Cell Envelope

Question 1

Why does Gram Negative Bacteria have LPS (lipopolysaccharide) in its outer membrane?

Answer 1

• Replaces phospholipid in outer leaflet
• Serves as defensive layer - protects against antibiotics, bacteriophages and antimicrobial peptides
• Strong lateral interactions between LPS molecules
• PROINFLAMMATORY
• LPS is amphipathic (both hydrophobic and hydrophilic regions) - can anchor firmly within outer lipid layer of bacterial cell
• help prevent passive diffusion of substances like antibiotics and detergents

Question 2

How is LPS pro-inflammatory and what role does TLR4 play?

Answer 2

• LPS interacts w/ receptors on macrophages + B-cells to trigger cytokine and chemokines release when body is fighting bacteria infection
• Binds to TLR4 (protein on surface of immune cells)
• Binding activates intracellular signaling pathways
• triggers up-regulation of pro-inflammatory cytokines
  -Excessive/prolonged inflammation -> pathologies
• can cause endotoxin shock from overproduction - type of bacterial toxin

Question 3

Describe the structure of LPS?

Answer 3

1. O-antigens/O-polysaccharide
   - highly variable between diff bacterial strains, allow host immune system to recognize and target specific bacterial types
   - varies in sugars, sequence, chemical linkage, substitution, ring forms used
   - outermost part: major antigen targeted by host antibody responses
   -3-5 sugars repeated <25 times
2. Core-oligosaccharide
   - structures much more limited
3. Lipid A
   - highly hydrophobic
   - v endotoxically active
   - conserved structure - recognised by host receptors like (TLR4)

Question 4

What are the 2 defining features of Gram-Positive Cell Walls?

Answer 4

a. Teichoic Acids
b. Cell-Wall-Anchored Proteins

Question 5

What are Teichoic Acids (in context of Gram-Positive Bacterium)?

Answer 5

• Negatively charged polymers
• 2 Types: Lipoteichoic acid (membrane-anchored) and Well Teichoic Acid (peptidoglycan-anchored)

Question 6

What are the functions of Teichoic Acids?

Answer 6

• Binds to receptors and surfaces
• Negative surface charge (due to phosphate groups) - allows to bind positively charged ions (cations)
• Growth and division
  (involved in regulation of cell division - influence proper localization + assembly of cell wall machinery) (also help coordinate peptidoglycan synthesis - essential for building cell wall during growth)
• Host cell recognition
• Provide protection from harmful molecules (AMPs and Glycopeptide antibiotics)
• Cation homeostasis (bind to cationic groups + are receptors for phage particles)

Question 7

What are some modifications to Teichoic Acids?

Answer 7

• Lots of variation in modifications
  1. D-Alanine
  BENEFIT: increased resistance to host defences, antimictobrial peptides, glycopeptide antibiotics
  (more common in chronic infections)
  CON: can reduce ability to adhere to host cells + establish infection

2. Glycosylation
   BENEFIT: Increased protection from immune system
   Con: may increase susceptibility to bacteriophages

Question 8

What are cell-wall anchored proteins?

Answer 8

• Surface proteins that are covalently attached to peptidoglycan cell wall via sortase enzymes
• Synthesized in cytoplasm
• translocated across cytoplasmic membrane
  -Covalently anchored to pentaglycine cross-bridge of peptidoglycan (Staphylococcys aureus) (crucial for various bacterial processes, including colonization, biofilm formation, and pathogenesis)

Question 9

What are the Functions of Sortase-Anchored Surface Proteins?

Answer 9

• Bacterial adhesion
• Invasion of mammalian cells
• Binding to plasma proteins
• Immune evasion
• Inducing inflammation (interact w/ host immune receptors + trigger signaling pathways -> release of cytokines)
• Biofilm formation
  Ex. Listeria monocytogenes, staphylococci, streptococci, enterococci

Question 10

What are some similarities between Capsules, Extracellular Polysaccharides and Biofilms?

Answer 10

Outermost layer of protection beyond outer membrane + cell wall

Common structure, biogenesis and export pathways

Assist in adhesion to solid surfaces

Protect against antibiotics, antimicrobial peptides and host immune responses

Make infections hard to treat

Question 11

What are Capsules? Where are they found?

Answer 11

• Loacted on outside of bacterial cell wall (outermost layer) and envelopes entire cell
• Connect to the peptidoglycan in Gram-negative bacteria or the plasma membrane in Gram-positive bacteria via covalent attachments to either phospholipid or lipid-A molecules
• Polysaccharide layer w/ high water content
• Is a type of glycocalyx (sticky sugar coat)
• Resists phagocytosis

Question 12

What are the functions of the capsule?

Answer 12

• Barrier to toxic hydrophobic molecules (i.e detergents) - due to high water content
• Protects cell against desiccation/drying out
• Resistance to bacteriophage (can mask receptors present on cell surface - prevents phages from directly binding to receptors)
• Evade host defenses (Avoid detection -> hide cell envelope structures (escape phagocytosis);
  Avoid destruction -> prevent antibody-mediated lysis )

Question 13

What is Bacillus Anthracis and why is the capsule significant?

Answer 13

• The capsule functions as the virulence factor
• inhibits phagocytosis + allows survival of anthrax and decimation of healthy cells
• Prevents immune cells like macrophages from engulfing + destroying bacteria
• Invasive bacterial pathogens are often encapsulated and based upon serotype (grouping bacteria based on cell surface structures)

Question 14

What are EPS and how do they relate to biofilms?

Answer 14

• Extracellular Polysaccharides form the biofilm matrix (3-dimensional network around + between bacterial cells)
• considered fundamental component
• Acts as a “glue like” material that binds bacterial cells
• Made up of polysaccharides, proteins, lipids, nucleic acids
  -Provides sheltered environment for bacterial cells against antimicrobial agents
  -Stability of matrix is ensured by non-covalent bonding between EPS that involves weak physiochemical forces
  -Matrix provides structural scaffold for biofilm – stable + resilient

Question 15

Bacteria in biofilm are:

Answer 15

1. Impervious to phagocytosis by neutrophils and macrophages
2. Resistant to antimicrobial peptides + complement
3. Semi-dormant - difficult to inhibit w/ antibiotics

Question 16

How do biofilms complicate Cystic Fibrosis?

Answer 16

• caused by P. aeruginosa
  -Bacteria often grows as biofilms in lungs
• Biofilms contribute to severity and persistance of infection by protecting bacteria from antibiotics + immune responses
  -Polysaccharide Alginate helps biofilm structure

Question 17

What are characteristics of S-layers (Surface layers)?

Answer 17

• 2D crystalline surface layers that are oblique, square or hexagonal
• Serve as a protective barrier w/ selective permeability
• Composed of proteins or glycoproteins
  -Usually a single protein
• Coats entire bacterial cell surface
• Found in gram-positive, gram-negative bacteria and Archae

Question 18

What are functions of S-layers in bacteria?

Answer 18

1. Acts as molecular sieve - absorb molecules based on size and morphology of pores
2. Protection
   -physical barrier against various environmental factors
   - resistant to bacteriophage, complement, phagocytosis
3. Adhesion to host cells
   - directly interact w/ various substrates + host cells