Bacterial Pathogens and Diseases II (Endotoxins)

Question 1

Describe the membranous layers of bacteria

Answer 1

All bacteria have an inner membrane

Gram +ve have an outer peptidoglycan layer

Whereas Gram -ve have an extra outer membrane

Question 2

Describe the structure of Gram -ve outer membrane

Answer 2

Outer membrane: bilayered, outer leaflet distinct in that it has lipopolysaccharide (endotoxin)

Question 3

Where is gram-ve peptidoglycan layer situated?

Answer 3

Within periplasmic space, gram -ve have the peptidoglycan layer

Question 4

What are the 3 components of lipopolysaccharides (LPS)?

Answer 4

Lipid A component
Polysaccharide Core
O-side Chain

Question 5

Describe the lipid A component of LPS

Answer 5

Phosphorylated glucosamines attached to long chain fatty acids.

No. and type of fatty acid vary by species.

Hydrophobic

Main toxigenic component of endotoxin

Question 6

Outline the structure of the polysaccharide core of LPS

Answer 6

Keto Deoxy Octanoic acid (KDO) and heptose

Relatively constant between species.
Hydrophilic

Question 7

Describe the structure of the O-side chain

Answer 7

Repeat units of tri, tetra or pentasaccharide sugars.
Highly variable between species
Hydrophilic

Question 8

How do LPS molecules protect against hydrophobic molecules?

Answer 8

LPS molecules non covalently cross bridged by Ca and Magnesium ions – provides a barrier to hydrophobic molecules including bile salts

Question 9

Outline characteristics of endotoxins

Answer 9

• Endotoxin is lipopolysaccharide (LPS)
• Lipid A active component; not immunogenic
• O antigen is highly immunogenic + immune specific
• Only gram-ve bacteria
• Heat stable
• Not converted to toxoids
• Major initiator of sepsis pathway

Question 10

What is sepsis?

Answer 10

Life threatening organ dysfunction caused by dysregulated host response to infection by innate immunity

Question 11

Which cells are active in sepsis?

Answer 11

Sepsis driven by innate immune response:

• macrophages
• monocytes
• NK cells
• Dendritic cells

Question 12

How does the innate immune response initiate sepsis?

Answer 12

Receptors recognise bacterial components (PAMPs) and/or danger signals (DAMPs) to activate cytokine transcription that will then recruit an inflammatory response

Question 13

What are PAMPs?

Answer 13

pathogen associated molecular patterns e.g. endotoxin

Question 14

What are DAMPs?

Answer 14

damage associated molecular patterns from damaged host cells

Question 15

How is DAMPs/PAMPs detection by innate immune cells mediated?

Answer 15

Cell membrane receptors
- toll-like receptors (TLR)

Cytosol receptors
- NOD-like and RIG-I like receptors

Question 16

What is the effect of PAMPs/DAMPs recognition by immune cells?

Answer 16

Pro-inflammtory Cytokine production (TNF-a, IL-1, IL-6)

Inflammasomes induce IL-1b and IL-18 production
- causes rapid apoptosis

Question 17

What are PRRs?

Answer 17

pattern recognition receptors

• recognise PAMPs
  e. g. TLRs

Question 18

Which TLR is commonly paired with sepsis?

Answer 18

TLR4 are the PRRs that pair mostly with sepsis LPS

Question 19

Outline a similarity between endo- and exotoxins

Answer 19

Both endo- and exotoxins have the ability to produce proinflammatory cytokines either through lymphocytes (exotoxins) or lipopolysaccharides (endotoxins)

Question 20

Outline how E.coli endotoxin activates TLR4

Answer 20

1. Lipid A component of LPS is recognised by MD-2
2. MD-2+LPS bind to TLR4 molecule on the cell surface
3. Causes dimerization of TLR4 causing intracellular
   signalling

Question 21

How does the TLR4 complex form when endotoxin binds?

Answer 21

MD2 has a coreceptor binding protein CD14 that enables the formation of the complex on TLR4

Question 22

What is the effect of TLR4 activation?

Answer 22

The binding to TLR4 causes activation of an intracellular cascade that eventually leads to NF-Kꞵ activation

Question 23

What is the significance of NF-Kꞵ activation?

Answer 23

NF-Kꞵ activation leads to production of proinflammatory cytokines

Question 24

What is the effect of cytokines on immune cells?

Answer 24

Increase number, lifespan and activation state of innate immune cells
Cause fever

Question 25

Describe how cytokines effect endothelial cells

Answer 25

Increase adhesion molecules and chemokine expression by endothelial cells

Question 26

What effect does cytokines have on acute phase proteins?

Answer 26

Increase acute phase protein such as complement , fibrinogen and CRP

Question 27

Outline the effects of cytokines that lead to a thrombus formation

Answer 27

1. Causes neutrophils to release extracellular traps (NETs) made of DNA and antimicrobial proteins that form a scaffold for platelet activation. 2. Cause release of microparticles by activated platelets 3. Increase tissue factor expression by blood monocytes

Question 28

What is the purpose of thrmobus formation by cytokines?

Answer 28

Formation of a thrombus (immunothrombosis) traps microbes within this Attracts and activates further leukocytes.

Question 29

How do we define all the cytokine actions occurring at once?

Answer 29

All these effects together are called septicemia or septic shock

Question 30

Why is there such a drastic immune response to LPS?

Answer 30

Very potent immune response even in presence of small amounts of LPS as gram -ve bacteria in the bloodstream is life threatening and can be lethal

Question 31

What are the common symptoms cuased by LPS?

Answer 31

LPS causes the following symptoms: - Iron deficiency - Fever - Hypoglycaemia - DIC Thrombosis - Hypotension - Shock

Question 32

What effect does LPS have on macrophage activity?

Answer 32

LPS activate macrophages to produce TNF (IL-1): - causes a fever - Decreases Fe; liver tries to collate Fe so bacteria can’t grow - Causes endothelial cells to express receptors

Question 33

How does LPS effect mast cells?

Answer 33

LPS causes mast cells to degranulate producing mediators

Question 34

How do LPS cause liver damage?

Answer 34

Endotoxins also lead to liver damage due to release of acute phase proteins reducing glucose levels

Question 35

What is the effect of LPS Platelet activation?

Answer 35

Also activates platelets causing: Increased vascular permeability; neutrophil recruited to damage site are able to get into tissue; can cause low BP ⇒ shock Disseminated intravascular coagulation (DIC) ⇒ thrombosis

Question 36

How does LPS lead to thrombosis?

Answer 36

Complement pathway can also be activated as well as clotting mechanisms ⇒ hypotension and thrombosis

Question 37

Outline the signs and symptoms of sepsis

Answer 37

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

Question 38

What is bacteraemia?

Answer 38

Bacteria present in blood but have short lifespan due to phagocytosis

Question 39

What is septicaemia?

Answer 39

Long lasting harmful bacteria in blood unable to be phagocytosed by macrophages Septic shock mortality: 50-85%

Question 40

What is the aim of sepsis dysregulation?

Answer 40

Achieves rapid control of localised and minor infections

Question 41

Why does sepsis dysregulation not always occur?

Answer 41

However dyregulation may pass threshold → systemic injury Tissue factor; initiation of thrombin formation from prothrombin + clotting Tissue factor pathway; extrinsic

Question 42

How does sepsis damage mitochondria?

Answer 42

Production of reactive oxygen species (ROS) – Hydroxyl and nitric oxide – damages cellular proteins, DNA and lipids and impairs mitochondria

Question 43

What are the consequences of complement pathway activation in sepsis?

Answer 43

Complement activation (esp. C5a) – increase ROS, granulocyte enzyme release, endothelial permeability and tissue factor expression

Question 44

How does sepsis lead to DIC thrombosis?

Answer 44

Widespread immune thrombosis leading to disseminated intravascular coagulation (DIC) with impaired microvascular function and organ dysfunction

Question 45

What is the consequence of mitochondrial damage in sepsis?

Answer 45

Mitochondrial damage leads to decreased intracellular ATP and cells enter a state of hibernation – exacerbates organ dysfunction

Question 46

How is sepsis mediated?

Answer 46

Sepsis is actively negatively regulated by anti-inflammatory cytokines e.g. IL-10

Question 47

What are the regulatory effects of IL-10 for sepsis?

Answer 47

suppresses proinflammatory cytokines; IL-6 and ɣ-interferon Produces receptors for proinflammatory cytokines (TNF and IL-1) to bind and downregulate them - antagonis

Question 48

What causes meningococcal sepsis?

Answer 48

Caused by Neisseria meningitidis | Gram negative diplococcus

Question 49

What are the different types of meningococcal sepsis?

Answer 49

Serotypes A,B,C, Y, W135 - varying O side chains

Question 50

What is the effect of meningococcal sepsis?

Answer 50

Can cause disease ranging from meningitis to life threatening meningococcal sepsis. > meningococcus is very effective in sepsis