Medical Microbiology: Bacterial Pathogens and Diseases II (Endotoxins)

Question 1

Describe the structure of a Gram negative bacterial cell wall

Answer 1

• Outer membrane - Contains lipopolysaccharides (endotoxins) and porins
• Inner membrane
• Inbetween outer and inner membrane is the periplasm which contains peptidoglycans

Question 2

Describe the structure of a lipopolysaccharide

Answer 2

• Lipid A
  
  • Contains phosphorylated glucosamines attached to long chain fatty acids
  • No. and type of fatty acid vary by species
  • Hydrophobic
• Polysaccharide core
  
  • Ketodeoxyoctanoic acid (KDO) and heptose
  • Relatively constant between species
  • Hydrophilic
• O-side chain
  
  • Repeat units of tri, tetra or pentasaccharide sugars.
  • Highly variable between species and straisn of same species
  • Hydrophilic

Question 3

What are some of the characteristics of endotoxins?

Answer 3

• Endotoxin is lipopolysaccharide (LPS)
• Lipid A is the active component – not immunogenic (vaccine can’t be produced against it)
• O-side chain/O-antigen is highly immunogenic and immune specific
• Found only in gram negative bacteria
• Heat stable
• Can’t be converted into toxoids
• Major initiator of the sepsis pathway

Question 4

What is sepsis?

Answer 4

• Life threatening organ dysfunction caused by a dysregulated host response to infection

Question 5

What immune system cells are involved in sepsis?

Answer 5

• Macrophages
• Monocytes
• Granulocytes
• Natural killer cells
• Dendritic cells - Antigen presenting cells

Question 6

What do the cells involved in sepsis detect and what is this detetction mediated by?

Answer 6

• These cells detect 2 things:
  
  • Pathogen associated molecular patterns (PAMP’s) such as endotoxin
  • Damage associated molecular patterns (DAMP’s) from damaged host cells
• The detection of PAMPs and DAMPs are mediated via
  
  • Cell membrane receptors – toll-like receptors (TLR) and C-type lectin receptors
  • Cytosol receptors - NOD-like receptors and RIG-I-like receptors

Question 7

What is the effect of the immune system cells involved in sepsis detecting PAMPs and DAMPs?

Answer 7

• Production of pro-inflammatory cytokines such as: TNFα, IL-1, IL-6
• Activation of inflammasomes to produce IL-1β and IL-18 that cause rapid programmed cell death

Question 8

How do endotoxins cause the production of inflammatory cytokines?

Answer 8

• Lipid A component of exotoxin (LPS) is recognised by the protein MD-2
• LPS-bound MD-2 binds to toll-like receptor 4 (TLR4) molceule on cell surface of macrophage
  
  • Co-receptor binding protein CD14 also binds to TLR4
• This leads to dimerization of TLR4 which leads to intracellular signalling
  
  • Activation of NF-kB
  • Leads to production of TNF-α and other pro-inflammatory cytokines

Question 9

What are the effects of pro-inflammatory cytokines?

Answer 9

1. Increase number, lifespan and activation state of innate immune cells
2. Increase adhesion molecule and chemokine expression by endothelial cells - This focuses immune response to area of infection
3. Increase acute phase protein such as complement, fibrinogen and CRP release from liver
4. Cause fever
5. Causes neutrophils to release extra-cellular traps (NETs) made of DNA and antimicrobial proteins that forms a scaffold for platelet activation
6. Cause release of microparticles by activated platelets
7. Increase tissue factor expression by blood monocytes
   * Last 3 effects cause formation of a thrombus (immunothrombosis) - traps microbes which attracts and activates further leucocytes

Question 10

What are some of the effects of sepsis on the body?

Answer 10

• Increased vascular permeability
• Hypotension leading to hypovolaemic shock
• Fever
• Disseminated intravascular coagulation (DIC)
• Multiple organ failure

Question 11

What happens when a endotoxin causes disregulation of the inflammatory immune response?

Answer 11

• Production of reactive oxygen species (ROS), e.g. Hydroxyl and nitric oxides - These damage cellular proteins, DNA and lipids and impairs mitochondria
• Complement activation (esp. C5a) – increase ROS, granulocyte enzyme release, endothelial permeability and tissue factor expression
• Widespread immunothrombosis leading to disseminated intravascular coagulation (DIC) with impaired microvasculature function and organ dysfunction
• As a reult of all these you get mitochondrial damage - This leads to decreased intracellular ATP and cells enter state of hibernation – exacerbates organ dysfunction

Question 12

How can sepsis be resolved?

Answer 12

• Process of sepsis resolution is active not passive
• Anti-inflammatory cytokines (e.g. IL-10) produced early in process
  
  • This suppresses production of IL-6 and γ-interferon
  • Also stimulates production of soluble TNF receptor and IL-1 receptor antagonist
  • This results in TNF binding to recptor and not cells and IL-1 not having receptor to bind to
• Autophagy of PAMP’s and DAMP’s – removal by macrophages
• Damaged cells – undergo apoptosis and engulfment by macrophages

Question 13

What is Meningococcal Sepsis?

Answer 13

• Caused by Neisseria meningitidis which is a Gram negative diplococcus bacteria
• There are multiple strain types/serotypes - e.g. A,B,C,Y, W135
• Neisseria meningitidis can cause disease ranging from meningitis to life threatening meningococcal sepsis

Question 14

What makes meningococcus (Neisseria meningitidis) so effective in sepsis?

Answer 14

• Has a lipooligosaccharide instead of lipooligsaccharide - has a very short O-side chain
  
  • This means terminal part of structure is similar to human erythrocyte antigen
  • This makes it hard to detect compared to erythrocytes
• Constantly sheds blebs when inside body (large chunks of outer cell membrane) which produces large amounts of LOS

Question 15

Explain how all these symptoms are called by Meningococcal sepsis

Answer 15