Macromolecular Machineries in Cell‐Wall Recycling

Abstract

Peptidoglycan, the major constituent of bacterial cell wall, is a huge polymeric macromolecule composed of glycan strands covalently linked by interconnecting peptide stems and critical for survival of bacteria. Peptidoglycan is constantly edited, biosynthesised and degraded, during essential bacterial processes such as division, elongation or insertion of the cellular machinery (e.g. flagella, pili) into cell wall. Degradation fragments are recovered, especially in Gram‐negative bacteria, by active transport and recycled to rebuild the wall. In some important Gram‐negative pathogens, β‐lactam resistance is directly linked to peptidoglycan recycling. The recycling process requires orchestration of different lytic enzymes and transporters from periplasm, the inner membrane, to cytoplasm. Recent structural and functional studies have provided relevant insights into enzymatic regulation, substrate specificity and activity of the lytic machineries involved in recycling with relevant implications in antibiotics resistance.

Key Concepts

  • Peptidoglycan recycling is an essential process in Gram‐negative bacteria that recovers self‐degradation products of cell wall in the cytoplasm to reutilise them to rebuild the wall.
  • PG recycling is important in cell‐wall biosynthesis and in bacterial communication in many bacteria, and in antibiotics resistance in some pathogens from Enterobacteriaceae and Pseudomonadaceae families.
  • PG recycling involves a large number of enzymes and transporters spanning from periplasm, inner and outer membranes, and cytoplasm.
  • Lytic transglycosylases (LTs) are periplasmic and, typically, modular enzymes that initiate PG recycling by non‐hydrolytic degradation of glycan chains. They are classified in six families. Several LTs are found in each Gram‐negative bacterium.
  • The Slt structure reveals how, under the effect of β‐lactam antibiotics, the cell wall is repaired by degradation of aberrant nascent PG chains and how it initiates the resistance phenotype.
  • MltF is a modular membrane‐bound LT that experiences allosteric activation triggered by muropeptides.
  • PG amidases are present in periplasm and cytoplasm. Activity in periplasm is regulated by cellular location, protein–protein interactions and oligomeric state. An activation mechanism has been discovered for cytosolic enzymes.
  • Anhydro‐muropeptides are the key molecules to activate the AmpR to induce AmpC β‐lactamase in response to β‐lactam challenge.
  • Regulation (in space and time) of the catalytic processes as well as of interaction between different partners is essential in PG recycling enzymes.

Keywords: cell‐wall recycling; antibiotic resistance; lytic transglycosylases; peptidoglycan amidases; NagZ glucosaminidase; Gram‐negative bacteria

Figure 1. Cell‐wall organisation. Gram‐negative (a) and Gram‐positive (b) cell‐wall architecture. Alternative linked units of N‐acetylglucosamine (NAG, in pale green) and N‐acetylmuramic acid (NAM, in dark green) build the peptidoglycan layer. (c) Detailed scheme of peptidoglycan chemical structure with the cross‐linked peptide stems and different lytic enzymes degrading the peptidoglycan in cell‐wall recycling.
Figure 2. The cell‐wall recycling process and its connection with β‐lactam resistance in Gram‐negative bacteria. Peptidoglycan recycling process is indicated by orange arrows and β‐lactam resistance pathway by red arrows. Degradation of PG is performed by LTs and PG amidases (e.g. AmpDh2). Degradation fragments cross the inner membrane through anhNAM‐specific permease AmpG or by peptide transporter Opp. In the cytoplasm, fragments are further degraded by NagZ, AmpD and LdcA enzymes for de novo synthesis of PG. When β‐lactam antibiotic is present in periplasm, d,d‐transpeptidation (TP) by PBPs is blocked and aberrant nascent PG chains are produced. Slt cleaves this abnormal nascent PG by endo and exolytic activities, producing the anhydro products. Increased amounts of anhydro muropeptides from nascent PG chains activates AmpR resulting in expression of AmpC β‐lactamase that will be exported to periplasm.
Figure 3. Cell‐wall repair by Slt and induction of antibiotics resistance in Pseudomonas. (a) Lipid II reaches the periplasm and is polymerised by the transglycosylase (TG) domain to generate the nascent PG, which undergoes the cross‐linking reaction in the transpeptidase (TP) domain of a PBP. Apo conformation of Slt is inactive. Below, crystal structure of apo Slt (PDB code 5OHU). (b) β‐Lactam inhibits TP resulting in long aberrant PG chains that serve as substrate for the endolytic cleavage by Slt. Below, detailed view of the crystal structure Slt in complex with 4(NAG–NAM(pentapeptide)) (yellow sticks) (PDB code 6FCU). (c) Once PG is cleaved in the middle, the Slt exolytic reaction occurs. Below, detailed view of the crystal structure of Slt in complex with NAG–NAM(tetrapeptide)–NAG–anhNAM(tetrapeptide) (in green sticks) (PDB code 6FCR). (d) Final disaccharide NAM–anhNAM–pentapeptide products are generated and reached the cytoplasm through AmpG permease, where they induce AmpC β‐lactamase production that subsequently neutralises the antibiotic. Below, detailed view of the crystal structure of Slt in complex with NAG–anhNAM(pentapeptide) (in orange sticks) (PDB code 6FBT). Lee et al. . Reproduced with permission of National Academy of Sciences.
Figure 4. Muropeptide‐induced activation of the catalytic activity in MltF. Proposed model for the muropeptide‐induced activation of the catalytic activity in MltF is depicted as a cartoon (top) and associated crystallographic structures (below). (a) MltF inactive form has the active site blocked. (associated structure PDB code 5A5X). (b) Peptides such as those released by periplasmic amidases (as AmpDh2) bind to the ABC module of MltF generating a large conformational change in the CM. Crystal structures of several steps are shown with a peptide inside the ABC (PDB codes 5AA1, 5AA2, 5AA3 and 5AA4). (c) Upon activation, MltF exposes the catalytic cleft allowing the entrance of the PG chains to perform catalysis. Associated structure of MltF with a muropeptide (green spheres) and with the inhibitor Bulgecin A (yellow spheres) is shown (PDB code 4P0G).
Figure 5. Crystal structures of representative lytic transglycosylases, glucosaminidase NagZ, peptidoglycan amidases and l,d‐carboxypeptidases involved in PG recycling. Protein structures are displayed in ribbon representation with the ligands showed in spheres (peptide stem coloured in green and glycan chains in yellow). Domains are coloured differently and labelled. (a) MltC (subfamily 1B) in complex with tetrasaccharide (PDB code 4CFP). (b) MltE (subfamily 1C) in complex with chitopentaose (PDB code 4HJZ). (c) EtgA (subfamily 1G) (PDB code 4XP8). (d) MltA (Family 2) in complex with chitohexaose (PDB code 2PI8). (e) SltB3 (Family 3) in complex with anhydromuropeptide and NAG (PDB code 5AO7). (f) MltG (Family 5) (PDB code 2R1F). (g) NagZ in complex with NAG and anhydromuropeptide products (PDB code 5G3R). (h) AmiC (PDB code 4BIN). (i) AmpDh2 dimer in complex with tetrasaccharide 2(NAG–NAM) (PDB code 4BPA) and with pentapeptide (PDB code 4BOL) superimposed. (j) AmpDh3 tetramer in complex with a trisaccharide pentapeptide (PDB code 4BXD) and with anhNAM‐pentapeptide (PDB code 4BXE) superimposed. (k) AmpD in complex with anhNAM‐pentapeptide (PDB code 2Y2B). (l) carboxypeptidase LdcA dimer in its apo form (PDB code 1ZL0).
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Further Reading

Mayer C (2012) Bacterial Cell Wall Recycling. Wiley.

Alcorlo M, Martinez‐Caballero S, Molina R and Hermoso JA (2017) Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases. Current Opinion in Structural Biology 44: 87–100.

Rivera I, Molina R, Lee M, Mobashery S and Hermoso JA (2016) Orthologous and paralogous AmpD peptidoglycan amidases from Gram‐negative bacteria. Microbial Drug Resistance 22 (6): 470–476.

Dominguez‐Gil T, Molina R, Alcorlo M and Hermoso JA (2016) Renew or die: the molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram‐negative pathogens. Drug Resistance Updates 28: 91–104.

Dik DA, Fisher JF and Mobashery S (2018) Cell‐wall recycling of the Gram‐negative bacteria and the nexus to antibiotic resistance. Chemical Reviews 118 (12): 5952–5984.

Dik DA, Marous DR, Fisher JF and Mobashery S (2017) Lytic transglycosylases: concinnity in concision of the bacterial cell wall. Critical Reviews in Biochemistry and Molecular Biology 52 (5): 503–542.

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Batuecas, María T, and Hermoso, Juan A(Apr 2019) Macromolecular Machineries in Cell‐Wall Recycling. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028397]