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Infection And Immunity > ADP-ribosylating toxins > Flashcards

ADP-ribosylating toxins Flashcards

1
Q

what are Bacterial toxins?

A
  • major virulence factors for some bacteria

- damage cells, tissues, organs, etc.

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2
Q

what are the 2 classes of toxins?

A
  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
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3
Q

what is Diphtheria?

A
  • 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
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4
Q

describe the Diphtheria toxin

A
  • 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)
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5
Q

explain Processing of diphtheria toxin

A

• 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’

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6
Q

explain Internalisation of diphtheria toxin

A

• 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

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7
Q

Mode of action of diphtheria toxin

A
  • 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
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8
Q

What is Cholera?

A

• 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

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9
Q

what did S.N. De demonstrated in 1959?

A

cell free extracts of V. cholerae caused diarrhoea

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10
Q

when was cholera toxin (CT) purified?

A

1969 84-kDa

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11
Q

how many cholera serotypes were found

A
  • 140

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

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12
Q

where is CT encoded?

A
  • filamentous phage
    • Phage can integrate in chromosome or replicate as a plasmid
    • Receptor is the toxin co-regulated pilus (TCP)
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13
Q

describe the Cholera toxin

A

• 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

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14
Q

describe Internalisation of cholera toxin

A
  • 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
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15
Q

Mode of action of cholera toxin

A
  • 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.
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16
Q

what happens In normal circumstances When a receptor is stimulated:

A
  • 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
17
Q

what happens in cells exposed to cholera toxin

A

• 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

18
Q

explain Regulation of Cholera Toxin

A
  • 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
19
Q

what are the Low/High Cell Density Phenotypes

A

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

High Density
Low adherence
Dispersal

20
Q

what are the Functions of Secretion

A
  • 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
21
Q

name 3 ADP-ribosylating toxins

A
  • Pseudomonas aeruginosa exotoxin A  Ef-2
  • P. aeruginosa exoenzyme S  ras gene family
  • E. coli heat labile enterotoxin  stimulatory G protein
22
Q

summarise ADP-ribosylating toxins

A
  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

Infection And Immunity (16 decks)

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