MICR 221 Lecture 9: Prokaryotic Cell Envelope - PDF

Antibiotic Use in Canada 2019: 213,000 kg of antibiotics intended for use in humans (Canada) Most are β-lactam antibiotics Penicillins, cephalosporins, carbapenems 145,000 kg Target...

Antibiotic Use in Canada 2019: 213,000 kg of antibiotics intended for use in humans (Canada) Most are β-lactam antibiotics Penicillins, cephalosporins, carbapenems 145,000 kg Target cell wall 1 Data from: Canadian Antimicrobial Resistance Surveillance System Report 2021 Lecture 9: The Prokaryotic Cell Envelope: Part I Jan. 23, 2025 2 Lecture Learning Outcomes After this lecture, students will be able to describe… The lipids and proteins in the cytoplasmic membrane The function of peptidoglycan in the cell wall The structure of peptidoglycan, and how it is synthesized by PBPs and other enzymes How antibiotics (e.g., β-lactams, vancomycin) target peptidoglycan, and how bacteria resist these antibiotics 3  Prokaryotic Cell Envelope Cytoplasmic membrane and all layers that surround it Includes cell wall Peptidoglycan, outer membrane (Gram- negatives), others Also includes layers outside cell wall (e.g., capsule) Roles include: Controls what enters, exits cell Protects against stresses, antibiotics, immune cells, etc 4 Image from: https://micro.magnet.fsu.edu/cells/bacteriacell.html  Cytoplasmic Membrane Lipid bilayer Lipid composition depends on conditions (e.g., temperature) Protein-rich Semipermeable barrier Water, very hydrophobic substances diffuse through Hydrophilic substances (ions, most nutrients) do not 5 Image from: Prescott’s Microbiology, 11th Edn  Bacterial Cytoplasmic Membrane Mainly phospholipids Fatty acids attached to glycerol by ester bonds Glycerol bonded to phosphate group Phosphate may have substituent (e.g., ethanolamine) Amphipathic: polar and non- polar regions 6 Image from: Prescott’s Microbiology, 11th Edn  Cytoplasmic Membrane Protein Functions Transporters Import substances (e.g., nutrients) Export substances (e.g., EPS for biofilms, toxins) Signal transduction (detect external stimuli) Energy transduction Electron transport chain (ETC) enzymes generate H+ gradient across membrane (proton motive force, PMF) PMF powers ATP synthesis, transport, etc 7 Image from: https://www.nature.com/scitable/topicpage/cell-membranes-14052567/ Osmolarity and the Cytoplasmic Membrane Bacteria are usually in a hypotonic environment More solutes in cell than outside Water drawn into the cell (osmotic pressure) Water influx causes swelling, can cause osmotic lysis How do bacteria survive in hypotonic conditions? 8 Bacterial Cell Wall Most bacteria have a cell wall Layer(s) outside cytoplasmic membrane Peptidoglycan (PG) is major component Gram-positives: thick Gram-negatives: thin Some cell walls have additional layers Gram-negative outer membrane 9  Peptidoglycan (PG) Strong Determines cell shape Protects against osmotic lysis (pushes against membrane) Elastic Stretches, contracts in response to osmotic pressure Porous, mesh-like (nutrients, waste can pass through) 10 Image from: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/peptidoglycan  Osmotic Stabilization of the Cell If PG is disrupted, cell is more susceptible to osmotic lysis in a hypotonic environment PG targeted by antibiotics, immune system Bacteria can survive PG degradation in isotonic conditions Lose shape, forming spheroplasts (Gram-negative) or protoplasts (Gram-positive) Swell and lyse if moved to hypotonic conditions 11 Image from: Prescott’s Microbiology, 11th Edn  Mycoplasmas Small (~0.2 μm), pleomorphic bacteria Some are intracellular parasites Lack a cell wall Osmotically sensitive Incorporate sterols from host into cytoplasmic membrane Increases stability Osmotic pressure is lower inside other cells ~ Isotonic 12 Image from: Prescott’s Microbiology, 11th Edn Peptidoglycan and Antibiotics PG is a very good antibiotic target On or near surface Made by most bacteria Not made by human cells Many antibiotics target PG β-Lactam antibiotics (e.g., penicillins) Vancomycin How do they target PG? Target PG chemical structure Target enzymes that make PG 13  Peptidoglycan Structure PG made of sugars and amino acids PG backbone is a long glycan strand with repeating disaccharide units NAM: N-acetylmuramic acid NAG: N-acetylglucosamine Every NAM bears a peptide chain Glycan strands connected to each other through peptide cross-links 14 Image from: Prescott’s Microbiology, 11th Edn Peptidoglycan Biosynthesis: Lipid II Many steps needed to make PG Lipid II: key PG precursor Contains “monomeric” PG subunit: NAM-NAG disaccharide Pentapeptide Bound to membrane by undecaprenol Lipid II synthesis starts with UDP-NAG UDP (uridine diphosphate) activates NAG 15 Making Lipid II Assembled in cytoplasm, flipped across membrane 16 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 (antibiotic) inhibits alanine racemase, D-Ala-D-Ala ligase 17  Penicillin-Binding Proteins Lipid II incorporated into PG by penicillin-binding proteins (PBPs) Located in periplasm Bacteria need PBPs for: Cell growth Cell division Cell wall recycling Many PBPs have a glycosyltransferase domain Makes glycan strand Transpeptidase domain makes peptide cross-links 18 Image from: https://www.xtal.iqfr.csic.es/grupo/xjuan/projects/cell-wall-remodeling/cell-wall-remodeling.html PBP Glycosyltransferase Activity PBP adds lipid II disaccharide to glycan backbone of PG Extends glycan backbone NAM activated by pyrophosphate Releases undecaprenyl pyrophosphate 19 Undecaprenol Recycling Undecaprenyl pyrophosphate is then recycled Dephosphorylated, then flipped to cytoplasm Bacitracin (antibiotic) Binds to undecaprenyl pyrophosphate Blocks dephosphorylation 20  Lysozyme PG glycan backbone can be degraded Bacterial enzymes for cell wall remodeling Antimicrobial enzymes Lysozyme: part of innate immune system Saliva, tears, milk, mucous secretions Cleaves NAM-NAG bond Weakens cell wall Lysozyme more effective against Gram-positives PG is more exposed Gram-negatives have outer membrane 21 Image from: Prescott’s Microbiology, 11th Edn  Peptidoglycan Structure PG strands are helical Peptides extend from glycan backbone PBPs cross-link peptides from different strands 22 Image from: Prescott’s Microbiology, 11th Edn Peptidoglycan Peptide Chains Pentapeptide attached to NAM sugar Amino acid sequence can vary Contains D-amino acids (e.g., D-Ala) Diamino acid in third position L-lysine or meso-diaminopimelic acid (meso-Dap) 23 Peptidoglycan Cross-Linking (Gram-Negatives) Gram-negatives: cross-link between residue 3 (amino group of diamino acid) and residue 4 (carbonyl) Amide bond Releases terminal D-alanine 24 Peptidoglycan Cross-Linking (Gram-Positive) Gram-positives: peptide chains cross-linked through interpeptide bridge Bridge attached to diamino acid Composition varies (e.g., pentaglycine in S. aureus) Releases terminal D-alanine 25 Transpeptidation Mechanism Cross-links are formed by PBP transpeptidase domain First, PBP forms complex with peptide Then, diamino acid reacts with complex, forms amide bond Transpeptidase domain is targeted by β-lactam antibiotics 26  β-Lactam Antibiotics β-Lactams: penicillins, cephalosporins, carbapenems Most widely used antibiotics Four-membered β-lactam ring Penicillin G first in clinical use Made by Penicillium mold Narrow-spectrum Duchesne observed Penicillium antibiotic properties (1896) Re-discovered by Fleming (1928) Found antibiotic activity due to secreted product 27 Image from: Prescott’s Microbiology, 11th Edn β-Lactams Block Transpeptidation β-Lactams inhibit PBP transpeptidase activity β-lactam ring reacts with serine in PBP: Blocks PBP from forming cross-links Weakens PG, leading to cell lysis β-Lactams are bactericidal 28 β-Lactam Resistance and β-Lactamases β-Lactamases: enzymes that degrade β-lactams Major antibiotic resistance mechanism Serine β-lactamases (SBLs): serine reacts with β-lactam SBLs can hydrolyze complex (cf. PBPs) Hydrolysis product is inactive 29 β-Lactamase Inhibitors β-lactams are co-prescribed with β-lactamase inhibitors E.g., Augmentin: amoxicillin and clavulanic acid Inhibitors stop SBLs from degrading β-lactams Most inhibitors react with SBL serine, blocking active site 30 β-Lactam Resistance and MRSA Methicillin-resistant Staphylococcus aureus (MRSA) Major cause of healthcare-associated infections MRSA mecA gene encodes PBP2a β-Lactam-resistant PBP Active site is shielded, only opens when bound to PG 31  Vancomycin Glycopeptide antibiotic Made by Streptomyces (soil bacteria) Used for Gram-positive infections Antibiotic of last resort vancomycin peptidoglycan pentapeptide Binds to D-Ala-D-Ala in PG peptide chain, blocks PBPs 32 Image from: https://pdb101.rcsb.org/motm/192 Vancomycin Resistance Vancomycin resistant microbes are now common E.g., vancomycin-resistant enterococci (VRE) Changes to PG structure can confer resistance E.g., replace D-Ala with D-lactate (D-Lac) or D-serine Weakens vancomycin binding by ~1000-fold PBPs can still form cross-links 33 Reminders Midterm Exam 1: in class on Jan. 27 Accommodated exams administered by Exam’s Office Check Ventus Multiple choice and short answer questions Lectures 1 - 8 Nothing from the lecture today Zoom review session today, 11 – 12 PM Lab 1 Assignment Section 002: due Jan. 23, 2:30 PM Lab 2 Section 002: Jan. 23 at 2:30 PM Complete Lab 2 Pre-Lab Quiz before 34