ADP-ribosylating toxins Flashcards
what are Bacterial toxins?
- major virulence factors for some bacteria
- damage cells, tissues, organs, etc.
what are the 2 classes of toxins?
- endotoxin, LPS component on surface of - Gram negative bacteria
- 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
Dispersal
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
- Toxins have various roles in disease
- Diphtheria toxin is a two component toxin (synthesized from a single precursor) that transports an effector protein into the cytoplasm and inhibits translation
- 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
