MICR 221 - Lecture 10: Prokaryotic Cell Envelope PDF

Antibiotics and Cell Wall Structure Bacterial susceptibility to an antibiotic measured by minimum inhibitory concentration (MIC) Lowest antibiotic concentration that fully inhibits growth Penicillin G MIC Colistin MIC Escherichia coli 32 μg/mL...

Antibiotics and Cell Wall Structure Bacterial susceptibility to an antibiotic measured by minimum inhibitory concentration (MIC) Lowest antibiotic concentration that fully inhibits growth Penicillin G MIC Colistin MIC Escherichia coli 32 μg/mL 1 μg/mL Staphylococcus aureus 0.5 μg/mL 128 μg/mL E. coli is Gram-negative; S. aureus is Gram-positive Cell wall structure can have a major impact on antibiotic susceptibility 1 Lecture 10: The Prokaryotic Cell Envelope: Part II Jan. 28, 2025 2 Lecture Learning Outcomes After this lecture, students will be able to describe… The structure of LPS, and how it contributes to the functions of the Gram-negative outer membrane How and why bacteria modify the structure of LPS The functions of proteins in the outer membrane and periplasm The structure and function of the teichoic acids in the Gram-positive cell wall How the mycobacterial cell wall differs from typical Gram-positives and Gram-negatives 3  Gram-Negative Cell Wall Gram-negatives have additional lipid bilayer outside PG Outer membrane 4 Image from: Prescott’s Microbiology, 11th Edn Gram-Negative Outer Membrane Asymmetric bilayer Outer leaflet: lipopolysaccharide (LPS) LPS only in Gram-negatives Inner leaflet: phospholipids Outer membrane is an impermeable barrier Excludes antibiotics, host defenses, etc Membrane proteins are needed for transport, detecting stimuli, etc 5 Lipopolysaccharide (LPS) Chemical properties contribute to barrier function Negatively charged Amphipathic (hydrophilic and hydrophobic) Sterically bulky Three parts: Lipid A Core polysaccharides O-Antigen 6 Lipid A (LPS) Embedded in outer membrane Anchors LPS to membrane Glucosamine sugars attached to fatty acids Phosphorylated (negative charges) Responsible for toxicity (endotoxin) Released when cells lysed Causes fever, inflammation Can lead to septic shock, multiple organ failure 7 Core Polysaccharide (LPS) Links lipid A and O-antigen Made of sugars Branched Contains Kdo (2-keto-3- deoxyoctulosonate) Anionic sugar Negatively charged Phosphorylated Kdo sugars 8 O-Antigen (LPS) All LPS in a cell has same repeating 3 - 5 sugar unit Number of repeats varies LPS size heterogeneous Structure varies between strains Used for classification E.g., E. coli O157:H7 O157: type of O-antigen Antigenic Potential vaccine target Bacteria can change structure to evade immune response (e.g., antibodies) 9 Divalent Cations Phosphates, Kdo sugars cause electrostatic repulsion Divalent cations (e.g., Ca2+, Mg2+) stabilize outer membrane Cross-bridge adjacent LPS molecules Neutralize repulsion Cation-binding molecules (e.g., EDTA) weaken outer membrane 10  Outer Membrane Functions Permeability barrier Prevents toxic substances from reaching targets inside cell Substances must pass through steric bulk, hydrophilic and hydrophobic regions O-antigen core polysaccharide lipid A inner leaflet 11 Image from: https://doi.org/10.1038/s41467-020-14655-9 Outer Membrane Functions Hydrophobic antibiotics can easily cross cytoplasmic membrane Travel through outer membrane is much harder Charged lipid A, core, and divalent cations Hydrophilic O-antigen, core Gram-positives more susceptible to many antibiotics 12 Outer Membrane Functions Bacteria live in environments with degradative enzymes Gastrointestinal (GI) tract: proteases, lipases Enteric bacteria typically Gram-negative Immune system: lysozyme Enzymes are large, unable to cross outer membrane Outer membrane itself not degraded by most enzymes 13 Outer Membrane Functions Bacteria live in environments with detergents E.g., bile acids in GI tract Detergents solubilize lipid bilayers Amphipathic, often negatively charged Cytoplasmic membrane is easily solubilized Outer membrane resistant to detergents LPS keeps detergent kept away from hydrophobic core (steric effects, hydrophilicity, charge) 14 Divalent Cations and LPS Structure Divalent cations (e.g., Mg2+) needed for cross-bridging If Mg2+ is sufficient, LPS is made with “normal” lipid A Phosphates cross-bridged by Mg2+ 15 Impact of Mg2+ Limitation on LPS If Mg2+ is limited, some bacteria modify lipid A with 4- aminoarabinose (4AA) 4AA forms cross-bridges with phosphate groups 16 Colistin and Lipid A Colistin: cationic lipopeptide antibiotic Specifically kills Gram-negatives Binds to lipid A phosphate groups Lipid tail permeabilizes lipid membranes 17 Mg2+ Limitation and Colistin Resistance Mg2+ levels impact bacterial susceptibility to colistin If Mg2+ plentiful, cells are sensitive If Mg2+ limited, lipid A might be modified with 4AA 4AA prevents colistin from binding to lipid A Bacteria are resistant 18 MCR-1 and Colistin Resistance mcr-1 (mobilized colistin resistance) gene confers resistance to colistin Plasmid-encoded; easily spread by HGT MCR-1 attaches phosphoethanolamine to lipid A Positive charge repels colistin 19  Outer Membrane Proteins Outer membrane is protein-rich (50% by mass) Mostly lipoproteins, β-barrel proteins Porins, receptors needed for import Secretion systems, efflux pumps needed for export Proteins build the outer membrane (LPS, proteins) Proteins anchor outer membrane to PG 20 Image from: https://doi.org/10.1038/nrmicro1322  Proteins: Nutrient Uptake Porins form channels in outer membrane Allow nutrients to enter β-Barrel proteins Most are trimeric >250,000 porins/cell Side view: Top view: Center is water-filled channel Structure provides some selectivity 21 Image from: Prescott’s Microbiology, 11th Edn  Proteins: Outer Membrane Assembly LPS is assembled in cytoplasmic membrane Must cross periplasm, outer membrane Difficult due to LPS amphipathicity Lpt proteins form Lpt pathway Transports LPS to outer membrane LptD (β-barrel protein) translocates LPS through membrane 22 Image from: Prescott’s Microbiology, 11th Edn  Peptidoglycan-Associated Proteins Outer membrane is destabilized if not attached to PG Vesiculation Proteins anchor outer membrane to PG Braun’s lipoprotein: Fatty acid chain is embedded in outer membrane Covalently attached to PG Most abundant protein in E. coli Some porins, lipoproteins bind to PG non-covalently 23 Image from: Prescott’s Microbiology, 11th Edn  Gram-Negative Periplasm Region between cytoplasmic and outer membrane 20 - 40% of cell volume PG occupies entire periplasm Cytoplasmic membrane pushed against PG by osmotic pressure Proteins attach outer membrane to PG Protein-rich space 24 Image from: Prescott’s Microbiology, 11th Edn  Periplasmic Proteins Nutrient transport proteins Deliver nutrients to cytoplasmic membrane Catabolic enzymes Proteases, lipases, etc Components of cellular structures (e.g., pili, flagella) Penicillin-binding proteins Antibiotic resistance enzymes (e.g., β-lactamases) 25 Image from: David Goodsell, @dsgoodsell  Gram-Positive Cell Wall No outer membrane Thick PG layer (20 - 80 nm; < 10 nm in Gram-negatives) Teichoic acids Small periplasmic space 26 Image from: Prescott’s Microbiology, 11th Edn  Teichoic Acids Major component of Gram-positive cell wall ~50% by weight (Bacillus subtilis) Linear polymers of glycerol or ribitol Joined by phosphates 20-30 glycerol units (B. subtilis) Phosphate groups negatively charged Glycerol/ribitol can have glucosamine or D- alanine substituents R group in figure Positively charged 27 Image from: Prescott’s Microbiology, 11th Edn  Teichoic Acids Wall teichoic acid (WTA) Teichoic acid in figure Attached to NAM or peptide in PG Extends beyond PG surface Lipoteichoic acid (LTA) Attached to glycolipids in cytoplasmic membrane Associated with PG 28 Image from: Prescott’s Microbiology, 11th Edn  Functions of Teichoic Acids Anchor cell wall to cytoplasmic membrane Bind to cations Minimize repulsion Control access to cell wall surface Regulate where PG is degraded during cell division D-Alanylation protects against antibiotics, host defenses 29 Image from: doi: 10.1146/annurev-micro-092412-155620  Teichoic Acids and Infections Contribute to pathogenesis Adhesion, biofilm formation Colonization of host tissues Release can cause severe inflammatory response Recognized by innate immune system Microbe-associated molecular pattern (MAMP) Also: LPS, PG Activate pattern recognition receptors on immune cells E.g., toll-like receptors (TLRs) 30 Image from: https://www.invivogen.com/when-you-cant-do-it-alone-tlr2-heterodimers-and-innate-immunity  Mycobacteria Group of Gram- positive bacteria E.g., Mycobacterium tuberculosis Difficult to stain Gram-staining: do not appear pink or purple Acid-fast staining needed Heat needed to stain cell wall Decolorize with acid-alcohol Mycobacteria retain stain, other cells decolorized Mycobacteria have unusual cell walls 31 Image from: Prescott’s Microbiology, 12th Edn  Mycobacterial Cell Wall Considered Gram- positive, but have an outer membrane Membrane made of mycolic acids, not LPS Arabinogalactan layer between PG and outer membrane Sugar polymers Covalently connects mycolic acids to PG 32 Image from: Prescott’s Microbiology, 11th Edn  Mycobacterial Outer Membrane Outer membrane is an asymmetrical bilayer Inner leaflet: mycolic acids glycolipids Outer leaflet: mixture of lipids No phospholipids mycolic acid Membrane is very hydrophobic, highly impermeable Impenetrable (e.g., to stains, many antibiotics) Porins needed for small molecules to enter Mycolic acid 33 Image from: Prescott’s Microbiology, 11th Edn Reminders Bacteriology Quiz 3 opens Jan. 30 at 3 PM Lectures 9 - 11 Office hour: Jan. 28, 12 – 1 PM, Botterell 449 Lab 1 Assignment Section 004: due Jan. 30 at 2:30 PM Sections 003, 005: due Jan. 31 at 2:30 PM Lab 2 Section 004: Jan. 30 at 2:30 PM Sections 003, 005: Jan. 31 at 2:30 PM Complete Lab 2 Pre-Lab Quiz before 34