Exam 1: Cell Walls

Christian Gram

• Had a mixed culture.

• Wanted to put them into groups → he developed a gram strain.

  • Differential strain.

• Steps:

  1. Heat-fix a smear of bacteria and expose to crystal violet for one minute.

  2. Add iodine solution for one minute.

     • Iodine is a mordant.

       • Makes the solution sticky.

  3. Decolorize with alcohol for 20-30 seconds.

     • Gram-positive cells keep the purple color.

     • Gram-negative cells lose the purple color.

       • Outer surface is membrane.

         • More susceptible to alcohol.

  4. Counterstain with safranin for 1-2 minutes.

     • Attaches a red-pink color to gram-negative cells.

     • Also attached to gram-positive, but is out-competed by the purple.

• Occurs because of the different membranes.

Peptidoglycan

• Peptid → peptide.

• Glycan → sugar.

• Mesh-like polymer composed of identical subunits.

  • N-acetylglucosamine (NAG or G).

  • N-acetylmuramic acid (NAM or M).

    • NAM has peptides/amino acids attached.

  • Order goes NAM → NAG → NAM → NAG…

    • Linked by glycosidic bonds.

  • Several different amino acids.

• Chains of linked peptidoglycan subunits are cross-linked by peptides.

• Builds front to back, and then it layers.

  • By peptide bonds.

Peptidoglycan monomer

• D-amino acids.

  • Peptidases recognize L-amino acids.

    • With peptidoglycan having D, it is resistant to peptidases.

  • D-glutamic acid.

  • D-alanine.

    • D is only found in peptidoglycan.

• Meso-Diaminopimelic acid (DAP).

  • Found in gram-negative bacteria only.

• L-Lysine.

  • Found in gram-positive peptidoglycan.

Gram negative peptidoglycan structure

• D-alanine in one strand connects with DAP to link the peptides.

  • Covalently linked.

• D-amino acids are unique.

Gram positive peptidoglycan structure

• Uses interbridge to attach peptides.

  • Five amino acids.

    • Uses the same five.

  • Links to L-Lysine.

Synthesis

• Peptidoglycan precursor made in cytosol.

  • M-G pentapeptide.

• Brought to membrane, interacts with bactoprenol.

  • Bactoprenol is stuck in the membrane because it has over 50+ carbons.

• Bactoprenol flips it across the membrane → flippase.

  • From inside to outside.

• Autolysins break existing bonds to allow for the peptides to enter.

• Transglycosylase glues in backbone.

Transpeptidation

• Chemical reaction that forms the peptide cross-links.

• Penicillin binding protein (transpeptidase) connects peptides.

Antimicrobial target

• Penicillin.

  • Binds to penicillin binding protein and inhibits it.

  • Cannot perform transpeptidation.

    • Make cell wall weak.

  • Works best with gram-positive interactions.

• Vancomycin.

  • Treats resistance infections.

    • Last line of defense.

• Bacitracin.

  • Triple antibiotic ointment.

• Cycloserine.

  • Treats tuberculosis.

• Fosfomycin.

  • Treats urinary tract infections.

Autolysins

• Secreted by the cell in a small amount.

• In order to grow, the bacterium needs to digest own peptidoglycan.

• Autolysins digest peptidoglycan.

• New material can then be added.

Gram positive bacteria review

• Cell wall is on superficial surface.

  • On the outside.

• Thick peptidoglycan.

  • Many layers.

• Teichoic/lipoteichoic acids.

  • Lipoteichoic acid goes to membrane, teichoic does not.

    • Both extend off the surface.

• Sortase: attaches secreted proteins covalently to the cell wall.

  • Attaches the teichoic acid to the cell wall.

  • Only in gram positive bacteria.

Gram negative bacteria review

• Two membranes.

  • Outer.

    • Has LPS.

      • The core polysaccharide is going to be the same in all strains of the species.

      • Lipid A is an attachment point.

        • Called an endotoxin because it is attached.

      • O-specific polysaccharide is variable.

        • Main focus.

    • Has porins.

  • Inner.

• Cell wall is in the space between two layers.

  • Called the periplasm.

  • Has less layers of peptidoglycan.

Impact of cell wall and outer membrane

• Support cell shape.

• Protect from toxic substances.

  • The membranes for gram negative and positive will work differently.

• Osmotic protection:

  • Isotonic solution → happy cells.

  • Hypotonic solution → lysis; water moves in, more salt inside.

  • Hypertonic solution → plasmolysis; water leaves, more salt outside.

• Contribute to pathogenicity.

  • LPS = endotoxin.

    • Considered a mitogen.

      • Non-specifically turns on immune cells.

  • Peptidoglycan.

    • Activates complement.

• Immune system recognition.

  • Pathogen-associated molecular patterns (PAMPs).

    • Only found on microbes.

  • Recognized by pattern recognition receptors (PRRs).

    • Results in a number of defense responses.

• Target for antimicrobials.

Sec secretion

• One-step secretion process.

  • Something is secreted through one membrane.

• Goes through the periplasm, then through the membrane.

Two-step secretion

• Mainly with gram-negative bacteria.

  • Outer membrane and periplasm.

• Most secreted proteins moves through the general secretory pathway (type 2).

• Type 2: sec = secretes linear proteins (unfolded).

• Type 5: tat = secretes folded proteins.

  • Autotransporter.

    • Some of the protein is used to create the opening for secretion.

One-step secretion (across outer membrane)

• Type 1:

  • Spans both membranes.

  • One-step system.

• Type 3, 4, and 6:

  • Has needle-like structures.

  • Spans both membranes and host membrane.

    • Three membranes.

  • Type 3 is protein.

  • Type 4 is microbial DNA.

  • Type 6 has two forms:

    • Retracted vs. contracted.

      • Shoots the secretion through.

Archaeal cell walls

• No peptidoglycan.

• Different cell wall structures.

  • Pseudomurein.

    • Fake peptidoglycan.

  • Complex polysaccharides.

  • Protein, glycoprotein (S-layers).

    • Most have S-layers.

• Psuedomurein vs. peptidoglycan:

  • NAG in peptidoglycan vs. N-acetyltalosaminuronic acid in psuedomurein.

  • Different linkages.

  • L-amino acids.

    • No D-amino acids in psuedomurein.

Fungal cell walls

• Simpler.

• Polysaccharides.

  • Chitin.

  • Cellulose.

  • Glucan.