Bacterial Cell Walls and Antibiotics

• In 2019, 213,000 kg of antibiotics were intended for use in humans in Canada
• Most antibiotics are β-lactam antibiotics.
• Penicillins, cephalosporins, and carbapenems make up 145,000 kg of antibiotic use and target the cell wall

Lecture Learning Outcomes

• After the lecture, students will be able to describe lipids and proteins in the cytoplasmic membrane
• After the lecture, students will be able to describe the function of peptidoglycan in the cell wall
• After the lecture, students will be able to describe the structure of peptidoglycan and how it's synthesized by PBPs and other enzymes.
• After the lecture, students will be able to describe how antibiotics target peptidoglycan and how bacteria resist these antibiotics.

Prokaryotic Cell Envelope

• The prokaryotic cell envelope consists of the cytoplasmic membrane and all layers that surround it.
• The cell wall, peptidoglycan, outer membrane (in Gram-negatives), and other external layers like capsules are included
• Roles include controlling what enters and exits the cell and protecting against stresses, antibiotics, and immune cells

Cytoplasmic Membrane

• The cytoplasmic membrane is a lipid bilayer whose composition depends on conditions such as temperature
• The cytoplasmic membrane is protein-rich
• The cytoplasmic membrane is a semipermeable barrier: water and hydrophobic substances diffuse through while hydrophilic substances do not

Bacterial Cytoplasmic Membrane

• Bacterial cytoplasmic membranes are mainly composed of phospholipids
• Fatty acids are attached to glycerol by ester bonds
• Glycerol is bonded to a phosphate group, which may have a substituent like ethanolamine.
• The membrane is amphipathic, containing polar and non-polar regions

Cytoplasmic Membrane Protein Functions

• Transporters import substances like nutrients and export substances like EPS for biofilms and toxins
• Proteins are involved in signal transduction, detecting external stimuli
• Electron transport chain (ETC) enzymes generate a H+ gradient, also known as proton motive force (PMF), across the membrane for energy transduction
• PMF powers ATP synthesis and transport

Osmolarity and Cytoplasmic Membrane

• Bacteria are usually in a hypotonic environment
• They have more solutes in the cell than outside, causing water to be drawn into the cell (osmotic pressure)
• Water influx causes swelling and can lead to osmotic lysis
• Bacteria survive in hypotonic conditions through certain adaptations

Bacterial Cell Wall

• Most bacteria have a cell wall as the outer layer outside the cytoplasmic membrane
• Peptidoglycan (PG) is the major component of the cell wall.
• Gram-positives have a thick peptidoglycan layer
• Gram-negatives have a thin peptidoglycan layer
• Some cell walls have the additional layer of a Gram-negative outer membrane

Peptidoglycan

• Peptidoglycan is a strong and elastic structure that determines cell shape.
• Protects against osmotic lysis by pushing against the membrane.
• It stretches and contracts in response to osmotic pressure; it is porous, allowing nutrients and waste to pass through

Osmotic Stabilization of the Cell

• If Peptidoglycan is disrupted, the cell becomes more susceptible to osmotic lysis in a hypotonic environment
• Peptidoglycan is targeted by antibiotics and the immune system.
• Bacteria can survive peptidoglycan degradation in isotonic conditions
• In isotonic conditions bacteria lose shape and form spheroplasts (Gram-negative) or protoplasts (Gram-positive)
• In hypotonic conditions bacteria swell and lyse if moved

Mycoplasmas

• Bacteria without a cell wall measuring 0.2um in size
• Some are intracellular parasites.
• They are osmotically sensitive.
• They incorporate sterols from hosts into the cytoplasmic membrane increasing stability
• Osmotic pressure is lower inside other cells ~ isotonic

Peptidoglycan and Antibiotics

• Peptidoglycan structure is a good antibiotic target, because it's easily accessed on the cell surface
• Peptidoglycan is made by most bacteria, not made by human cells
• Many antibiotics target peptidoglycan, including ẞ-Lactam antibiotics like penicillins, and also vancomycin
• Antibiotics target Peptidoglycan's chemical structure, and the enzymes that create it

Peptidoglycan Structure

• The peptidoglycan structure consists of sugars and amino acids.
• Peptidoglycan backbone consists of a long glycan strand w/repeating disaccharide units
  • NAM: N-acetylmuramic acid
  • NAG: N-acetylglucosamine
• Each NAM bears a peptide chain
• Glycan strands are connected by peptide cross-links

Peptidoglycan Biosynthesis: Lipid II

• Lipid II is a precursor containing "monomeric" PG subunits: NAM-NAG disaccharide and pentapeptide, bound to membrane by undecaprenol
• Lipid II synthesis starts with UDP-NAG as UDP activates NAG

Making Lipid II

• Assembled in cytoplasm, flipped across membrane

D-Alanine Synthesis and Incorporation

• Lipid II contains D-alanine
• Alanine racemase makes D-Ala
• D-Ala-D-Ala ligase makes D-Ala-D-Ala
• Cycloserine inhibits alanine racemase and D-Ala-D-Ala ligase

Penicillin-Binding Proteins

• Lipid II is incorporated into PG by penicillin-binding proteins (PBPs) located in the periplasm
• Bacteria require PBPs for cell growth, cell division, and cell wall recycling
• Many PBPs have a glycosyltransferase domain which builds glycan strands
• Transpeptidase domains create peptide cross-links

PBP Glycosyltransferase Activity

• PBP adds lipid II disaccharide to glycan backbone of peptidoglycan
• Extends glycan backbone
• NAM activated by pyrophosphate

Undecaprenol Recycling

• Undecaprenyl pyrophosphate is then recycled and dephosphorylated, then flipped to cytoplasm
• Bacitracin (antibiotic) binds to undecaprenyl pyrophosphate and blocks dephosphorylation

Lysozyme

• The peptidoglycan glycan backbone can be degraded bacterial enzymes and antimicrobial enzymes
• Lysozyme is part of the innate immune system found in saliva, tears, milk, and mucous secretions
• Cleaves NAM-NAG bond and weakens cell wall
• Lysozyme is more effective against Gram-positives
• Gram positives have more exposed peptidoglycan
• Gram-negatives have outer membranes

Peptidoglycan Structure

• PG strands are helical
• Peptides extend from the glycan backbone
• PBPs cross-link peptides from separate strands

Peptidoglycan Peptide Chains

• Pentapeptide attaches to NAM sugar with an amino acid
• The sequence of peptide chains can vary and contain D-amino acids, like D-Alanine
• Diamino acids exist in third position, which can be comprised of L-lysine of meso-diaminopimelic acid (meso-Dap)

Peptidoglycan Cross-Linking (Gram-Negatives)

• In gram-negatives, cross Linking is acheived a direct link (amide bond)
• Cross-link between residue 3 (amino group of diamino acid) and residue 4 (carbonyl)
• Forms Amide bond
• Releases terminal D-alanine

Peptidoglycan Cross-Linking (Gram-Positive)

• In gram-positives, peptide chains are cross-linked through Interpeptide bridge
  • Bridges are attached to a diamino acid
  • Composition of chains varies (e.g., pentaglycine in S. aureus)
  • Terminal D-alanine is released

Transpeptidation Mechanism

• Cross-links are formed by PBP transpeptidase domain
• PBP forms complex with peptide first
• Diamino acid reacts with complex and forms the amide bond

Beta-Lactam Antibiotics

• Including Penicillins, Cephalosporins, and Carbapenems
• Are the most widely used antibiotics and feature a four-membered Beta-lactam ring
• Penicillin G was the first in clinical use, made by the Penicillium mold and has Narrow-spectrum
• In 1896, Duchesne observed Penicillium antibiotic properties
• In 1928, Fleming re-discovered the antibiotic, finding activity from a secreted product

Beta-Lactams Block Transpeptidation

• Beta-Lactams inhibit PBP transpeptidase activity
• The beta-lactam ring reacts with serine in PBP
• Prevents PBP from forming cross-links, weakening PG, which leads to cell lysis
• Beta-Lactams are thus bactericidal

Beta-Lactam Resistance and Beta-Lactamases

• Beta-Lactamases: are enzymes that degrade Beta-Lactams which is a major antibiotic resistance mechanism
• Serine Beta-Lactamases (SBLs) can cause hydrolysis when serine reacts with Beta-lactam
• Hydrolysis product is inactive

Beta-Lactamase Inhibitors

• Beta-lactams are often co-prescribed with Beta-Lactamase Inhibitors
  • Augmentin is an example: amoxicillin and clavulanic acid
• Inhibitors like Augmentin stop SBLs from degrading beta-lactams
• Most Inhibitors will react with SBL serine, blocking the active site

Beta-Lactam Resistance and MRSA

• CAuses Methicillin-resistant Staphylococcus aureus (MRSA)
  • Is a major cause of healthcare-associated infections
• The MRSA mecA gene encodes PBP2a to cause resistance to beta-lactam antibiotics
  • The active site is shielded and will only open when bound to PG

Vancomycin

• A Glycopeptide antibiotic
  • Made by Streptomyces (soil bacteria)
  • Antibiotic of last resort for Gram-positive infections
• Binds to D-Ala-D-Ala in PG peptide chain, blocks PBPs

Vancomycin Resistance

• Vancomycin resistant microbes are now common, for instance, vancomycin-resistant enterococci (VRE)
• Changes to PG structure can confer resistance
• Such as replacing D-Ala with D-lactate (D-Lac) or D-serine to weaken vancomycin binding by ~1000-fold
• PBPs can still form cross-links

Explore the structure of bacterial cell walls, their main components like peptidoglycans, and the action of antibiotics. Learn about the targets of antibiotics like penicillin, cephalosporins and carbapenems, and processes like peptidoglycan biosynthesis. Questions cover the impact on cell shape, osmotic lysis, and the role of Penicillin-Binding Proteins.