BioM24 Peptidoglycan: Structure, Function, and Biosynthesis

Peptidoglycan Structure

Peptidoglycan is a crucial component of bacterial cell walls, providing strength, support, shape, and acting as a barrier.

  • Gram-positive bacteria: Have a thick peptidoglycan layer outside the cell membrane.
  • Gram-negative bacteria: Have a thin peptidoglycan layer between the inner and outer membranes, residing in the periplasmic space.

Peptidoglycan Function

Peptidoglycan provides:

  • Strength: Counteracts turgor pressure (2-6 atm).
  • Support: Anchors various proteins, including efflux systems, secretion systems, importers, lipoproteins (e.g., Braun lipoprotein), outer membrane proteins, sortase substrates, wall-binding proteins, inner membrane proteins, periplasmic filaments, cell morphology apparatus, and other wall polymers (e.g., teichoic acids).
  • Barrier: Prevents entry of surfactants, antimicrobials, defense peptides, bacteriophages, other bacteria, predators, ions, and nutrients; protects against osmolarity changes.
  • Shape: Determines cell shape (e.g., Caulobacter crescentus, E. coli, Helicobacter pylori).
    • Specific proteins (Csd1, CcmA, Csd2, Csd3 in H. pylori) contribute to curvature and twist, influencing the helical rod shape.

Peptidoglycan Terminology & Composition

  • Interchangeable terms: Murein, sacculus, wall, peptidoglycan.
  • Repeating units: (Disaccharide-peptide)n.
  • Composition:
    • N-acetylglucosamine (NAG)
    • N-acetylmuramic acid (NAM)
    • Peptide stem (L-Ala, D-Glu, DAP or L-Lys, D-Ala, D-Ala)
  • Linkage: β-1,4-glycosidic bond between NAG and NAM (target of lysozyme).
  • Location: Present in most bacteria, absent in Archaea, Mycoplasma, and L-forms (but present in Planctomyces and Chlamydiae).
  • Size: Total mass of 3x1063 {x} 10^6 kDa (e.g., E. coli chromosome is ~1.5x1061.5 {x} 10^6 kDa).
  • Length: Gram-negative bacteria have smaller peptidoglycan lengths compared to Gram-positive bacteria (up to 400 units long).

Peptide Stem Variations

The peptide stem varies between species:

  • E. coli: L-Ala – D-Glu – DAP – D-Ala – D-Ala.
  • S. aureus: L-Ala – D-Glu(NH2) – L-Lys – D-Ala – D-Ala with a pentaglycine bridge (Gly5).

Crosslinking (Transpeptidation)

  • Peptides can be crosslinked with the loss of the terminal D-Ala via transpeptidation.
  • The Gly5 bridge in S. aureus allows for a high degree of crosslinking.
  • Gram-negative bacteria typically have less crosslinking.

Reaction:

L−Ala−D−Glu−DAP−D−Ala−D−Ala+L−Ala−D−Glu−DAP−D−Ala−D−Alatranspeptidase→L−Ala−D−Glu−DAP−D−Ala+D−Ala−DAP−D−Glu−L−AlaL-Ala - D-Glu - DAP - D-Ala - D-Ala + L-Ala - D-Glu - DAP - D-Ala - D-Ala {transpeptidase} \rightarrow L-Ala - D-Glu - DAP - D-Ala + D-Ala - DAP - D-Glu - L-Ala

Peptidoglycan Biosynthesis

The biosynthesis of peptidoglycan involves several steps:

  1. Cytoplasmic Unit Building: Occurs in the cytoplasm where there are plenty of metabolites and ATP for synthetic use.
  2. Export to Periplasm: The unit is exported to the periplasm.
  3. Final Assembly: Final assembly of peptidoglycan occurs across the membrane.

Sugar Synthesis

  • Precursors: Fructose-6-P, Glucosamine-6-P
  • Enzymes: GlmS, GlmM, GlmU, MurA, MurB
  • Products: UDP-NAG, UDP-NAM
  • Inhibitors: MurA is inhibited by terreic acid and fosfomycin (warfare!).

Peptide Synthesis

  • Amino acids are sequentially added to UDP-NAM using homologous enzymes (MurC-F).

Reactions:

  • UDP−NAM−COO−+ATP+L−Ala→MurCUDP−NAM−L−Ala+ADP+PUDP-NAM-COO^- + ATP + L-Ala \xrightarrow{MurC} UDP-NAM-L-Ala + ADP + P

  • UDP−NAM−L−Ala+ATP+D−Glu→MurDUDP−NAM−L−Ala−D−Glu+ADP+PUDP-NAM-L-Ala + ATP + D-Glu \xrightarrow{MurD} UDP-NAM-L-Ala-D-Glu + ADP + P

  • UDP−NAM−L−Ala−D−Glu+ATP+L−Lys→MurEUDP−NAM−L−Ala−D−Glu−L−Lys+ADP+PUDP-NAM-L-Ala-D-Glu + ATP + L-Lys \xrightarrow{MurE} UDP-NAM-L-Ala-D-Glu-L-Lys + ADP + P

  • UDP−NAM−L−Ala−D−Glu−L−Lys+ATP+D−Ala−D−Ala→MurFUDP−NAM−L−Ala−D−Glu−L−Lys−D−Ala−D−Ala+ADP+PUDP-NAM-L-Ala-D-Glu-L-Lys + ATP + D-Ala-D-Ala \xrightarrow{MurF} UDP-NAM-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala + ADP + P

  • Inhibitors: D-cycloserine inhibits D-Ala & D-Ala.

  • Park Nucleotide: Significant structural homology among MurC, MurD, MurE, and MurF.

  • Induced fit mechanism.

  • In S. aureus, Gly5 is added via FemX1A2,3B4,5 using tRNA-Gly.

Inner Membrane Synthesis

  • Enzymes: MraY and MurG
  • MraY: Transfers UDP-NAM-pentapeptide to undecaprenyl-phosphate, forming lipid I.
  • MurG: Adds UDP-NAG to lipid I, forming lipid II.

Reactions:

  • UDP−NAM−pentapeptide+undecaprenyl−phosphate→MraYlipidI+UMPUDP-NAM-pentapeptide + undecaprenyl-phosphate \xrightarrow{MraY} lipid I + UMP

  • lipidI+UDP−NAG→MurGlipidII+UDPlipid I + UDP-NAG \xrightarrow{MurG} lipid II + UDP

  • MraY has 10 transmembrane helices and is integral to the membrane. Its structure was determined in 2013. Many inhibitors exist, and the active site is presumed to be in/at the membrane. Parallels exist between MraY action and eukaryotic Asn-glycoprotein synthesis.

  • MurG is membrane-associated and has a known structure with a solvent-exposed active site. Murgocil is a potential inhibitor.

  • Tunicamycin inhibits all N-glycosylation

Transport

  • A flippase (MurJ/MviN) is required to transport lipid II from the cytoplasm to the periplasm.
  • FtsW/RodA may also have flippase activity.

Final Assembly

  • Glycosyltransferase (GT): Polymerizes lipid II to the growing peptidoglycan chain (chainn + lipid II → chainn+1 + UPP).
  • Transpeptidation (TP): Crosslinks the peptidoglycan chains (chain + wall → insertion + D-Ala).
  • GT is membrane-bound, uses a catalytic glutamate, and is inhibited by moenomycin lipid.
  • TP is solvent-exposed, uses a catalytic serine, and is inhibited by β-lactams (e.g., penicillin).
  • Bifunctional PBPs possess both GT and TP activity.

Glycosyltransfer Mechanism

  1. GT deprotonates.
  2. Chain is transferred.
  3. Longer chain shifts back, lipid II attacks the chain.
  4. NAM-NAG is increased by one unit.
  5. Lipid II reloads, initiating another reaction.
  6. UPP is lost.
  • Moenomycin blocks the GT reaction as it mimics the chain.

Transpeptidation Mechanism

  • Attacking peptide attacks free stem peptide (e.g., NAM-L-Ala-D-Glu-L-Lys-).
  • Crosslink forms (e.g., D-Ala-Gly).

Beta-Lactam Antibiotics

  • β-lactam ring opens and forms a covalent adduct, inactivating PBPs.
  • The lack of PBP function weakens the cell wall, leading to lysis.
  • The D-Ala-D-Ala : β-lactam equivalence was predicted by the "Tipper-Strominger" hypothesis.

The cell wall synthesis machinery can malfunction due to beta-lactam antibiotics, leading to:

  • Damaged TP, uncrosslinked strand made by GT, synthase stalls
  • Misincorporation of glycans

Beta-Lactamases

  • Beta-lactamases are enzymes that inactivate beta-lactam antibiotics.
  • Serine β-lactamases form stable or labile acyl-enzymes.

Mechanisms:

  • Stable Acyl-Enzyme: The enzyme is inactivated, leading to cell lysis. Clavulanic acid forms a stable acyl-enzyme that inactivates the beta-lactamase.

  • Labile Acyl-Enzyme: The enzyme is regenerated, restoring its function.

Other Inhibitors of Peptidoglycan Biosynthesis

  • Vancomycin: Binds D-Ala-D-Ala termini, preventing crosslinking. Resistance occurs via incorporation of D-Ala-D-Lac or D-Ala-D-Ser.
  • Nisin: Binds free lipid II via PP part, inducing pore formation.
  • Bacitracin: Binds free UPP, interfering with UPP recycling.

Peptidoglycan Models

Classic Circumferential Model: Horizontal Layers
Scaffold Perpendicular Model: Radiating chains

PG Synthesis Organization for Cellular Shape

  • MreB: Bacterial actin homolog; cytoplasmic filament. MreBCD path recruits PBPs for even spacing of PG elongation, resulting in rod shape.
  • FtsZ: Bacterial tubulin homolog; constrictable ring at septum, recruits PBPs to synthesize new poles.

Remodelling & Recycling

  • Peptidoglycan is in constant flux, involving:
    • Recycling of tetrapeptides & NAM-NAG-tetrapeptides via Opp and AmpG.
    • Bypass of first three Mur ligase reactions via MplA.
    • Detection via NOD proteins.

Braun Lipoprotein

  • Consists of a 20AA signal peptide and a 58AA mature protein.
  • Located in the outer membrane (OM).
  • Contains a very unusual N-terminal Cys, linked to fatty acids.

L-Forms

  • Organisms that usually have peptidoglycan but have lost it due to specific circumstances (e.g., prevention of PG synthesis, growth media with matched osmolarity).
  • Generate via UPP biosynthesis KO. Wall-less division (blebbing), model for ancient progenitor?

Peptidoglycan and Infection

  • Lysozyme cleaves PG, leading to bacterial cell death and release of PG fragments.
  • PG fragments are recognized as pathogen-associated molecular patterns, triggering pro-inflammatory innate immune responses via TLR2 and Nod2.
  • Many mucosal pathogens modify PG around the lysozyme cleavage site to avoid degradation.

PG Modifying Enzymes

  • PG modifying enzymes can function as weapons in bacteria:bacteria interactions, through contact-dependent inhibition (CDI) systems.

PG Modification as a Signaling Agent

  • Peptidoglycan modifications can serve as signaling agents, influencing processes like attachment, invasion, niche formation, lysis, septation, and growth.

Turgor Pressure

Turgor pressure is the pressure from a point, pushing out = all rods, spheres, cocci etc.

References & Further Reading

  • Cytoplasmic Stages PG synthesis: Barreteau FEMS Microbiol Rev 2008 1-40
  • Lipidic Stages PG synthesis: Bouhss FEMS Microbiol Rev 2008 1-26
  • PG Structure: Vollmer FEMS Microbiol Rev 2007 1-19
  • GT reaction: Lovering Curr Opin Str Biol 2008 18(5)
  • PBPs & TP reaction: Macheboeuf FEMS Microbiol Rev 30 2006 673-91
  • Small paper (letter!) on arguments of 3D models: Young TRENDS in Microbiol 14 (4) 2006
  • Cell wall antibiotics: Schneider Int J Med Micro 300 2010 161-169