How Bacterial Toxins Contribute to Disease and Dissemination

Question 1

What are Bacterial Toxins

Answer 1

Substances that are released/secreted from
the bacterium that causes damage to the host

(generally) regarded as critical in disease progression

Question 2

What do bacterial toxins play a significant role in

Answer 2

Nutrient acquisition

Transmission

Immune evasion

Competition

Question 3

What are Endotoxins

Answer 3

They are lipopolysaccharides (LPS)

They are relatively non-specific and trigger inflammatory responses which result in:

• fever
• septic shock
• organ failure
• death

Question 4

What are LPS

Answer 4

Components of the Gram-negative cell wall that are vital to the structure and functional integrity of the bacterial outer-membrane

Can be released during the division or death of a bacterial cell (following antibiotic or immune system exposure)

Question 5

What are the LPS components

Answer 5

• O-antigen: highly variable (serological specificity, exposed to the immune system)
• Lipid A: Disaccharide backbone which is highly conserved. Acyl chain length/substitution pattern is the primary determinant of its endotoxicity

Question 6

What do Endotoxins trigger

Answer 6

Toll-like receptor engagement and activation

Inflammasome activation

Complement activation

Question 7

What is the contribution of LPS in disease

Answer 7

Dysregulation/manipulation of the immune system

Our bodies recognise PAMP - evolved to be super-sensitive to LPS and therefore induced hyper-inflammatory conditions to eliminate bacteria from the bloodstream

Question 8

What are exotoxins

Answer 8

They are actively secreted from the bacterium

They are often classified based on their binding/target site, mode of action, structure or generic function

Question 9

What are exotoxin’s mode of action

Answer 9

ADP-ribosyltransferase (Cholera toxin)

Beta-barrel pore forming toxins e.g., S. aureus alpha haemolysin

Question 10

What are exotoxins target/binding site

Answer 10

Cholesterol e.g., streptolysin O

Elongation factor 2 e.g., diphtheria toxin

Question 11

Where/what does the exotoxin act on

Answer 11

Cytolytic, dermonecrotic, enterotoxin, leucocidin, neurotrotoxin, superantigen

Question 12

What is the structure of Bacterial Exotoxins

Answer 12

Can be single proteins or organised into oligomeric protein structures.

Organised with distinct AB structure-function properties

Question 13

What does the A domain of an Exotoxin include

Answer 13

Encodes an active (catalytic) activity

Examples:
- Ribosylation
- Glucosylation
- Proteolysis
- Non-covalent modification of host proteins
- modification through direct binding of host proteins.

Question 14

What does the B domain of Exotoxins include

Answer 14

2 functional domains:

1) Receptor binding domain

2) Translocation domain

Question 15

What is the structure of a Binary toxin

Answer 15

2 independent polypeptide chains: non-associated AB toxins (anthrax toxin)

Question 16

What is the structure of an oligomeric toxin

Answer 16

Multimeric complex consisting of two or more non-covalently linked subunits/domains (cholera toxin)

Question 17

What is the structure of a protoxin

Answer 17

Secreted in an inactive form (proenzyme) which can be converted into an active form by proteolytic enzyme (Iota toxin of Clostridium perfringens)

Question 18

What is the structure of a pore-forming toxin

Answer 18

monomeric or bi-component molecules which bind to specific cells and oligomerise on he surface resulting in a cell membrane pore

Question 19

What is Cholera

Answer 19

Bacteria: Gram-negative Vibrio cholerae

Mode of transmission: faecal-oral route

Symptom: voluminous diarrhoea (rice water stool)

Causes of death:
- dehydration
- acidosis
- hypovolemic shock

Question 20

What is the cholera toxin (CTX)

Answer 20

• Classical AB(5) structure
  Heterodimeric subunit composed of 2 polypeptide chains (CTX-A1 and CTX-A2) and a pentameric CTX-B subunit composed of 5 identical polypeptide chains.
• CTX-B specifically binds to receptors on host cells (intestinal epithelial cells)
• Receptors: GM1 gangliosides (Primary) or histo-blood group antigens (Secondary) - binding promotes the endocytosis of the toxin

Question 21

What is the CTX internalisation process

Answer 21

1) Endosomal compartment - trafficked to the Golgi and then to the ER

2) In the ER CTX dissociates, CTX-A1 is exported out of the cytosol and activated by the ADP ribosylation factor 6 (ARF6)

3) ARF6-CTX-A1 activates adenyl cyclase (AC) by catalysing ADP ribosylation of a G protein-coupled receptor (GPCR)

4) AC catalyses the conversion of ATP to cyclic adenosine monophosphate (cAMP), increasing the intracellular cAMP conc.

5) Activation of protein kinase A (PKA) phosphorylates the CFTR chloride channel proteins, ultimately resulting in the release of electrolytes (Cl-, HCO3-, Na+, K+) and water into the intestinal lumen, causing the secretory diarrhoea characteristic of cholera.

Question 22

What is the Botulinum toxin

Answer 22

Bacteria: Gram-positive, anaerobic, spore-forming Clostridium botulinum

It is a neurotoxin

Prevalent in soli and marine sediments

Botulism can occur as a result of food contamination (canned homemade, preserved, or fermented)

Question 23

What is the structure of the Botulinum toxin

Answer 23

Binary AB toxin: N-terminal is a zinc dependent metalloprotease which is linked to the C-terminal B fragments by a disulphide bond

Question 24

How does the BoNT interfere with normal neurotransmitter release

Answer 24

Blocks the release of acetylcholine at the neuromuscular junction causing flaccid paralysis

Question 25

What are the two specific receptors found on nerve cells that BoNT binds to:

Answer 25

- Polysialoogangliosides (complex sugar molecules on nerve cells) - Synaptic vesicle proteins (Synaptotagmin & Synaptic Glycoprotein 2)

Question 26

What happens when BoNT binds to the nerve cells

Answer 26

It is taken up via endocytosis and the light chain of the BoNT cleaves the SNARE proteins (which are needed for neurotransmitter release)

Question 27

How does BoNT cause paralysis

Answer 27

By preventing the SNARE proteins from working, BoNT blocks the release of acetylcholine, a neurotransmitter essential for muscle contraction Without acetylcholine, muscles cannot contract, leading to flaccid paralysis

Question 28

How do normal neurotransmitters work

Answer 28

1) Acetylcholine is released at the neuromuscular junction via the assembly of the SNARE complex 2) Acetylcholine is released into the synaptic cleft and then binds receptors on the muscle cell 3) Acetylcholine is the primary neurotransmitter of the parasympathetic nervous system that enable smooth muscle contraction

Question 29

How does the BoNT help Clostridium botulinum

Answer 29

There is the consumption of infectious spores while grazing The spores would germinate and cause fulminant infection resulting in animal death This results in release of nutrients and spores that can inoculate the soil and potentially promote plant growth and lead to transmission to a new host BoNT may have some specific activity in the soil that is undiscovered - it would constitute the selective pressure to retain such a lethal factor

Question 30

What are Leukotoxins: Leukocidins

Answer 30

Produced prominently by S. aureus Responsible for a wide range of infections which differ in severity

Question 31

How do Leukotoxins help the bacteria

Answer 31

- Releases nutrients from cells (haemoglobin from erythrocytes - Target and kill immune cells - Transmission: pus

Question 32

What is the Enterotoxin: Shiga toxin

Answer 32

Released by: Gram-negative Escherichia coli common commensal in the bovine gut Responsible for the morbidity and mortality in humans (Enterohaemorrhagic E. coli (EHEC)) Possesses Shiga toxin-encoding prophage - key role in disease progression EHEC transmitted to humans from ruminants - contaminated foods, water animals, infected persons, contaminated surfaces.

Question 33

What is the structure of the Shiga toxin

Answer 33

AB5 toxin - enzymatically active monomeric A subunit non-covalently attached to pentamer of identical B subunits

Question 34

How does the shiga toxin it interact with the host cell

Answer 34

It is iron regulated - restricted at high Fe concentrations and therefore toxin production occurs in the distal small intestine and colon Interacts with cell membrane glycolipid globotriaocylceramide (Gb3) - initiates endocytosis and internalisation

Question 35

What is the process of internalisation of the shiga toxin

Answer 35

1) The toxin localises to early and recycling endosome 2) Retrograde trafficking machinery shuttles toxin from endosomal compartment through the Golgi and ER 3) The transfer requires clathrin and retromer proteins that are important in generating membrane curvatures and retrograde tubules

Question 36

What is retro-translocation

Answer 36

This is a host cell intracellular pathway that translocates A subunit from the ER into the cytosol. Here, furin cleaves the C terminal A domain (endosome) but they remain linked by disulphide bonds In the ER lumen, disulphide bonds are reduced and the A domain is liberated

Question 37

What does the Shiga toxin target

Answer 37

It facilitates the endohydrolysis of the N-glycosidic bonds at one specific adenosine residue at position 4324 It targets the 28S rRNA of eukaryotic ribosomes It inhibits aminoacyl tRNA binding to the A site of the 60S ribosomal subunit - prevents the peptide elongation, protein synthesis and results in cell death.

Question 38

What are the alternative roles of the shiga toxin

Answer 38

- Bacteria evolve toxins to compete or establish a niche, outside human hosts - These 'virulence factors' provide a competitive advantage only in this niche and are therefore positively selected for.

Question 39

What is the shiga toxin's natural environment

Answer 39

Soil, water and bovine gastrointestinal tract