Bacterial Pathogens and Disease II- Exotoxins

Question 1

What is a gram negative bacterial cell wall like?

Answer 1

Its features are the peptidoglycan in between the inner and out membrane
Luckily embedded in the outer membrane is the lipopolysaccharide consisting of three parts:
- O-polysaccharide
- Core polysaccharide
- Lipid A

Question 2

What are the three features of the lipopolysaccharide?

Answer 2

LIPID A.
- Phosphorylated glucosamines attached to long chain fatty acids.
- Number 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 penta-saccharide sugars.
- Highly variable between species
- Hydrophilic

Question 3

What are the characteristics of endotoxins?

Answer 3

Endotoxin is lipopolysaccharide (LPS)
LPS molecules non covalently cross bridged by Ca and Magnesium ions – provides a barrier to hydrophobic molecules including bile salts.
Lipid A is the active component. – not immunogenic so will not cause an immune response
O antigen is highly immunogenic and immune specific.
Found only in gram negative bacteria.
Heat stable
Not converted to 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

How are immune cells involved in sepsis?

Answer 5

Sepsis primarily driven by the innate immune system response.
- macrophages,
- monocytes,
- granulocytes,
- natural killer cells
- dendritic cells.
These cells detect:
- pathogen associated molecular patterns (PAMP’s) such as endotoxin,
- damage associated molecular patterns (DAMP’s) from damaged host cells.
This detection mediated via:
- cell membrane receptors – toll-like receptors (TLR) and C-type lectin receptors.
- cytosol receptors - NOD-like receptors, RIG-I-like receptors.
Effect:
- Production of pro-inflammatory cytokines TNFα, IL-1, IL-6
- via inflammasomes to produce IL-1β and IL-18 that cause rapid programmed cell death

Question 6

How does sepsis occur?

Answer 6

If there is disproportionate activation of a macrophage from the detection of LPS.
So if we have an infection producing a ton of LPS it can ruin the balance leading to a dysregulated immune response as seen in sepsis

Question 7

How are endotoxins detected?

Answer 7

CD14, TLR4 and MD2 are the combination of receptors that interact between them to detect endotoxins.
In this example, LPS molecule with the long fatty acid of E. coli which is detected by binding to CD14 and then transferred from CD14 to TLR4 by MD-2
The fatty acid chains gets inserted into the globular protein MD-2 and that is then delivered as a complex to TLR4
There is dimerisation of different TLR4s which then induces downstream signalling of generally two types:
- MYD88- where the detection of the complex (of CD14,MD2 and TLR4) leading onto signals dependent on the MYD88 pathway
· This leads to the translocation to the nucleus of the TF NF-kB leading onto the activation of inflammatory cytokine genes (specifically tumour necrosis factor alpha (TNF-a) and b

Question 8

What are the effects of pro-inflammatory cytokines?

Answer 8

Increase number, lifespan and activation state of innate immune cells.
Increase adhesion molecule and chemokine expression by endothelial cells.
Increase acute phase protein such as complement , fibrinogen and CRP.
Cause fever.

Causes neutrophils to release extra-cellular traps (NETs) made of DNA and antimicrobial proteins that forms a scaffold for platelet activation.
Cause release of microparticles by activated platelets
Increase tissue factor expression by blood monocytes

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

Question 9

What happens during the dysregulation of sepsis?

Answer 9

Production of reactive oxygen species (ROS) – Hydroxyl and nitric oxide – damages 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. 
Mitochondrial damage leads to decreased intracellular ATP and cells enter state of hibernation – exacerbates organ dysfunction.

Question 10

What happens during the resolution of sepsis?

Answer 10

Active process- not passive
Anti-inflammatory IL-10 produced early in process:
- Supresses production IL-6 and g-interferon
- Stimulates production of soluble TNF receptor and IL-1 receptor antagonist
Autophagy of PAMP’s and DAMP’s- removal
Damaged cells- undergo apoptosis and engulfment of macrophages

Question 11

What kind of bacteria causes meningococcal sepsis?

Answer 11

Gram negative diplococcus
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 makes meningococcus so effective in sepsis?

Answer 12

This bacteria has evolved to produce an incredibly active LPS that are released from the membrane instead of embedded in the form of Blebs

Causes:
Local but disseminated coagulation
Oedema associated with inflammation
And in some circumstances, organ failure