Prokaryotic Cell Envelope: LPS, Teichoic Acids & Antibiotics

• Lecture is part 2 of the Prokaryotic Cell Envelope
• Lecture Date: Jan 28, 2025

Lecture Learning Outcomes

• Understanding the structure of LPS and its contribution to the Gram-negative outer membrane functions
• Understanding how and why bacteria alter the structure of LPS.
• The functions of proteins in the outer membrane and the periplasm.
• Knowing the structure/function of the teichoic acids in the Gram-positive cell wall
• Comparing the mycobacterial cell wall to typical Gram-positives and Gram-negatives.

Antibiotics and Cell Wall Structure

• Bacterial susceptibility is measured by minimum inhibitory concentration (MIC).
• MIC is the lowest concentration of an antibiotic that completely inhibits growth.
• E. coli is Gram-negative, and S. aureus is Gram-positive.
• MIC Values:
  • E. coli: Penicillin G MIC is 32 µg/mL, Colistin MIC is 1 µg/mL.
  • Staphylococcus aureus: Penicillin G MIC is 0.5 µg/mL, Colistin MIC is 128 µg/mL.
• Cell wall structure has a large impact on antibiotic susceptibility

Gram-Negative Cell Wall

• Gram-negative bacteria have an additional lipid bilayer outside the peptidoglycan layer in their cell walls called the outer membrane.

Gram-Negative Outer Membrane

• Asymmetric bilayer: outer leaflet of lipopolysaccharide (LPS), inner leaflet made of phospholipids.
• Outer membrane is an impermeable barrier and excludes antibiotics, host defenses, etc.
• Membrane proteins are required for transport and stimulus detection.
• LPS only exists in Gram-negative bacteria.

Lipopolysaccharide (LPS)

• Chemical properties contribute to its barrier function
• Negatively charged
• Amphipathic (hydrophilic and hydrophobic)
• Sterically bulky
• Has three parts:
  • Lipid A
  • Core polysaccharides
  • O-antigen

Lipid A (LPS)

• Embedded in the outer membrane and anchors LPS to the membrane
• Consists of glucosamine sugars attached to fatty acids, and is phosphorylated (negative charges).
• Responsible for toxicity (endotoxin) in lysed cells
• Responsible for causing fever, inflammation, and can cause septic shock/multiple organ failure.

Core Polysaccharide (LPS)

• Links Lipid A to the O-Antigen
• Made of sugars, is branched, and contains Kdo (2-keto-3-deoxyoctulosonate, an anionic sugar).
• Negatively charged
• Phosphylated

O-Antigen (LPS)

• All LPS in a cell has a repeating 3-5 sugar unit where the number of repeats varies
• Structure varies between strains
• Used for classification
  • E. coli O157:H7, where O157 is the type of O-antigen
• Antigenic
  • Potential vaccine target
  • Bacteria can change structure to evade immune response (e.g., antibodies).

Divalent Cations

• Phosphates and Kdo sugars cause electrostatic repulsion
• Divalent cations (e.g., Ca2+, Mg2+) stabilize the outer membrane by cross-bridging adjacent LPS molecules and neutralizing repulsion.
• Cation-binding molecules (e.g., EDTA) weaken the outer membrane.

Outer Membrane Functions

• Permeability Barrier
  • Prevents toxic substances from reaching targets inside the cell
• Substances must pass through steric bulk, hydrophilic and hydrophobic regions.
• Hydrophobic antibiotics can easily cross the cytoplasmic membrane whereas travel through the outer membrane is harder
  • Due to: Charged lipid A, core, divalent cations and hydrophilic O-antigen, core
• Gram-positives are more susceptible to many antibiotics
• Bacteria live in environments with degradative enzymes
  • Gastrointestinal (GI) tract: proteases, lipases
    • Enteric bacteria are typically Gram-negative
  • Immune system: lysozyme
• Enzymes are too large to cross the outer membrane
• The outer membrane itself is not degraded by most enzymes
• Bacteria live in environments with detergents
  • E.g., bile acids in the GI tract
• Detergents solubilize lipid bilayers
  • Amphipathic, often negatively charged.
• Cytoplasmic membrane is easily solubilized
• Outer membrane is resistant to detergents
  • LPS keeps detergent kept away from the hydrophobic core (steric effects, hydrophilicity, charge)

Divalent Cations and LPS structure

• Divalent cations (e.g., Mg2+) are needed for cross-bridging
• If Mg2+ is sufficent, LPS is made with "normal" lipid A
  • Phosphates are cross-bridged by Mg2+.
• If Mg2+ is limited, some bacteria modify lipid A with 4-aminoarabinose (4AA)
  • 4AA forms cross-bridges with phosphate groups.

Colistin and Lipid A

• Colistin is a cationic lipopeptide antibiotic
  • Specifically kills Gram-negatives.
  • Binds to lipid A phosphate groups and the lipid tail permeabilizes lipid membranes.
• Mg2+ levels impact bacterial susceptibility to colistin.
  • If Mg2+ is plentiful, cells are sensitive.
  • If Mg2+ is limited, lipid A might be modified with 4AA and 4AA prevents colistin from binding to lipid A, so the bacteria are resistant.
• mcr-1 (mobilized colistin resistance) gene confers resistance to colistin
  • Plasmid encoded, easily spread by HGT
  • MCR-1 attaches phosphoethanolamine to lipid A, ultimately the now resulting positive charge repels colistin

Outer Membrane Proteins

• The outer membrane is protein-rich (50% by mass)
  • mostly lipoproteins, β-barrel proteins
• Porins, receptors are needed for import
• Secretion systems, as well as efflux pumps, are needed for export
• Proteins build the outer membrane (LPS, proteins)
• Proteins anchor the outer membrane to PG

Proteins: Nutrient Uptake

• Porins form channels in the outer membrane that enable nutrients to enter
  • β-Barrel proteins form the porins; they are mostly trimeric ->250,000 porins/cell
• Center of the porins contain a water-filled channel where the structure provides some selectivity.

Proteins: Outer Membrane Assembly

• LPS is assembled in the cytoplasmic membrane
• It must cross the periplasm and outer membrane (difficult because of LPS amphipathicity)
• Lpt proteins form the Lpt pathway
  • Transports LPS to outer membrane
• LptD (β-barrel protein) translocates LPS through the membrane

Peptidoglycan-Associated Proteins

• If the outer membrane is not attached to PG, it is destabilized and vesiculation can occur
• Proteins anchor the outer membrane to PG
• Braun's lipoprotein contains a fatty acid chain embedded in the outer membrane
  • It is covalently attached to PG
  • Most abundant protein in E. coli
• Some porins and lipoproteins bind to PG non-covalently

Gram-Negative Periplasm

• Region between the cytoplasmic and outer membrane, occupying 20-40% of cell volume
• PG occupies the entire periplasm
• The cytoplasmic membrane is pushed against PG by osmotic pressure
• Proteins attach the outer membrane to PG
• Protein-rich space

Periplasmic Proteins

• Nutrient transport proteins that deliver nutrients to the 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)

Gram-Positive Cell Wall

• No outer membrane
• Thick PG layer (20 - 80 nm; PG layer is < 10 nm in Gram-negatives)
• Contains Teichoic acids
• Small periplasmic space

Teichoic Acids

• A major component of the Gram-positive cell wall
  • ~50% by weight (Bacillus subtilis)
• Linear polymers of glycerol or ribitol joined by phosphates; typically about 20-30 glycerol units (B. subtilis)
• Phosphate groups negatively charged
• Glycerol/ribitol can have glucosamine or D-alanine substituents
  • R group figure is positively charged
• Wall teichoic acid (WTA): teichoic acid is attached to NAM or peptide in PG and extends beyond PG surface.
• Lipoteichoic acid (LTA) is attached to glycolipids in the cytoplasmic membrane and is associated with PG.

Functions of Teichoic Acids

• Anchor the cell wall to cytoplasmic mambrane
• Bind to cations and minimize repulsion
• Control access to the cell wall surface
  • Regulate where PG is degraded during cell division
• D-alanylation protects against antibiotics, host defenses.
• Contribute to pathogenesis
  • Adhesion, biofilm formation, and colonization of host tissues
  • Their release can cause severe inflammatory response
• Recognized by the innate immune system as microbe-associated molecular patterns (MAMPs)
  • They are also activators of pattern recognition receports on imune cellse.g., toll-like receptors (TLRs)

Mycobacteria

• Group of Gram-positive bacteria
  • Mycobacterium tuberculosis
• Difficult to stain because Gram-staining usually does not appear as pink or purple under a microscope
• Need acid-fast staining
  • Heat needed to stain cell wall
  • Decolorize with acid-alcohol--mycobacteria retain stain and other cells decolorize Unusual cell walls

Mycobacterial Cell Wall

• Considered Gram-positive, but have an outer membrane
• The membrane is made of mycolic acids, not LPS
• Arabinogalactan between PG and outer membrane
  • Sugar polymers
  • Covalently connects mycolic acids to PG

Mycobacterial Outer Membrane

• Outer membrane is an asymmetrical bilayer with an inner leaflet of mycolic acids and an outer leaflet of mixture of lipids, with no phospholipids
• Very hydrophobic and highly impermeable
  • Impenetrable from stains and many antibodies/antibiotics
• Porins (specialized channels) are needed for small molecules to enter

Explore the structure of LPS and its role in Gram-negative outer membrane functions. Understand bacterial cell wall structures, including teichoic acids in Gram-positive bacteria and the unique mycobacterial cell wall. Also, examine the impact of cell wall structure on antibiotic susceptibility, using MIC values for E. coli and S. aureus.