are Peptidoglycan Processing Exo-β- N-Acetylmuramidases with Distinct Substrate Specificities

Abstract

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-β-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-N-acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His₆-fusions and characterized as exo-lytic β-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic β-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-β-N-acetylmuramidases. IMPORTANCE Two exo-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

Keywords: Bacteroidetes; CAZy glycosidase; MurNAc auxotrophy; N-acetylmuramic acid (MurNAc); carbohydrate metabolism; cell wall recycling; disaccharidase; exo-lytic muramidase; family GH171; glycoside hydrolase; pNP-MurNAc; peptidoglycan salvage.

FIG 1

Genomic organization of the exo-β-N-acetylmuramidase namZ of B. subtilis and the three putative namZ-like genes of T. forsythia. The namZ operon of B. subtilis strain 168 (NCBI Reference Sequence accession no. NC_000964.3) includes three genes (amiE, nagZ, and namZ) involved in PGN turnover/recycling and three genes (murQRP) required for the uptake of MurNAc and the transcriptional regulation of the operon: amiE encodes the exo-MurNAc-L-Ala amidase BsAmiE (6), nagZ encodes the exo-β-N-acetylglucosaminidase BsNagZ (20), and namZ encodes the recently identified and characterized exo-β-N-acetylglucosaminidase BsNamZ (in red) (7); furthermore, murP encodes the MurNAc-specific phosphotransferase system (PTS) transporter BsMurP, murQ encodes the MurNAc 6-phosphate etherase BsMurQ, and murR encodes the putative transcriptional regulator of the operon (8). Within the genome of T. forsythia strain ATCC 43037 (NCBI Reference Sequence: NZ_JUET00000000.1), three orthologous namZ-like family GH171 glycosidases (CAZy GH171; www.cazy.org) were identified, TfnamZ1 (Tanf_08370), TfnamZ2 (Tanf_00660), and TfnamZ3 (Tanf_00855). According to the KEGG Database (31), the TfnamZ1 cluster of T. forsythia includes 9 genes (Tanf_08345 to Tanf_08385), with Tanf_08375, Tanf_08380, and Tanf_08385, encoding the MurNAc transporter TfMurT (16), the MurNAc kinase TfMurK (14), and the MurNAc 6-phosphate etherase TfMurQ (16), respectively, which are involved in MurNAc transport and catabolism. The genes upstream of TfnamZ1 (Tanf_08370) encode the inner membrane permease TfAmpG (Tanf_08365), which was recently shown to transport the disaccharides GlcNAc-MurNAc and GlcNAc-anhMurNAc (18) and a putative lytic transglycosylase, which we named TfLytB (Tanf_08360). The functions of the further upstream genes are unknown: Tanf_08350 and Tanf_08355 encode two putative glycosyltransferases (Gtf), and Tanf_08345 a putative xanthan lyase. The gene TfnamZ2 (Tanf_00660) is organized in a gene cluster with three other genes, Tanf_00675, Tanf_00670, and Tanf_00665, which encode an uracil DNA glycosylase, LPS assembly protein, and a hypothetical protein, respectively. The third namZ ortholog of T. forsythia, TfnamZ3 (Tanf_00855) is a monocistronic gene. Genes that code for proteins of a yet uncharacterized function are indicated by a question mark or are presented in gray, characterized proteins are shown in black, and the namZ genes are shown in red.

FIG 2

Specificity of NamZ proteins for the chromogenic substrates pNP-GlcNAc and pNP-MurNAc. The recombinant enzymes TfNamZ1 and TfNamZ2 were assayed, together with two recombinant enzymes from B. subtilis 168 of known activity. The β-N-acetylmuramidase BsNamZ cleaves pNP-MurNAc, and the β-N-acetylglucosaminidase BsNagZ cleaves pNP-GlcNAc, as evidenced by the development of a yellow color upon the release of para-nitrophenol. TfNamZ1 and TfNamZ2, both showed exo-β-N-acetylmuramidase activity, i.e., they specifically cleaved pNP-MurNAc, and showed no β-N-acetylglucosaminidase activity, i.e., no cleavage of pNP-GlcNAc.

FIG 3

Cleavage of MurNAc-GlcNAc by BsNamZ, TfNamZ1, and TfNamZ2. MurNAc-GlcNAc, generated by digestion of S. aureus peptidoglycan (PGN) with SaAtl^AM, SaAtl^Glc, and BsNagZ, was incubated for 20 h at 37°C with the enzymes BsNamZ, TfNamZ1, TfNamZ2, or buffer (as control). The remaining substrate (MurNAc-GlcNAc, green) in the reaction mixture and the reaction products (MurNAc, blue; GlcNAc, red) were quantified using LC-MS. The relative metabolite concentrations (% area under the curve, AUC, of the respective mass peak) represent the mean from three biological replicates. Data were analyzed with one-way ANOVA with Dunnett’s multiple comparison test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01, *, P < 0.05; ns, P > 0.05; ns, nonsignificant.

FIG 4

TfNamZ1 and TfNamZ2 show differences in product formation when incubated with PGN-derived glycans from S. aureus. Peptide-free PGN glycans were generated by digestion of PGN sacculi isolated from S. aureus strain USA300 with the S. aureus amidase SaAtl^AM. The PGN glycan strands were incubated for 20 h at 37°C with TfNamZ1 or TfNamZ2 recombinant enzymes, or buffer as a control (as indicated). Subsequently, the TfNamZ1 digested glycan strands were further digested with BsNagZ (TfNamZ1+BsNagZ). The metabolite concentrations (peak intensities; cps) in the reaction mixtures were analyzed by LC-MS. (A) Base peak chromatograms (gray lines) and extracted ion chromatograms (EIC) corresponding to GlcNAc-MurNAc (G-M; orange), with [M+H]⁺ = 497.198 m/z, are presented. (B) EICs, corresponding to GlcNAc (red) and MurNAc (blue), with [M+H]⁺ = 222.097 m/z and [M+H]⁺ = 294.118 m/z, respectively, were plotted.

FIG 5

Substrate specificity of TfNamZ1 with PGN-derived glycans from B. subtilis. PGN glycan strands from B. subtilis were generated by digestion of PGN sacculi isolated from B. subtilis 168 with the B. subtilis amidase BsCwlC. The enzyme reactions were heat inactivated, and the generated B. subtilis glycan strands were incubated with TfNamZ1 or buffer as a control. Subsequently, the TfNamZ1 digested glycan strands were further digested with BsNamZ (TfNamZ1+BsNamZ) or with BsNagZ (TfNamZ1+BsNagZ). Enzyme reactions were analyzed by LC-MS in positive ion mode. (A) Data are presented as base peak chromatograms (BPC) 200–1300 m/z (gray lines) and as extracted ion chromatogram (EIC), corresponding to GlcNAc-MurNAc (orange) and GlcN-MurNAc (green) with [M+H]⁺ = 497.198 m/z and [M+H]⁺ = 455.187 m/z, respectively, shown as intensities (cps). (B) Data are presented as EICs (intensity; cps), corresponding to GlcNAc (red) and MurNAc (blue), with [M+H]⁺ = 222.097 m/z (red) and [M+H]⁺ = 294.118 m/z (blue), respectively.