L1peptidoglycan

Basic Architecture of Bacterial Cells

• Diagrams of Gram-positive and Gram-negative bacterial cells illustrate differences in cell wall structure.

Surface Structures

• Pili (fimbriae) and flagella are surface structures found in bacteria.

  • Shorter pili surround the cell surface.

  • Longer flagella located at cell poles or distributed over the cell surface.

Cell Walls

• Focus on the cell wall (peptidoglycan layer).

• Gram-positive cells:

  • Thick peptidoglycan forms the outermost layer.

• Gram-negative cells:

  • Thinner peptidoglycan located between inner and outer membranes, creating a periplasmic space.

  • Outer membrane is absent in Gram-positive species.

Electron Micrographs

• Thin section electron micrographs show:

  • Gram-positive: Thicker peptidoglycan layer as the outermost structure.

  • Gram-negative: Outer membrane with a unique structure, mainly composed of lipopolysaccharides (LPS).

Peptidoglycan Structure

• Composed of two main components:

  • Glycan: Made from N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM).

  • Peptide Chains: Provide structural rigidity and enable cross-linking.

Significance of Peptidoglycan

• Conservation: Sugar components are conserved across bacterial species, while peptide components are more variable.

• peptide cross-linking is essential for structural integrity.

Arrangement of Peptidoglycan

• Diagrams show individual peptidoglycan strands of alternating NAG and NAM.

• Peptides attached only to NAM residues allowing for crosslinking and mesh-like structure.

Peptidoglycan Mesh Structure

• Peptidoglycan forms a highly resistant mesh surrounding the bacterial cell.

• Boiling E. coli in detergent results in a deflated peptidoglycan structure under microscopy.

• High rigidity required for bacterial cell viability.

Peptidoglycan Biosynthesis

• Summary of steps:

  • Formation of Lipid I from disaccharide NAG-NAM with linked pentapeptide.

  • Transport across membrane via undecaprenol pyrophosphate.

  • Incorporation into peptidoglycan by glycosyltransferase and transpeptidase enzymes.

Antibiotic Targeting of Peptidoglycan

• Peptidoglycan is a primary target for antibiotics due to its essential role in cell viability and bacterial specificity.

• Penicillins and other B-lactams target peptidoglycan synthesis:

  • Structure features a B-lactam ring.

  • B-lactams block transpeptidase enzymes, hindering cross-linking and resulting in cell death.

Penicillin Discovery

• Discovered by Alexander Fleming in 1928 from Penicillium fungi.

• Highly effective against Gram-positive bacteria due to accessibility of peptidoglycan.

Structural Variability Between Gram-positive and Gram-negative

• Access to peptidoglycan differs due to the presence of outer membranes in Gram-negative.

• Later derivatives of penicillin developed to penetrate Gram-negative cells.

Role of B-Lactam Ring and Bacterial Resistance

• B-lactam ring resembles D-Alanine-D-Alanine terminus crucial for bacterial transpeptidation.

• Resistance can occur through the action of B-lactamase enzymes that cleave the B-lactam ring.

Peptidoglycan Synthesis Process

• Occurs in the cytoplasm with enzyme-mediated steps:

  • UDP-GlcNAc converted to UDP-MurNAc by MurA and MurB enzymes.

  • Sequential addition of amino acids by ligase enzymes (MurC-F).

  • Lipid I formation at inner membrane and conversion to Lipid II via MurG.

  • Final incorporation into peptidoglycan and cross-linking by transpeptidases involves the release of terminal D-Ala residue.

Proteins Covalently Attached to Peptidoglycan

Gram-positive Bacteria:

• In Gram-positive bacteria, a variety of surface proteins are covalently linked to peptidoglycan, which play roles in pathogenic processes.

• Enzyme Responsible: The enzyme responsible for this covalent attachment is sortase A.

  • Function: Sortase A recognizes a sorting signal (usually a specific peptide sequence) on the surface proteins and cleaves this signal. It then links the N-terminal carboxyl group of the sorting signal to the side-chain amine at the third position of the peptide stem, effectively anchoring the protein to the peptidoglycan. This mechanism allows for the organization of surface proteins that are crucial for the bacteria's adherence and virulence.

Gram-negative Bacteria:

• In contrast, covalent attachment of surface proteins is less understood, but Braun's lipoprotein has been identified as the only known protein covalently linked to peptidoglycan in Gram-negative species. This lipoprotein connects the outer membrane to the peptidoglycan layer, stabilizing the overall cell wall structure.

Variation at Position 5 of the Peptide Stem

• Traditionally, position 5 of the peptide stem is occupied by D-Ala. However, in some strains with natural or acquired resistance to vancomycin, alternative amino acids can occupy this position, such as D-Lactate (D-Lac) or D-Serine (D-Ser).

• Clinical Significance: The replacement of D-Ala with these alternative amino acids reduces the binding affinity for vancomycin, an antibiotic that targets D-Ala-D-Ala structures. This modification is clinically significant as it provides bacteria with a mechanism of resistance against vancomycin, making infections caused by resistant strains harder to treat and control, leading to increased morbidity and mortality in affected patients.

The enzyme responsible for coupling proteins to peptidoglycan (PG) in Gram-positive bacteria is sortase A.

How it Works: Sortase A recognizes a sorting signal, which is usually a specific peptide sequence on the surface proteins. It cleaves this sorting signal to allow for the linkage of the N-terminal carboxyl group of the sorting signal to the side-chain amine at the third position of the peptide stem. This effectively anchors the protein to the peptidoglycan. This mechanism is crucial as it organizes surface proteins that are important for bacterial adherence and virulence.

Summary of Peptidoglycan Structure and Architecture

Overview

• The peptidoglycan (murein) sacculus is a crucial and unique structural element in the cell wall of most bacteria.

• Composed of glycan strands cross-linked by short peptides, it forms a closed, bag-shaped structure around the cytoplasmic membrane.

• The diversity in composition and peptide sequence of peptidoglycan varies significantly among different bacterial species and can change under different growth conditions.

• Limited biophysical data are available about its thickness, elasticity, and porosity.

Functions of Peptidoglycan

• Cell Integrity: Peptidoglycan preserves cell integrity by withstanding turgor pressure. Inhibition of its biosynthesis or degradation leads to cell lysis.

• Shape Maintenance: It contributes to the maintenance of defined cell shape and serves as a scaffold for anchoring other cell envelope components like proteins and teichoic acids.

• Biosynthesis Absence: Peptidoglycan is absent in certain bacteria like Mycoplasmas and Chlamydiae, although some genes necessary for its biosynthesis exist in Chlamydia.

Chemical Structure and Characteristics

• Peptidoglycan is characterized by linear glycan strands composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by β-1,4 bonds.

• Each MurNAc is substituted by a peptide stem, usually containing L-Ala, D-Glu, meso-A2pm (or L-Lys), D-Ala, and D-Ala.

• Cross-linking generally occurs between the D-Ala at position 4 and a diamino acid at position 3, either directly or through a short peptide bridge.

Variability and Modifications

• Structural features vary across species and even under different growth conditions, affecting peptide composition, glycan strands, and interpeptide bridges.

• Glycan strands undergo secondary modifications such as N-deacetylation, O-acetylation, and others, indicating their complex nature.

• Average Lengths: Glycan chain lengths vary, with Bacilli having lengths of 50–250 disaccharide units and Staphylococcus aureus around 18 units.

Protein Attachment

Gram-positive Species

• Surface proteins crucial for pathogenic processes are covalently linked to peptidoglycan.

• Enzyme: Sortase A, which cleaves a sorting signal in surface proteins and links them to the peptide stem of peptidoglycan.

Gram-negative Species

• The primary protein covalently attached is Braun's lipoprotein, which connects the outer membrane to the peptidoglycan layer, stabilizing the structure.

Cross-linking in Peptidoglycan

• Two types of cross-linking are observed:

  1. 3-4 Cross-linking: Extends from position 3 of one peptide unit to position 4 of another.

  2. 2-4 Cross-linking: Observed among certain coryneform bacteria.

• The degree of cross-linking varies based on species and growth state, with about 25–35% in E. coli and over 90% in S. aureus.

Biophysical Properties

• Peptidoglycan sacculi have unique biophysical properties, allowing them to withstand high turgor pressures while being flexible.

• Pore sizes allow diffusion of large molecules. Radii of mean pores are around 2 nm, permitting passage of proteins of certain molecular weights.

Modelling Structure

• Understanding the architecture of peptidoglycan is essential for biological processes, growth, and response to antibiotics.

• Models must accommodate variations and dynamic growth conditions in bacteria, focusing on glycan strand orientation, cross-linking, and structural diversity.

Conclusions

• Despite significant knowledge regarding peptidoglycan, research is ongoing to better understand its complex structure and adaptations, relevant to bacterial physiology and pathogenesis. Determining the precise molecular architecture of peptidoglycan will enhance understanding of bacterial growth mechanisms and antibiotic resistance.