Bacterial Pathogens and Disease II- Endotoxin Flashcards
Structure of gram-negative bacterial cell wall
Thin peptidoglycan layer but has an extra outer membrane as another protective layer compared to gram positive cell wall. That membrane has a periplasmic space to pass things through. These cells tend to have more control of what goes in and out of the bacteria.
· Outer membrane: bilayered; outer leaflet distinct in that it has lipopolysaccharide endotoxin (gram positive bacterial cell wall doesn’t have lipopolysaccharide)
· Outer membrane contains porins- OmpC, D, F, which allow movement of substances into and out of the cell
Structure of Lipopolysaccharide (LPS)
Lipid A Component
- phosphorylated glucosamines attached to long chain fatty acids
- number and type of fatty acids vary by species
- hydrophobic (repels water soluble substances)
Polysaccharide Core Component
- ketodeoxyoctanoic acid (KDO) and heptose
- relatively constant between species
- hydrophilic
O-Side Chain Component
- repeat units of tri, tetra or pentasaccharide sugars
- highly variable between species
- hydrophilic
What is an endotoxin?
lipopolysaccharide (LPS) of the gram negative bacterial cell wall
What is the active component of LPS?
Lipid A
Can you make a vaccine to lipid A?
Lipid A is not immunogenic, meaning you can’t make a vaccine against it
Characteristics of an endotoxin (LPS)
- Lipid A is the active component (not immunogenic)
- O antigen is highly immunogenic and immune specific
- Heat stable
- Not converted to toxoids
What does an endotoxin initiate?
Sepsis
What is sepsis?
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection
What drives sepsis?
Sepsis is driven by the innate immune system response, consisting of:
- macrophages
- monocytes
- granulocytes
- natural killer cells
- dendritic cells
These cells detect:
- pathogen associated molecular patterns (PAMPs) such as endotoxin
- damage associated molecular pattern (DAMPs) from damaged host cells
This detection leads to production of:
- pro-inflammatory cytokines TNF⍺, IL-1, IL-6
- pro-inflammatory cytokines IL-1β and IL-18 via inflammasomes, and these cytokines cause rapid apoptosis
What is the detection of PAMPs and DAMPs mediated by?
This detection is mediated via:
· Cell membrane receptors: toll-like receptors (TLR) and C-type lectin receptors
· Cytosol receptors: NOD-like receptors, RIG-I-like receptors
MOA of endotoxins
1) Endotoxin (LPS) binds CD14 receptor on macrophage surface
2) MD-2 then takes the LPS from the CD14 and passes it to the toll-like receptor 4 (cell membrane receptor)
3) Toll-like receptor starts to undergo dimerisation
4) This sets up the trigger for the production of pro-inflammatory cytokines
What is the effect of pro-inflammatory cytokines?
1) Increase number, lifespan and activation state of innate immune cells (positive feedback loop)
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) Cause 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
5+6+7 lead to formation of a thrombus (immunothrombosis)- microbes are trapped within this and attract and activate further leucocytes
What happens if the endotoxin infection is severe and pro-inflammatory cytokines are not controlled?
If the infection is severe enough, it may pass a threshold of being controlled locally and it starts to spread to the entire body, becoming a systemic injury → sepsis dysregulation
Sepsis dysregulation
· Production of reactive oxygen species (ROS)- hydroxyl and nitric oxide- damages cellular proteins, DNA and lipids and impairs mitochondria
· Complement activation (especially 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
Mitochondrial damage- leads to decreased intracellular ATP and cells enter state of hibernation- exacerbates organ dysfunction
Sepsis Resolution
Early on in the process of sepsis, IL-10 is produced, which is an anti-inflammatory cytokine:
- suppresses the production of pro-inflammatory cytokines IL-6 and 𝛾-interferon
- stimulates production of soluble TNF receptors which bind IL-10 rather than binding to the cells, and therefore it is taken out of the system
- stimulates production of IL-1 receptor antagonist, therefore IL-1 can’t bind to its receptor and cause any further damage.
Macrophages also remove all the PAMPs and DAMPs from the system, removing the stimulus for sepsis.
Damaged cells during this entire process undergo apoptosis and engulfment by macrophages.
This whole process is very active is not a passive process.
Why is meningococcus (Neisseria meningitidis) so effective in sepsis?
Has a different structure from the main gram-negative organisms:
· Lipid A has 6 fatty acid chains making it a highly effective endotoxin
· Very short O-antigen called a lipooligosaccharide (unlike a LPS which has long O-antigen) which is very similar to carbohydrates expressed in human cells, meaning it mimics human cells, and therefore the immune response to it is not very effective
· Produces blebs (chunks of outer cell membrane being shedded)- therefore the meningococcus is shedding huge amounts of lipoligosaccharides (endotoxin) to the environment which drives septicaemia
What does meningococcal sepsis cause?
Oedema
-due to increased endothelial permeability
Disseminated Intravascular Coagulation (DIC)
