Uncovering the cell wall and amino sugar metabolism of Tannerella forsythia - Impact on the adaptation to the oral habitat

• Applicant: Professor Dr. Christoph Mayer
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314202130

• Final Report Year: 2025

Final Report Abstract

Tannerella forsythia is an anaerobic, Gram-negative oral bacterium belonging to the Bacteroidota phylum. It is implicated in chronic and aggressive forms of periodontitis, which is an inflammatory disease affecting 750 million people worldwide. In late stages of periodontitis progression, T. forsythia, as part of the red complex consortium of periodontal pathogens along with Porphyromonas gingivalis and Treponema denticola, leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. Earlier axenic culturing studies revealed that T. forsythia is auxotrophic for N-acetylmuramic acid (Mur- NAc). MurNAc is a characteristic and essential carbohydrate component of the glycoconjugate peptidoglycan (PGN), which constitutes the cell walls of bacterial. Whole genome sequencing rationalises the MurNAc auxotrophy, since it revealed that the bacterium lacks the generally essential genes encoding the canonical MurA/MurB enzymes, required for the de novo synthesis of PGN precursors. Thus, this bacterium critically depends on cohabiting bacteria in the oral biofilm for the provision of MurNAc. We were able to show that T. forsythia accepts soluble PGN-derived fragments as well as polymeric PGN as sources of exogenous MurNAc. We discovered two unique, presumably periplasmic, exolytic β-N-acetylmuramidases (NamZ1 and NamZ2), which cleave exogenous PGN glycan strands imported into the periplasm at the non-reducing ends, generating N-acetylglucosamine (GlcNAc)-MurNAc disaccharides and MurNAc, respectively. Whereas all lysozymes (muramidases), characterised so far, cleave within the PGN glycan chains (i.e. they have endo-lytic activity), these novel enzymes cleave off sugar entities from one end of the PGN chain (i.e. they have exo-lytic activity).The NamZ enzymes constitute an entirely novel class of lysozyme-related glycosidases (classified as family GH171 of glycosidases; see CAZy data base: www.cazy.org/GH171.html)). Furthermore, we identified two inner membrane transporters: (i) AmpG that imports the disaccharides generated by NamZ1 and (ii) MurT that imports MurNAc generated by NamZ2. Metabolism of these sugars involves two intracellular MurNAc kinases (MurK1 and MurK2) that were biochemically characterised and their crystal structures solved. Our study unraveled important aspects of the complex PGN salvage metabolism of T. forsythia and thereby identified how this periodontal pathogen thrives in the oral microbial community by means of peptidoglycan degradation. The results of this work may pave new avenues for antibacterial treatment of periodontal disease.

Publications

• Crystal Structure of the N-Acetylmuramic Acid α-1-Phosphate (MurNAc-α1-P) Uridylyltransferase MurU, a Minimal Sugar Nucleotidyltransferase and Potential Drug Target Enzyme in Gram-negative Pathogens. Journal of Biological Chemistry, 290(17), 10804-10813.
  Renner-Schneck, Michaela; Hinderberger, Isabel; Gisin, Jonathan; Exner, Thomas; Mayer, Christoph & Stehle, Thilo
  
• Identification of a Novel N -Acetylmuramic Acid Transporter in Tannerella forsythia. Journal of Bacteriology, 198(22), 3119-3125.
  Ruscitto, Angela; Hottmann, Isabel; Stafford, Graham P.; Schäffer, Christina; Mayer, Christoph & Sharma, Ashu
  
• Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS. BIO-PROTOCOL, 7(15).
  Borisova, Marina & Mayer, Christoph
  
• N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism. Frontiers in Microbiology, 9.
  Hottmann, Isabel; Mayer, Valentina M. T.; Tomek, Markus B.; Friedrich, Valentin; Calvert, Matthew B.; Titz, Alexander; Schäffer, Christina & Mayer, Christoph
  
• Bacteria's different ways to recycle their own cell wall. International Journal of Medical Microbiology, 309(7), 151326.
  Mayer, Christoph; Kluj, Robert Maria; Mühleck, Maraike; Walter, Axel; Unsleber, Sandra; Hottmann, Isabel & Borisova, Marina
  
• Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis. BMC Microbiology, 19(1).
  Mayer, Valentina M. T.; Hottmann, Isabel; Figl, Rudolf; Altmann, Friedrich; Mayer, Christoph & Schäffer, Christina
  
• Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG. BMC Microbiology, 20(1).
  Mayer, Valentina M. T.; Tomek, Markus B.; Figl, Rudolf; Borisova, Marina; Hottmann, Isabel; Blaukopf, Markus; Altmann, Friedrich; Mayer, Christoph & Schäffer, Christina
  
• Peptidoglycan Salvage Enables the Periodontal Pathogen Tannerella forsythia to Survive within the Oral Microbial Community. Microbial Physiology, 31(2), 123-134.
  Hottmann, Isabel; Borisova, Marina; Schäffer, Christina & Mayer, Christoph
  
• NamZ1 and NamZ2 from the Oral Pathogen Tannerella forsythia Are Peptidoglycan Processing Exo-β- N -Acetylmuramidases with Distinct Substrate Specificities. Journal of Bacteriology, 204(3).
  Borisova, Marina; Balbuchta, Katja; Lovering, Andrew; Titz, Alexander & Mayer, Christoph
  
• N-acetylmuramic acid recognition by MurK kinase from the MurNAc auxotrophic oral pathogen Tannerella forsythia. Journal of Biological Chemistry, 299(9), 105076.
  Stasiak, Aleksandra Cecylia; Gogler, Karolin; Borisova, Marina; Fink, Phillipp; Mayer, Christoph; Stehle, Thilo & Zocher, Georg