Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis

Abstract

Background Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the “red complex” of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N -acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N -acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. Results We investigated T. forsythia’s peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo- N -acetylmuramidase, exo- N -acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS 2 ) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso -type. Conclusion T. forsythia peptidoglycan is of the A1γ-type like that of E. coli . Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso -DAP instead of LL-DAP, as reported previously. Electronic supplementary material The online version of this article (10.1186/s12866-019-1575-7) contains supplementary material, which is available to authorized users.

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

“…After digestion of PGN with the N -acetylmuramidase mutanolysin, the PGN composition was determined by LC-MS, revealing the presence of GlcNAc-MurNAc-peptides (G-M-tri/tetra) and dimers thereof as typical digestion products. For E. coli [ 13 ], as well as T. forsythia and P. gingivalis , the stem peptide is composed of Ala, Glu, m- DAP and Ala, referring to the PGN-type A1γ, as published previously [ 16 ]. Regarding the PGN of F. nucleatum , Vasstrand et al reported that lanthionine instead of m -DAP occurs in the stem peptide [ 27 , 28 ].…”

“…This study sought to investigate how the periodontal pathogen T. forsythia can benefit from cohabiting biofilm bacteria to overcome its MurNAc auxotrophy . T. forsythia possesses a conventional Gram-negative PGN of the A1γ-type [ 16 ]; thus, it is conceivable to assume that the bacterium procures the essential PGN building block MurNAc from the oral biofilm consortium. Currently it is unknown, which sources of MurNAc likely available in the oral cavity T. forsythia can access.…”

“…PGN sacculi from T. forsythia , P. gingivalis, F. nucleatum , and E. coli were isolated following a published protocol [ 16 , 26 ]. In short, biomass was harvested from bacterial cultures from the stationary growth phase and boiled in 8% SDS to lyse cells, followed by several washing steps with distilled water using ultracentrifugation (60,000 g, 30 min, 60 °C) and dialysis against distilled water.…”

“…In short, biomass was harvested from bacterial cultures from the stationary growth phase and boiled in 8% SDS to lyse cells, followed by several washing steps with distilled water using ultracentrifugation (60,000 g, 30 min, 60 °C) and dialysis against distilled water. Subsequently, PGN preparations were treated with α-amylase (24 mg ml − 1 ; Sigma) for 2 h at 37 °C and, subsequently, with pronase (10 mg ml − 1 ; Sigma) for 2 h at 60 °C to degrade contaminating carbohydrates and proteins, boiled, washed, and dried in a SpeedVac vacuum centrifuge [ 16 ].…”

“…PGN is a bag-shaped macromolecule (PGN sacculus) external to the bacterial cytoplasmic membrane that serves as a protection from adverse environmental effects and disruption due to high internal osmotic pressure and, thus, is essential for bacterial survival [ 14 ]. Despite its predictably unique PGN metabolism, T. forsythia possesses a common Gram-negative PGN structure of the A1γ-type [ 15 ] as was recently published by our group [ 16 ]: MurNAc and β-1,4-linked GlcNAc alternatingly form glycan backbone strands, terminating with a non-reducing 1,6-anhydroMurNAc residue (anhMurNAc) at the reducing end, with stem peptides composed of alanine (Ala), glutamic acid (Glu), meso -diaminopimelic acid ( m- DAP), and Ala, and likely a direct cross-linkage between m- DAP on the third and Ala on the fourth position of cross-linked stem peptides. This PGN type occurs also in P. gingivalis [ 16 ] and Escherichia coli [ 13 ], while in the PGN of F. nucleatum , m -DAP is replaced by lanthionine [ 17 ].…”