Cholera

Question 1

What type of bacteria causes cholera?

Answer 1

Gram-negative, anearobic bacteria

Question 2

What species of bacteria causes cholera?

Answer 2

Vibrio cholerae

Question 3

Where does Vibrio cholerae proliferate?

Answer 3

Water, which tends to be the source of infection

Question 4

How is one infected by the bacteria?

Answer 4

Intake of fecally contaminated water, food, or raw shellfish

Question 5

What animals is cholera most prevalent in?

Answer 5

Dogs, bison, cattle

Question 6

True or false. Avian and pig cholera have the same pathogen (Vibrio cholerae) as dog cholera.

Answer 6

False, the disease is caused by different pathogens

Question 7

What are the symptoms of cholera?

Answer 7

Severe diarrhea, vomiting, and if left untreated severe dehydration and death

Question 8

What causes the bacteria to produce the cholera toxin?

Answer 8

The bacteria anchor to the gut epithelium and the environment of the gut (pH, bile, amino acids) stimulates the production of the cholera toxin.

Question 9

Cholera toxin is a hetero-oligomer compromised of what three subunits and what is the function of each of these subunits?

Answer 9

5B subunit - for binding to specific receptor A2 subunit - serves as connection unit A1 subunit - toxic part that interacts with NAD

Question 10

Which subunit of the cholera toxin is the toxic part?

Answer 10

A1 subunit

Question 11

Which subunit of the cholera toxin serves as the connector?

Answer 11

A2 subunit

Question 12

Which subunit of the cholera toxin serves for binding to a specific receptor?

Answer 12

5B subunit

Question 13

How does cholera interact with gut epithelial cells?

Answer 13

By binding to glycolipid receptors (GM1 gangliosides) in lipid rafts.

Question 14

What is the name of the glycolipid receptors that cholera binds to and where are they located?

Answer 14

GM1 gangliosides in lipid rafts

Question 15

What happens after cholera toxin binds to GM1 glangioside receptor?

Answer 15

Complex is internalized and undergoes retrograde transport to the ER.

Question 16

Describe the retrograde transport cholera toxin undergoes after the cholera toxin/ganglioside complex is internalized.

Answer 16

After endocytosis, proteins move to early endosome that gets sorted into the retromer complex. The retromer complex then targets vesicles from the early endosome back to the golgi (mediated by SNARE proteins). Once it gets to the golgi, it gets passed back through the cisternae by COPI vesicles (vs. COP2 vesicles in normal transport) to the ER.

Question 17

What type of vesicles pass early endosomes targeted by the retromer complex through the cisternae of the golgi apparatus to the ER?

Answer 17

COPI vesicles (NOT COPII - COPII vesicles go the other way)

Question 18

What happens when the cholera toxin passes through the golgi retrograde to the ER and reaches the ER?

Answer 18

The A1 subunit dissociates from the rest of the toxin and gets released into the cytoplasm.

Question 19

Which subunits stay in the ER and which subunits of the cholera toxin get released to the cytoplasm after the toxin has gone through retrograde transport in COPI vesicles?

Answer 19

B5 and A2 subunits stay in the ER A1 subunit is released into the cytosol

Question 20

Specifically, what is the A1 subunit of the cholera toxin?

Answer 20

ADP ribosyltransferase

Question 21

What happens to A1 subunit of the cholera toxin once it is released into the cytosol?

Answer 21

It is refolded and exerts its toxic effects

Question 22

As an ADP ribosyltransferase, describe the mechanism of how the A1 subunit exerts its toxic effects on the cell.

Answer 22

It takes nicotinamide off of co-factor NAD and puts an ADP ribosyl group onto the modified protein. This is post-translational modification that can change the proteins enzymatic function.

Question 23

What is the specific target of the A1 subunit?

Answer 23

alpha subunit of the Gs G-protein

Question 24

What does the A1 subunit do to its target?

Answer 24

A1 subunit ADP ribosylates the alpha-subunit of the Gs-G protein. This inhibits the GTPase activity of the G-protein so it cannot switch itself off.

Question 25

What is the result of the inability of the Gs protein to switch itself off because of the A1 toxin?

Answer 25

Hyperactivation of adenylyl cyclase. This happens because the Gs-protein, being constantly active and GTP bound, binds to adenylyl cyclase which will take ATP and form cAMP which will activate protein kinase A (PKA).

Question 26

What is the function of PKA after it has been activated, and why do its effects become harmful in the presence of the cholera toxin?

Answer 26

PKA phosphorylates and activates the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). This opens the channel so we get movement of chloride out of the cell, with water following from basolateral side to the gut lumen. Excessive PKA activation due to the A1 subunit’s toxic effects that caused Gs protein to be constantly active can cause severe diarhea due to movement of water following chloride out of the cell and into the gut lumen.

Question 27

Efflux of chloride ions causes the efflux of ______ into the _____.

Answer 27

water; gut lumen

Question 28

List the basic steps of how one gets cholera.

Answer 28

1. Animal is infected with Vibrio cholerae 2. Vibrio cholerae produces cholera toxin in gut 3. Cholera toxin binds to GM1 gangliosides in lipid rafts of gut epithelial membrane 4. Cholera + ganglioside complex gets endocytosed 5. Cholera undergoes retrograde endocytosis to the ER 6. A1 subunit gets released into cytoplasm 7. ADP ribosylates the alpha Gs protein, permanently activating it 8. Induces activation of adenylyl cyclase 9. Production of cAMP and activation of PKA 10. PKA phosphorylates CFTR that opens chloride channel. 11. Chloride and water move into gut lumen