Biology 216 - Lect 36 - Cholera Toxin MOA
Last period - Approaches to study virulence; 
	Today specific application of approaches.
How does one determine MOA? Major expts from approx 
	10 classic papers (not just one person).
I. Original studies - Late 1800's V. cholerae isolated 
		from patients by Kitasato.
	Is there a toxin involved in pathogenesis of cholera? Criteria: 
	Supernatant factor cause disease.
	Originally - no toxin based on:  
	Supernatant -> mice i.p. no disease. Accepted for years. 
		Problem: Mouse i.p. inappropriate model.
	Post mortem little inflammation of Sm intestine, mucosa intact:
		Problem: Toxin may change metabolism without killing cells; 
	1959 - cell free sup gives positive rabbit ileal loop (ie toxin)
II. Purification and characterization of toxin (necessary before MOA) - 
	1969 Finkelstein and LoSpalluto; $75/mg
	Need: Hyperproducer (569B); Optimum media (Syncase); 
	Assay (Loops and Ouchterlony w/Ab to crude)
A. Ouchterlony: Ab to crude, and correlate pptn band with loop activity
B. Purification: AmSO4, DEAE, A5M, G75
	Two immunoreactive peaks when pure on G75, 
		only one gives loop response.
	Choleragen (84 Kd from column) - produces cholera: 
		Choleragenoid (56) - like that which produces cholera.
C. Molecular characterization (1976 - Ohtomo)
	Separate Gen on G75 in Urea/Formic acid 
		(disrupts H+ and hydrophobic bonds) - 
	SDS-PAGE analysis of subunits (Standard method of analysis):
	Conclusions: Gen = A (toxic subunit) + B; 
		Genoid = B; A = A1 (21kd) + A2 (7kd) held by disulfide bond)
		B = 5 subunits of 11kd non covalently linked
	EM of crystaline toxin = donut shape
		 (5 subunits surrounding another)
III. Mechanism of action
A. Historical foundation: 1968 - Using rabbit ileum stretched 
	as membrane observed:
	1. Add cAMP -> Cl- and CO3 transported serosal -> lumen
	2. Activators of Ad cyclase (vasopressin and theophillin)
		 cause same transport.
	3. Cholera crude culture filtrates -> same thing
	Conclusion: CT may activate Ad cyclase increasing cAMP. 
	1971 - same expt using pure CT.
B. Mid 1970s (Michael Gill) - Model for determining which 
	subunits affect Ad cyclase.
	Model: Pigeon erythrocyte lysate assay 
		(low cAMP and easy to obtain)
	CT (or subunits) + RBC (intact or lysate) ->
		 assay cAMP as measure of Ad cyclase.

Subunit	Intact RBC	RBC lysate
CT	+	+
B	--	--
A	--	+
A1	--	+
A2	--	--
A + B	+	+

	Conclusion: A1 is involved in activation of Ad cyclase, 
		and B necessary for internalization of A1
C. How does CT activate Ad cyclase (Gill and King - 
	used modification of pigeon RBC assay.
	1. Washed RBC (ghost/membrane w/Ad cyclase) + CT 
		+ ATP (substrate) -> no activity
		Conclusion: something in cytosol necessary for activity.
	2. Ghost + ATP + CT + (empty shelf) -> activity
		 (Add back of NAD, not NADP) restores activity.
		Conclusion: MOA similar to diphtheria toxin; 
		ADP-ribosylating toxin.
	3. CT + NAD -> hydrolysis of NAD to ADP-R + nicotinamide.
	4. What is target of ADP-ribosylation? (Gill, Meren 1978)
	RBC ghost + CT + 32P-NAD -> react 30 min, wash membranes 
		-> SDS-PAGE -> autoradiograph
	Coumassie stain multiple bands; Autoradiograph only 42 kd
		 (previously reported GTP-BP regulator of adenylate cyclase)
D. Receptor studies: Modified Ouchterlony: 
	GM1 ganglioside reacts with CT and B subunit.
E. Genomics
     Sequenced (1998)
     On lysogenic phage
     ctxA/ctxB operon - RBS efficiency