Bacterial Pathogens and Disease ENDOTOXINS

Question 1

Describe the endotoxin in the gram negative bacteria

Answer 1

• The endotoxin (the lipopolysaccharide) is an integral part of the gram negative bacterial cell wall
• It sits in the outer membrane (it is not secreted or released)

Question 2

What are three areas of a lipopolysaccharide?

Answer 2

• LIPID A - Hydrophobic (repels water soluble materials)
  
  • Phosphorylated glucosamines attached to long chain FAs
  • No. + type of FA varies in species
• POLYSACCHARIDE CORE- Hydrophillic
  
  • Ketodeoxyoctanoic acid (KDO) and heptose
  • Relatively constant between
• O-SIDE CHAIN - Hydrophillic
  
  • Repeat units of tri, tetra or pentasaccharides
  • Highly variable between species

Question 3

What are the characteristics of endotoxins?

Answer 3

• Endotoxin is a LPS
• Lipid A = active component → Not immunogenic
• O antigen is highly immunogenic and immune specific
• Only found in gram negative bacteria (exotoxins found in all bacteria)
• Heat stable (exotoxins are not heat stable)
• Not converted to toxoids (unlike exotoxins)
• Major initiator of the sepsis pathway

Question 4

Define sepsis

Answer 4

Life threatening organ dysfunction caused via a dysregulated host response to infection

Question 5

Describe the immune response that sepsis is primary driven with

Answer 5

• Sepsis is primarily driven by the innate immune system
  
  • Macrophages, monocytes, granulocytes, NKs, Dendritic cells
• Cells will detect:
  
  • PAMPs e.g endotoxins
  • DAMPs (damage associated molecular patterns) from damaged host cels
• Detection is mediated via
  
  • Cell membrane receptors (Toll like receptors and C-type lectin receptors)
  • Cytosol receptors (NOD-like receptors and RIG-I-like receptors)
• Effect
  
  • Production of inflammatory cytokines TNF-alpha, IL-1 and IL-6
  • Via inflammasomes to produce IL-1beta and IL-18 causing rapid programmed cell death

Question 6

Describe what happens when a endotoxin binds to a CD14 receptors on macrophages

Answer 6

• Will be transported to the toll like receptor via a molecule called MD2
• This will cause dimerization of two TLR4 receptors
• Will result in myddosome formation and activation of Nf-kB
• This allows production of TNF-alpha and other cytokines
• • Exotoxins will stimulate inflammatory cytokine release either directly through a superantigen (nonspecific bridging of the MHC Class II and T-cell receptor leading to cytokine production)

Question 7

What are the effects of pro-inflammatory cytokines?

Answer 7

1. Increase number, lifespand and activation state of innate immunity
2. Increase adhesion molecule and chemokine expression by endothelial cells
3. Increase acute phase protein such as complement, fibrinogen and CRP
4. Cause fever
5. Causes neutrophils to release extra-cellular traps 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

5+6+7 → Cause formation of a thrombus (immunothrombosis)– microbes trapped within this. → attracts and activate further leucocytes.

Question 8

How can sepsis become dysregulated?

Answer 8

• Noramlly the process described above will achieve rapid control of locallised and minor infections
• However the process may pass a threshold = systemic injury (occurs everywhere in the body)

Question 9

Describe the processes which occur in sepsis dysregulation

Answer 9

• Production of ROS ⇒ OH• and NO• = damage cellular proteins, DNA and lipids + impair mitochondria
• Complement activation (esp C5a) = increases ROS, granulocyte enzyme release, endothelial permeability and tissue factor expression
• Wide spread immunothrombosis leading to disseminated intravascular coagulation (DIC) with impaired microvasculature function and organ dysfunction
• Mitochondrial damage = decreased intracellular ATP and cells enter state of hibernation = exacerbates organ dysfunction

Question 10

How does the body try to resolve sepsis?

Answer 10

• Active (not passive process)
• Anti-inflammatory IL-10 produced early in process
  
  • Supresses IL-6 and gamma interferon
  • Production of soluble TNF-alpha receptor and and IL-1 receptor antagonist
    
    • TNF-alpha more likely to bind to this than TNF-a receptor on tissues preventing tissue necrosis
• Autophagy of PAMPs and DAMPs = removal of stimulus
• Damaged cells undergo apoptosis and engulfment by macrophages

Question 11

Describe meningococcal sepsis

Answer 11

• Caused by Neisseria meningitidis
• Gram negative diplococcus (therefore has endotoxin)
• Serotypes A,B,C, Y, W135
• Serotype A associated with large outbreaks in Sahel region of Africa – Meningitis belt.
• Serotype B,C and W135 found in UK – declined since introduction of MenC and now MenB vaccine.
• Can cause disease ranging from meningitis to life threatening meningococcal sepsis.

Question 12

What features does meningococcus have that make it so effective in sepsis?

Answer 12

• Six fatty acid chains = highly effective as endotoxin
• LOS - lipooligosaccharides= terminal part is similar to human erythrocyte antigen = mimicry of host antigen
• Sheds many polysaccharides in blebs = produce huge amounts of toxin