1. MECHANISM, TREATMENT AND PREVENTION FOR CHOLERA
PHM Fall 2019
Instructor: Chesa Dojo Soeandy
Coordinator: Jeffrey Henderson
MECHANISM, TREATMENT AND PREVENTION FOR CHOLERA
By: NAVDEEP MANGAT
SHAHRUKH RAHMAN
GEORGE CHEN
PARVINDER SAHOTA
Presented on: OCT 1, 2019

2. CONTENTS OVERVIEW OF CHOLERA GPCR MECHANISM
EFFECT OF CHOLERA TOXIN ON GPCR MECHANISM
TREATMENT
PREVENTION

3. CHOLERA Vibrio cholerae What is Cholera?
Infectious disease that causes severe diarrhea and dehydration
How is it caused?
Ingesting food and water that's contaminated by Vibrio cholerae
Epidemiology
Developing countries with poor sanitation
Limited outbreaks in developed countries
Vibrio cholerae

4. Cholera Epidemiology History
- Around 1850 John Snow looked into the cholera epidemic in London - He had a theory that waste dumped into rivers near waterwells would contaminate drinking water and spread disease - John mapped out infected victims and found almost all near the Broad Street Pump
- He convinced town officials to remove the handle off the pump to prevent people from using it - As a result the cholera epidemic immediately came to an end - This was strong evidence that the contaminated water caused cholera, but people were still skeptical of his theory

5. Discovery of Vibrio Cholerae
- In 1854 cholera had reached Florence Italy - Filippo Pacini was interested in cholera and performed autopsies on cholera patients - With his microscope he observed the intestinal mucosa - He found a rod shaped bacteria and categorized it as water born (Vibrio), which we now refer to as Vibrio Cholerae

6. GPCR MECHANISM
Before we can talk about cholera, we need to understand how the GPCR works. GPCR stands for G-protein coupled receptor. They are the largest and most diverse group of membrane receptors. The GPCR contains 7 transmembrane-spanning domains and the signal is transduced by heterotrimeric G-proteins. These 3 subunits are called: alpha, beta, gamma subunits.

7. GPCR is in the dormant state
Ligand binds to receptor and causes a conformational change
Receptor binds to the Gα subunit and alters the conformation of the coupled G-protein
The GPCRs that are located on the intestinal epithelial cells are in their resting, or ‘off’ state due to a GDP bound to the alpha subunit. The whole GPCR cascade begins when a ligand binds to the receptor. The receptor undergoes a conformational change and activates the trimeric G-protein. The receptor will then bind to the Gα subunit.

8. Leads to the separation of the Gα subunit and the Gβ-γ complex.
Conformational change promotes the release of GDP and binding of GTP to the Gα subunit
Leads to the separation of the Gα subunit and the Gβ-γ complex.
The alpha subunit undergoes a conformational change and the GDP dissociates. The GDP on the alpha subunit is replaced by a GTP, activating the Gα subunit. In addition, the alpha and the beta-gamma subunits split apart.

9. Gα subunit binds to adenylate cyclase (transmembrane protein) and activates the production of cyclic AMP (cAMP)
In intestinal epithelial cells, ↑ in [cAMP] -> activates Protein Kinase A (PKA) -> activates CFTR -> ↑ [Cl-] in lumen
The activated alpha subunit binds to adenylate cyclase and starts to produce cyclic AMP, which is a secondary messenger. Intracellular cAMP concentrations increase and initiate downstream affects. cAMP activates Protein Kinase A, which opens the Cystic fibrosis transmembrane conductance regulator, or CFTR channels, and increase chloride ion concentration in the lumen.

10. GTP is hydrolysed by GTPase to form GDP
Gα subunit dissociates from adenylate cyclase
Gα subunit and the Gβ-γ complex reassemble
GPCR is inactive
When the G-protein needs to be turned off, GTP is hydrolyzed back to GDP, and the alpha and beta-gamma subunits reassemble back to its resting state.

11. Because activated G-proteins alter the function of many downstream effectors, we need specific G-proteins for specific cascades. I won't be going through all of them but the one we are interested in is called the G-alpha 's' subunit, which stimulates adenylate cyclase.

12. Cholera Toxin
-The cholera toxin is made of 1 A subunit and 5 identical B subunits
-The A subunit is made up of an A1 peptide and an A2 peptide which are strongly held together by disulfide bonds

13. Cholera toxin binds to GM1 via B subunit Taken up by endocytosis
Travels in retrograde fashion to the endoplasmic reticulum
-Once the bacteria gets to the endothelial lining of the intestines, it releases CT, which binds to a GM1 receptor on the cell membrane, using the B unit that has a high affinity for GM1
-CT is taken up by endocytosis and travels to the endoplasmic reticulum in a retrograde or backward direction compared to normal proteins travelling from the ER

14. A1 peptide associates with Sec61 transporter and goes to G proteins
Protein disulfide isomerase (PDI) cleaves A subunit into A1 and A2 peptides
Mimics a misfolded protein, causing cell machinery to send it to cytosol for degradation
A1 peptide associates with Sec61 transporter and goes to G proteins
-Once at the ER, protein disulfide isomerase (PDI) cleaves the A subunit of CT into its A1 and A2 peptides
-The peptides mimic misfolded proteins, so they’re sent to the cytoplasm where they’re supposed to be degraded
-But instead, the A1 peptide associates with Sec61, a protein transporter, and is taken to the Gs alpha subunit

15. A1 peptide causes Gsα subunit to undergo ADP-ribosylation
Modified Gsα has internal GTPase activity inhibited
Locked in “on” state
Increased cAMP production
-A1 peptide takes ADP-ribose from an NAD+ molecule and adds it to the Gs alpha subunit in a process known as ADP-ribosylation
-This inhibits the alpha subunit’s internal GTPase activity, so that it can’t hydrolyze GTP back to GDP, leaving it in a permanent “on” state
-So this causes the Gs alpha subunit to continuously bind to and activate adenylate cyclase, leading to an increase in cyclicAMP production

16. Excess Cl- secreted into the intestinal lumen Electrolyte imbalance
Increased cAMP production → Increased PKA activity→ Increased activation of CFTR channels
Excess Cl- secreted into the intestinal lumen
Electrolyte imbalance
Cell responds by secreting excess water into the lumen
Cell absorption can’t keep up with excess secretion
Results in Cholera disease state
-The buildup of cyclicAMP then leads to an increased activation of Protein Kinase A, which then leads to more CTFR channels being activated
-This causes an excess amount of chlorine ions to be secreted into the intestinal lumen
-The cell will try to correct the electrolyte imbalance by secreting excess water into the lumen
-But cell absorption of water and ions can’t keep up with the continuous secretion
-This leads to the disease symptoms of cholera, such as metabolic acidosis from the electrolyte imbalance, severe watery diarrhea and vomiting, which can then lead to dehydration that can be life threatening

17. TREATMENT Minor: Oral rehydration solution Severe: Intravenous fluids
Antibiotics (eg. Doxycycline), in conjunction with hydration
Chlorpromazine

18. PREVENTION Cook food thoroughly, and avoid raw foods
Build improved water filtration systems
Ensure proper hygiene rules are followed
Vaccinations (Shanchol, Dukoral)

19. SUMMARY
Cholera is an infectious disease that causes severe diarrhea, dehydration, metabolic acidosis
Cholera toxins bind to plasma membrane of intestinal epithelial cells and lock the G-proteins in their GTP-bound active state
Results in: ↑ in intracellular [cAMP] -> activate Protein Kinase A (PKA) -> activate CFTR channels -> ↑ [Cl-] in lumen -> ↑ water and electrolytes in intestinal lumen -> eliminated in feces
The body loses high levels of water, electrolytes, bicarbonate
Treatment: Oral rehydration solution (minor ailment) or IV hydration (serious ailment)
Antibiotics can help treat severe symptoms
Prevention: Cook food well, improve water systems, vaccinations, proper hygiene

20. REFERENCES
Alhadeff, R., Vorobyov, I., Wool Yoon, H. and Warshel, A. (2018). Exploring the free-energy landscape of GPCR activation. [online] PNAS. Available at: [Accessed 27 Sep. 2019]. DOI: /pnas
Finkelstein, R. (2019). Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios. [online] Ncbi.nlm.nih.gov. Available at: [Accessed 25 Sep. 2019].
Frerichs, R.R. (n.d.). John Snow And The Broad Street Pump On The Trail Of An Epidemic. Retrieved from
Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature July;292(1):
Kaper J, Morris J, Levine M. Cholera. Clinical Microbiology Reviews Jan;8(1):46,
Lodish, H., Berk, A., Zipursky, S., Matsudaira, P., Baltimore, D. and Darnell, J. (2019). G Protein –Coupled Receptors and Their Effectors. [online] Ncbi.nlm.nih.gov. Available at: [Accessed 27 Sep. 2019].
The biology behind cholera. (n.d.). Retrieved from