Toxins: CT, PT, TcdA/B, CdtAB Flashcards
what are examples of toxins that alter signal transduction (2)
- cholera toxin
- pertussis toxin
what is one way that toxins can alter signal transduction
- toxins can target or alter cAMP production
what bacteria produces cholera toxin
- vibrio cholerae
what are the symptoms of cholera toxin (2)
- produce watery diarrhea
- lots of fluid secretion
cholera toxin structure (3)
- AB5 toxin
- one A
- five identical B subunits that form a pentamer
cholera toxin: B subunits
- binds to GM1 gangliosides
what is the cholera toxin’s mechanism of intoxication (7)
- B pentamer binds to receptor on host
- toxin is taken into endosome
- endosome fuses to Golgi
- retrograde transport of toxin from Golgi to the ER
- A subunit dissociates from pentamer
- A subunit enter the cytosol
- the A subunit ADP-ribosylates a heterotrimeric G protein
cholera toxin: how does the A subunit dissociate from the B subunit pentameter
- disulfide bonds which holds the toxin together are reduced
cholera toxin: how does the A subunit enter the cytosol
- enters via the Sec translocase
G proteins
- large family of proteins that control variety of cell functions through signal transduction
G proteins: function (2)
- bind GTP
- hydrolyze GTP to GDP
G protein subunits
- α, β, γ
G protein: α subunit
- binds GTP
G protein: Gαs (3)
- the “stimulatory” subunit
- activates adenylate cyclase
- increasing the production of cAMP
G protein: Gαi (2)
- the “inhibitory” subunit
- leads to decreased levels of cAMP in the cell
GTPase activity
- converts GTP –> GDP
G protein: stimulatory signal pathway (8)
- ligand interacts with its receptor
- heterotrimeric G protein recruited to the receptor
- dissociated of subunits from the receptor and each other
- Gαs binds GTP and activated adenylate cyclase, increased cAMP levels
- Gαs has GTPase activity
- leads to lower cAMP levels
- re-association of the subunits and GDP is lost
- cycle continues
G protein: inhibitory signal pathway (8)
- ligand interacts with receptor
- recruitment of heterotrimeric G protein to receptor
- dissociation of subunits from receptor and each other
- Gαi binds GTP and inhibits adenylate cyclase, reducing cAMP levels
- Gαi has GTPase activity
- leads to higher cAMP levels
- reassociation of the subunits and GDP is lost
- cycle continues
cholera toxin mechanism of intoxication (4)
- cholera toxin A subunit ADP-ribosylates Gαs
- causes continuous dissociation of the G protein heterotrimer
- leads to increased levels of cAMP
- cell is permanently “ON” excessively produces cAMP
CFTR (2)
- cystic fibrosis transmembrane conductance regulator
- a channel that plays a key role in cystic fibrosis
what is the result of the cholera toxin intoxication (4)
- phosphorylation of many proteins including CFTR
- stimulation of secretion of chloride ions from crypt cells in intestine
- inhibition of uptake of sodium and chloride ions from villous cells (intestine)
- stimulates cells to secrete fluids, leading to watery diarrhea
pertussis infection: method of transmission (2)
- aerosol infection
- transmission achieved by coughing
how does pertussis establish infection (2)
- bacterium binds mucin and ciliated epithelial cells that line trachea and nasopharynx
- bacteria replicate and colonize
how does pertussis maintain infection (2)
- evasion of the immune system
- production of several toxins
pertussis toxin: effects (2)
- chemokine suppression
- suppression of the recruitment of immune cells
what bacterium produced pertussis toxin
- bordetella pertussis
pertussis toxin: structure (4)
- AB5 toxin
- one A subunit
- four different B subunits
- subunits linked by disulfide bonds
pertussis toxin: A subunit
- S1
pertussis toxin: B subunit (4)
- S2
- S3
- 2xS4
- S5
how does the pertussis toxin bind to the host cells
- S2 and S3 bind to sialylated glycoproteins
pertussis toxin: mechanism of intoxication (7)
- pertussis toxin binds to host cell
- retrograde transport from endosome, to Golgi, to ER
- S1 gets translocated to cytosol
- S1 ADP-ribosylates Gαi part of Gαi-GDP-β-γ
- Gαi-GDP-β-γ cannot re-associate with receptor and GDP does not get released
- Gαi is inactivated
- adenylate cyclase activity cannot be turned off, resulting in increased cAMP levels
what are the consequences of the pertussis toxin intoxication (2)
- inhibition of chemotaxis of alveolar macrophages (AM), monocytes, NK cells, and neutrophils to site of infection
- an immune evasion mechanism
Clostridium difficile
- gram positive spore-former
how common is C. difficile (2)
- 5-10% of the population is naturally colonized as part of our microbiota
- 25% can be colonized in hospital setting
how does C. difficile behave after antibiotic treatment (2)
- germinates in the gut
- produces toxin once it germinates
what do C. difficile toxins do (3)
- damage mucosal cells in colon
- produce accumulation of dead cells, mucin, and fibrin
- produce pseudomembrane colitis
psuedomembrane colitis (2)
- when dead cells, mucin, and fibrin coalesce to form patchy lesions throughout the colon
- extremely painful
C. difficile: what toxins cause damage (2)
- TcdA and TcdB cause the most damage
- CdtAB causes damage to a lesser amount
how can patients acquire C. difficile infection (2)
- hospitalization
- antibiotics that wipe out natural microbiota
C. difficile: TcdA (3)
- structure
- target cell
- receptor
- single protein with multiple domains
- targets intestinal cells
- glycoprotein GP96 located on the apical side of colonic cells
C. difficile: TcdB target (3)
- structure
- target cell
- receptor
- single protein smaller than TcdA
- cells other than intestinal cells
- CSPG4 and FZD on basolateral side of cells
what are the components of TcdA toxin: A subunit
- active site
what are the components of TcdA toxin: B subunit (3)
- cleavage site
- translocation domain
- receptor binding domain
C. difficile: TcdA mechanism of intoxication
- entire protein endocytosed
- when pH changes, conformational change occurs in translocation domain
- “A” domain is translocated through the pore
- “A” is cleaved and released into cytosol
- A monoglucosylates small GTP-binding proteins
- G protein network is disrupted
- actin filaments depolymerize/breakdown
- tight junctions are destroyed, leading to diarrhea
- toxins also trigger apoptosis and pseudomembrane production
C. difficile: TcdA cleavage of A subunit (2)
- mechanism
- activated by…
- autoproteolysis
- activated by lnsP6
C. difficile: what small GTP-binding proteins does A subunit of TcdA affect (4)
- Rho
- Rac
- CDC42
C. difficile: how does A subunit of TcdA target the small GTP-binding proteins
- monoglucosyaltes on threonine-37 residue
apoptosis
- programmed cell death
heat-shock proteins (HSP)
- proteins active under stress to fold proteins
C. difficile: CdtAB structure (2)
- A subunit: CdtA
- B subunit: CdtB
C. difficile: what is the receptor for the CdtAB’s B subunit
- LSR
C. difficile: CdtAB mechanism of intoxication; adherence/invasion (4)
- CdtB cleaves by cellular protease
- CdtB oligomerizes and binds to its receptor
- CdtA now binds to oligomerized CdtB
- entire complex is taken up into cell
C. difficile: CdtAB mechanism of intoxication; post invasion (5)
- when endosome pH drops, CdtB forms pore in endosome membrane
- CdtA translocates through pore
- CtdA ADP-ribosylates G-actin
- ADP-ribosylated G-actin caps growing actin polymer
- released monomers are also ADP-ribosylated
how does actin polymerization occur (2)
- actin get polymerized at one end of the polymer
- actin monomers are released at the other end
C. difficile: what is the results of the CdtAB intoxication (2)
- actin is now trapped in ADP-ribosylated form
- complete breakdown of microfilament network occurs
how do you prevent C. difficile infection (2)
- limit antibiotic usage
- increase hygiene practices
CDI treatment (acute) (2)
- patients with diarrhea tested for TcdA and TcdB
- antibiotics can be used
when do CDI treatment fail
- failure to restore a stable, healthy microbiota after antibiotic treatment results in relapse
CDI treatment (chronic)
- fecal microbiota transplant (FMT)
