Bacterial Pathogens and Disease 2 – endotoxin

Question 1

Describe the structure of a gram negative bacterial cell wall

Answer 1

Gram negative bacterial cell wall:
• All bacteria have an inner wall- gram negative bacteria also have an additional membrane which contains lipopolysaccharide (toxin).
• In between inner and outer walls is periplasmic space with a peptidoglycan layer.
• Lipopolysaccharide- contains 3 sections.
• Lipid A- number and type of fatty acids vary by species of gram-negative bacteria. Is hydrophobic. 2 sugars (phosphorylated glucosamines) and long chain fatty acids- anchors structure to membrane (main toxigenic component).
• Polysaccharide core- rare sugars only found in gram negative bacteria (e.g. Ketodeoxyoctonoic acid (KDO) and heptose). Short. Stable across species.
• O chain- very variable and potent- sticks out from surface of bacteria. Repeats of different combinations of sugars (tri/tetra/pentasaccharides). Highly variable between strains of the same species. Antigenic variability means that antibodies may identify one strain and not another.
• The O chain protects the membrane to prevent the binding the complement.

Question 2

Describe the structure of a Lipopolysaccharide

Answer 2

Complex structure
Consist of 3 parts:
• Lipid A – involved in toxicity of molecule
• Core polysaccharides – relatively stable and not diverse
• O-side chain – this interacts with the immune system, different strains have different chains

Question 3

What is an endotoxin?

Where is it found and what is the active component of it?

Answer 3

• Endotoxin is lipopolysaccharide (LPS).
• Only found in gram negative bacteria.
• Heat stable.
• Not converted into toxoids.
• Lipid A is active component- not immunogenic (drives septic shock- major initiator of sepsis pathway). Can’t make vaccine against the lipids.
• O antigen- highly immunogenic and specific due to variability.
• Endotoxin is lipopolysaccharide (LPS)
• Lipid A is the active component. – not immunogenic.
• 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

Describe what occurs in sepsis

What drives it?

What do the cells detect?

How do they detect it and what is the effect?

Answer 4

o Life threatening organ dysfunction caused by a dysregulated host response to infection.
o daisregukation of innate immunity.
o Sepsis primarily driven by the innate immune system response.
­ Macrophages
­ Monocytes
­ Granulocytes
­ Natural killer cells
­ Dendritic cells
o These cells detect
­ Pathogen associated molecular patterns (PAMP’s) such as endotoxin
­ Damage associated molecular patterns (DAMP’s) from damaged host cells
o 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
o 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 5

Describe the mechanism of sepsis

Answer 5

1. Endotoxin is recognised by MD2 protein.
2. LPS and MD2 together bind to TLR4, on cell surface. CD14 is coreceptor binding
   protein.
3. Dimerization of TLR4.
4. Intracellular cascade.

Question 6

What are the effects of pro-inflammatory cytokines?

Answer 6

• High number of gram-negative bacteria in bloodstream can overwhelm the innate immune system.
• Overproduction of proinflammatory cytokines and dysregulate innate immune response (septic shock).
• The role of cytokines is to increase the number, lifespan and activate of innate immune cells, increase adhesion molecule and chemokine expression by endothelial cells, increase complement (C5a), fibrinogen and CRP, cause fever.
• Also, cause neutrophils to release extracellular traps (NETs) made of DNA and antimicrobial proteins to form a scaffold for platelet activation.
• Cause release of microparticles by activated platelets.
• Increase tissue factor expression by blood monocytes.
• Types of cytokines- tumour necrosis factor (TNF)- fever induction.
• Formation of immunothrombus with microbes trapped within which attract and activate further leucocytes.

1. Increase number, lifespan and activation state of innate immune cells.
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 (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 → formation of a thrombus (immunothrombosis)– microbes trapped within this. → attracts and activate further leucocytes.

Question 7

Describe the dysregulation that occurs in sepsis

Answer 7

• 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 8

How is sepsis resolved?

Answer 8

• Normally, sepsis would resolve but too much endotoxin LPS prevents this.
• Negative feedback is an active process, regulated by anti-inflammatory IL-10, produced early in the process.
• IL-10 suppresses IL-6 and γ-INF production- pro-inflammatory. Also stimulates TNF and IL-1 receptor antagonists.
• Autophagy of PAMPs and DMAPs (removal).
• Damaged cells undergo apoptosis and engulfment by macrophages.

Question 9

What is Meningococcal Sepsis?

Answer 9

• Gram-negative diplococcus called Neisseria meningitidis.
• Many different strains- A, B, C, Y, W.
• Antigenic variability in O side chain so antibodies must be unique.
• Each serotype is associated with different locations- A found in meningitis belt in Sehel region of Africa.
• B, C and W common in Europe but declined since introduction of MenC and MenB vaccine.
• Can cause meningitis and meningococcal sepsis.
• LPS from this bacteria is very dangerous and different from normal type. Only very short O antigen (not long)- lipooligosaccharide.
• Unusual to Neisseria species.
• Bacteria blebs its membrane, filled with lipid A component and LPS.
• Blebs overwhelm and cause endotoxic response.
• Very virulent with small infection.
• Septicaemia can result in red pinpricks on skin which coalesce to form skin haemorrhages due to DIC.
• Oedema is a sign of septicaemia.
• Caused by Neisseria meningitidis
• Gram negative diplococcus
• Serotypes A,B,C, Y, W135
• Serotype A associated with large outbreaks in Sahel region of Africa – Meningitis belt. (subsaharan belt of Africa, european cities B/C and now W).
• 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 10

What makes meningococcus so effective in sepsis?

Answer 10

• LOS – lipooligosaccharides - terminal part is structure similar to human erythrocyte antigen – mimicry of host antigen
• Hexa-acylated Lipid A – more toxic than penta-acylated
• Its lipopolysaccharide is diff from standard LPS, it lacks the O-antigen it is very short rather than long
• , los (lipooiigosaccharide), it is quite unique. still has a potent …. effect which can cause sepsis.
• blebs – lipid blobs of the membrane, full of lps. los, overwhelming effect