MECHANISM, TREATMENT AND PREVENTION FOR CHOLERA

1. PHM142 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 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 waterwellswould 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

7. GPCR is in the dormant state • Ligand binds to receptor and causes a conformational change • Receptor binds to the Gα subunitand alters the conformation of the coupled G-protein

8. 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.

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

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

12. Cholera Toxin

13. Cholera toxin binds to GM1 via B subunit • Taken up by endocytosis • Travels in retrograde fashion to the endoplasmic reticulum

14. 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

15. A1 peptide causes Gsα subunit to undergo ADP-ribosylation • Modified Gsα has internal GTPase activity inhibited • Locked in “on” state • Increased cAMP production

16. 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

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: https://www.pnas.org/content/115/41/10327 [Accessed 27 Sep. 2019]. DOI: 10.1073/pnas.1810316115 Finkelstein, R. (2019). Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios. [online] Ncbi.nlm.nih.gov. Available at: https://www.ncbi.nlm.nih.gov/books/NBK8407/ [Accessed 25 Sep. 2019]. Frerichs, R.R. (n.d.). John Snow And The Broad Street Pump On The Trail Of An Epidemic. Retrieved from https://www.ph.ucla.edu/epi/snow/snowcricketarticle.html Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 July;292(1):413-417. Kaper J, Morris J, Levine M. Cholera. Clinical Microbiology Reviews. 1995 Jan;8(1):46, 56-59. 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: https://www.ncbi.nlm.nih.gov/books/NBK21718/ [Accessed 27 Sep. 2019]. The biology behind cholera. (n.d.). Retrieved from https://bigpictureeducation.com/biology-behind-cholera