Lecture 33 - Clostridial Diseases

Question 1

Why are Clostridia hard to study?

Answer 1

Hard to culture, hard to genetically manipulate

Question 2

Features of genus Clostridium
1)
2)
3)
4)

Answer 2

1) Gram +
2) Anaerobic
3) Form heat-resistant endospores
4) Several pathogenic species, which cause disease through toxin secretion

Question 3

Neurotoxic pathogenic Clostridia
1)
2)

Answer 3

1) C tetani

2) C botulinum

Question 4

Enterotoxic pathogenic Clostridia
1)
2)

Answer 4

1) C difficile

2) C perfringens

Question 5

Histotoxic pathogenic Clostridia
1)
2)

Answer 5

1) C perfringens

2) C septicum

Question 6

Gas gangrene
1)
2)
3)

Answer 6

1) Caused by infection of wounds with histotoxic Clostridia (C perfringens most common)
2) C perfringens is the major cause of traumatic gas gangrene
3) Alpha-toxin is the causative agent

Question 7

Toxin that causes gas gangrene

Answer 7

Alpha toxin

Question 8

Alpha-toxin effects

Answer 8

Hydrolyses membrane phospholipids, destabilises cell membrane

Question 9

Alpha toxin mode of action
1)
2)

Answer 9

1) Sphingomyelinase, phospholipase activity

2) Hydrolyses phosphoreal choline from phosphatidyl choline in glycerol

Question 10

Enzymatic activity of alpha toxin

Answer 10

Phospholipase C activity

Question 11

Alpha-toxin structure

1) a, b
2) a, b

Answer 11

1) N-terminal alpha-helical domain
a) Three Zn2+ binding sites
b) Phospholipase C domain

2) C-terminal beta-sheet domain
a) Essential for toxicity
b) Binds to cell membrane

Question 12

Alpha-toxin C-terminal beta sheet domain structure

Answer 12

Structurally similar to eukaryotic C2 phospholipid binding domains

Question 13

Eukaryotic enzyme with a C2 phospholipid binding domain

Answer 13

Pancreatic lipase

Question 14

Results of tests for alpha toxin as a virulence determinant in C perfringens
1)
2)
3)

Answer 14

1) Mice injected in thigh with either wild type C perfringens, alpha-toxin - mutant, alpha-toxin - mutant with toxin+ plasmid
2) Wild type and toxin- mutant with toxin+ plasmid caused necrosis
3) Toxin- mutant caused no necrosis

Question 15

How does gas gangrene occur?
1)
2)

Answer 15

1) C perfringens spores enter wound

2) H2, CO2 produced, alpha toxin produced

Question 16

Significant disease caused by C difficile

Answer 16

Pseudomembranous colitis

Question 17

Toxins produced by C difficile

Answer 17

Toxin A and toxin B (both very large proteins)

Question 18

Pseudomembranous colitis appearance

Answer 18

Yellowish plaques of inflammatory cells, mucus and fibrin overlay normal gut mucosa

Question 19

How does C difficile cause disease?
1)
2)
3)

Answer 19

1) Broad-spectrum antibiotic therapy leads to disruption of normal microbiota
2) C difficile spores from environment invade, germinate and colonise intestinal tract
3) Secretion of toxins A and B

Question 20

Symptoms of C difficile disease

Answer 20

Spectrum from mild diarrhoea to fulminant pseudomembranous colitis

Question 21

When did hypervirulent C difficile strains first appear?

Answer 21

2002

Question 22

What type of toxins are toxins A and B?

Answer 22

Monoglucosyltransferases

Question 23

Action of toxins A and B
1)
2)

Answer 23

1) Glucosylate Rho family of proteins with N-terminal catalytic domain
2) Disrupt actin cytoskeleton

Question 24

Rho proteins

Answer 24

Very important signalling proteins in eukaryotic cells

Question 25

Mode of toxin A and B uptake
1)
2)
3)
4)
5)

Answer 25

1) Toxin taken into cell by endocytosis
2) Acidification of endosome causes conformational change in toxin
3) Autocatalytic subunit autocleaves
4) Translocation domain inserts into endosome membrane, catalytic domain escapes into cytoplasm
5) Catalytic subunit attaches glucose to threonine 37 of Rho

Question 26

What on Rho do toxin A and B attach glucose to?

Answer 26

Threonine 37

Question 27

How was it determined whether toxin A or toxin B were more important in C difficile infection?
1)
2)
3)

Answer 27

1) Made isogenic toxin A and toxin B C difficile mutants
2) Injected mutants into hamsters
3) Toxin B is essential for virulence (hamsters with toxB+ had same lifespan as wild type, hamsters with toxA+ lived longer)

Question 28

Tetanus symptoms

Answer 28

Rigid paralysis, often fatal

Question 29

Botulism symptoms

Answer 29

Flaccid paralysis, often fatal

Question 30

How many types of tetanus toxin are there?

Answer 30

One

Question 31

How many types of botulinum toxin are there?

Answer 31

Eight (BOnT/A - BOnT/H)

Question 32

Where does tetanus toxin act?

Answer 32

In spinal cord. Blocks transmission in muscle relaxation pathway

Question 33

Where does botulinum toxin act?

Answer 33

At the motor endplate

Question 34

Is botulism a disease?

Answer 34

No. An intoxication

Question 35

Type of enzyme that tetanus toxin and botulinum toxin are

Answer 35

Zinc endopeptidases

Question 36

Protein domain that both tetanus toxin and botulinum toxin share

Answer 36

Zincin domain

Question 37

What are zincin domains?

Answer 37

Protein domains that bind zinc

Present in botulinum toxin and tetanus toxin

Question 38

Structure of botulinum toxin and tetanus toxin
1)
2)
3)

Answer 38

1) N-terminal catalytic domain
2) Translocation domain, transmembrane helix
3) C-terminal binding domains

Question 39

Zinc-binding motif in botulinum and tetanus toxins

Answer 39

HExxH

Question 40

Mechanism of action of botulinum toxin
1)
2)
3)
4)
5)

Answer 40

1) Internalised at neuromuscular junction
2) Bind cell surface receptors, enter through pre-synaptic neuron membrane by endocytosis
3) Acidification of vesicle causes conformational change, catalytic subunit escapes into cytoplasm
4) Proteolysis of target SNARE proteins
5) Acetylcholine is prevented from being released by presynaptic neuron onto motor endplate

Question 41

Mechanism of action of tetanus toxin
1)
2)
3)
4)

Answer 41

1) Binds cell surface receptors, enters through endocytosis
2) Stays within endocytic vesicle, moves by intra-axonal retrograde transport to inhibitory neurons of spinal cord
3) Escapes endosome
4) Proteolysis of target SNARE proteins
5) Prevents neurotransmitter release

Question 42

SNARE proteins
1)
2)
3)

Answer 42

1) Involved in fusion of synaptic vesicle containing neurotransmitters and synaptic membrane
2) v-snares on synaptic vesicle bind to t-snares on synaptic membrane
3) Botulinum and tetanus toxins cleave SNARE proteins

Question 43

Difference between tetanus and botulinum toxins

Answer 43

Where they act

Mechanism of action is extremely similar