Lecture 33 - Clostridial Diseases Flashcards
Why are Clostridia hard to study?
Hard to culture, hard to genetically manipulate
Features of genus Clostridium 1) 2) 3) 4)
1) Gram +
2) Anaerobic
3) Form heat-resistant endospores
4) Several pathogenic species, which cause disease through toxin secretion
Neurotoxic pathogenic Clostridia
1)
2)
1) C tetani
2) C botulinum
Enterotoxic pathogenic Clostridia
1)
2)
1) C difficile
2) C perfringens
Histotoxic pathogenic Clostridia
1)
2)
1) C perfringens
2) C septicum
Gas gangrene
1)
2)
3)
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
Toxin that causes gas gangrene
Alpha toxin
Alpha-toxin effects
Hydrolyses membrane phospholipids, destabilises cell membrane
Alpha toxin mode of action
1)
2)
1) Sphingomyelinase, phospholipase activity
2) Hydrolyses phosphoreal choline from phosphatidyl choline in glycerol
Enzymatic activity of alpha toxin
Phospholipase C activity
Alpha-toxin structure
1) a, b
2) a, b
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
Alpha-toxin C-terminal beta sheet domain structure
Structurally similar to eukaryotic C2 phospholipid binding domains
Eukaryotic enzyme with a C2 phospholipid binding domain
Pancreatic lipase
Results of tests for alpha toxin as a virulence determinant in C perfringens
1)
2)
3)
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
How does gas gangrene occur?
1)
2)
1) C perfringens spores enter wound
2) H2, CO2 produced, alpha toxin produced
Significant disease caused by C difficile
Pseudomembranous colitis
Toxins produced by C difficile
Toxin A and toxin B (both very large proteins)
Pseudomembranous colitis appearance
Yellowish plaques of inflammatory cells, mucus and fibrin overlay normal gut mucosa
How does C difficile cause disease?
1)
2)
3)
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
Symptoms of C difficile disease
Spectrum from mild diarrhoea to fulminant pseudomembranous colitis
When did hypervirulent C difficile strains first appear?
2002
What type of toxins are toxins A and B?
Monoglucosyltransferases
Action of toxins A and B
1)
2)
1) Glucosylate Rho family of proteins with N-terminal catalytic domain
2) Disrupt actin cytoskeleton
Rho proteins
Very important signalling proteins in eukaryotic cells
Mode of toxin A and B uptake 1) 2) 3) 4) 5)
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
What on Rho do toxin A and B attach glucose to?
Threonine 37
How was it determined whether toxin A or toxin B were more important in C difficile infection?
1)
2)
3)
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)
Tetanus symptoms
Rigid paralysis, often fatal
Botulism symptoms
Flaccid paralysis, often fatal
How many types of tetanus toxin are there?
One
How many types of botulinum toxin are there?
Eight (BOnT/A - BOnT/H)
Where does tetanus toxin act?
In spinal cord. Blocks transmission in muscle relaxation pathway
Where does botulinum toxin act?
At the motor endplate
Is botulism a disease?
No. An intoxication
Type of enzyme that tetanus toxin and botulinum toxin are
Zinc endopeptidases
Protein domain that both tetanus toxin and botulinum toxin share
Zincin domain
What are zincin domains?
Protein domains that bind zinc
Present in botulinum toxin and tetanus toxin
Structure of botulinum toxin and tetanus toxin
1)
2)
3)
1) N-terminal catalytic domain
2) Translocation domain, transmembrane helix
3) C-terminal binding domains
Zinc-binding motif in botulinum and tetanus toxins
HExxH
Mechanism of action of botulinum toxin 1) 2) 3) 4) 5)
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
Mechanism of action of tetanus toxin 1) 2) 3) 4)
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
SNARE proteins
1)
2)
3)
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
Difference between tetanus and botulinum toxins
Where they act
Mechanism of action is extremely similar
