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Flashcards in ADP-ribosylating toxins Deck (22):

what are Bacterial toxins?

- major virulence factors for some bacteria
- damage cells, tissues, organs, etc.


what are the 2 classes of toxins?

1. endotoxin, LPS component on surface of - Gram negative bacteria
2. protein exotoxins
- ADP-ribosylating toxins: diphtheria, cholera
- neurotoxins: tetanus, botulinum
- enzyme toxins
- membrane-damaging toxins


what is Diphtheria?

• Tonsillar form begins with sore throat, mild fever
• Colonisation of throat - toxin causes localised necrosis of epithelial cells and inflammatory response
• Formation of pseudomembrane due to deposition of fibrin and dead cells
• Blocks larynx, may lead to asphyxiation
• Caused by Corynebacterium diphtheriae, aerobicGram positivenon-motilerod


describe the Diphtheria toxin

• 100 ng of toxin per kg body weight is lethal!
• Localised and systemic effects
• Highly immunogenic – formalin-treated toxin highly effective as vaccine
• Toxigenicity depends on presence of lysogenic phage in bacterial genome
• Toxin gene expression is iron-regulated
• Toxin inhibits protein synthesis in target cells
• Toxin receptor is a precursor of heparin-binding epidermal growth factor (HB-EGF)


explain Processing of diphtheria toxin

• Toxin synthesised as a 60 kDa protein
• Cleaved by proteases to yield 2 subunits linked by disulphide bond
• A: 21 kDa - heat stable, toxic activity
• B: 39 kDa - essential for binding
‘Subunit toxin’


explain Internalisation of diphtheria toxin

• Interaction between C-terminus of B-subunit and host receptor HB-EGF
• Internalisation by endocytosis of the toxin-receptor complex, followed by fusion with lysosome
• Acidification causes conformational change to B subunit, which then inserts into cell membrane
• Breakage of disulphide bond, A-subunit released
into cytoplasm


Mode of action of diphtheria toxin

• ADP-ribosylating toxin
• CleavesNAD+(nicotinamide adenine dinucleotide)
• TransfersADP-riboseto host protein EF-2 (elongation factor 2)
• ADP-ribose binds to a modified histidine residue
• Protein synthesis inhibited


What is Cholera?

• Vibrio cholerae, Gram negative, motile, curved rod
• Transmitted by contaminated water/food – 1000
organisms are sufficient to cause disease
• Profuse diarrhoea and vomiting 35–60 hours after infection
• Rapid dehydration, electrolyte imbalance and acidosis – treatment by oral rehydration therapy
• V. cholerae colonises small intestinal mucosa and secretes toxin
• The toxin is the major virulence factor - disrupts ion transport in intestinal epithelium


what did S.N. De demonstrated in 1959?

cell free extracts of V. cholerae caused diarrhoea


when was cholera toxin (CT) purified?

1969 84-kDa


how many cholera serotypes were found

- 140
- few cause disease – O1 (Classical and El Tor) and O139


where is CT encoded?

- filamentous phage
• Phage can integrate in chromosome or replicate as a plasmid
• Receptor is the toxin co-regulated pilus (TCP)


describe the Cholera toxin

• Toxin comprises 2 protein components:
- A protein, 27 kDa, 2 subunits, A1 (23 kDa) and A2,
5.5kDa, linked by a disulphide bond
- B protein is a pentamer of 56 kDa subunits
• ctxA and ctxB genes are in an operon, 2+ copies of the operon per chromosome
• Shine-Dalgarno sequence for ctxB more efficient than that for ctxA so 5 more B synthesised than A
• Experimental mutation of ctxB
SD sequence results in significantly reduced synthesis of CtxB protein


describe Internalisation of cholera toxin

• B subunit binds mono- sialylganglioside (GM1)
• Binding promotes insertion of A subunit into membrane
• A1-A2 disulphide bond reduced
• A1 released into cytoplasmic membrane
• A1 diffuses within membrane to locate target protein


Mode of action of cholera toxin

• The A1 subunit has ADP-ribosylating activity
• Target is a 42 kDa membrane protein called Gs
• Gs regulates adenylate cyclase, a membrane protein whose catalytic component is located on the cytoplasmic side of the membrane
• Adenylate cyclase (AC) converts ATP to cyclic AMP
• Cyclic AMP plays a key role in intracellular signalling pathways from receptors for hormones, cytokines, neurotransmitters, etc.


what happens In normal circumstances When a receptor is stimulated:

- Gs is activated
- binds GTP
- Gs-GTP complex activates adenylate cyclase (AC)
• Cyclic AMP (cAMP) generated, acts as a 2nd messenger
• Gs has intrinsic GTPase activity
- Gs-GTP converted to Gs-GDP
- Gs-GDP cannot stimulate adenylate cyclase, Stimulus decays


what happens in cells exposed to cholera toxin

• Gs undergoes ADP-ribosylation
• Intrinsic GTPase activity blocked
• Conversion of Gs-GTP to Gs-GDP inhibited
• Stimulation of adenylate cyclase activity continues
• Excess cAMP accumulates
- protein kinases activated
- phosphorylate cell surface proteins
- ion and fluid fluxes across intestinal membrane disrupted
- diarrhoea


explain Regulation of Cholera Toxin

• Tcp is encoded in the Vibrio Pathogenicity Island along with ToxT
• ToxT regulates both Tcp and CT, and activates its own expression
• ToxT is regulated by TcpP/TcpH and ToxS/ToxR plus others
• TcpP/TcpH are regulated by AphA/AphB which are subject to a quorum sensing response


what are the Low/High Cell Density Phenotypes

Low Density
Cholera toxin – release of nutrients TCP – adherence
VPS/Biofilm – adherence

High Density
Low adherence


what are the Functions of Secretion

• V. cholerae colonies duodenum and CT is secreted and enters enterocytes
• Elevates cAMP levels
• Activates CFTR channel protein leading to secretion of chloride ions
• Osmotic imbalance causes release of water (in worst cases 30-40 litres/day)
• Release of nutrients and breakage of tight junctions for penetration of tissues


name 3 ADP-ribosylating toxins

• Pseudomonas aeruginosa exotoxin A  Ef-2
• P. aeruginosa exoenzyme S  ras gene family
• E. coli heat labile enterotoxin  stimulatory G protein


summarise ADP-ribosylating toxins

1. Toxins have various roles in disease
2. Diphtheria toxin is a two component toxin (synthesized from a single precursor) that transports an effector protein into the cytoplasm and inhibits translation
3. Cholera toxin is a two component toxin that inserts an effector protein into the cell membrane and modulates the activity of cell regulators leading to changes in membrane function