1. Biochemistry, Treatment and Prevention
Cholera
Biochemistry, Treatment and Prevention
Malak Al-Ali
Jessica Attalla
Ramsha Sehar
Noor Al-Imari
PHM Fall 2018
Instructor: Ms. Maya Latif
Coordinator: Dr. J. Henderson
23 October PHM142H1

2. What is Cholera? Infectious disease that causes severe diarrhea
Caused by the ingestion of contaminated food and water containing the bacterium Vibrio cholerae
There are many strains of Vibrio cholerae but only strains O1 and O139 cause outbreaks
Researchers estimate that there are million cases of cholera worldwide each year
Symptoms: watery diarrhea, leg cramps and vomiting
takes between 12 hours to 5 days for symptoms to appear (if any)
Rapid loss of fluids leads to dehydration, shock and death
Hi everyone! This is our group - Noor, Ramsha, Jessica and I am Malak. We will be discussing the biochemistry, prevention and treatment of cholera. So what is cholera? Cholera is an infectious disease that results in severe diarrhea and it is caused by the ingestion of contaminated food and water containing the bacterium Vibrio cholerae. There are only 2 strains of the bacterium that causes outbreaks, namely the O1 and O139 strains. It is a problem mainly affecting developing countries and there is a reported 1.3 to 4 million cases worldwide. People do not usually show symptoms but in severe cases, symptoms would include watery diarrhea, leg cramping and vomiting. It is problematic because the rapid loss of fluids can lead to death (within a few hours).

3. What is Cholera?
Cholera breakout linked to lack of access to clean water and sanitation facilities
Spread easily because people began to rely on “community taps”
The worst outbreak has been occurring in Yemen since October 2016
Currently, Yemen is experiencing the worst cholera outbreak since October 2016 due to the ongoing war and cholera is linked with lack of access to clean water and and the destruction of sanitation facilities. Many people have started to rely on “community taps” for their water source and this has significantly contributed to the spread of the bacterium. Jessica will now discuss the mechanism of the bacterium Vibrio cholerae.

4. Mechanism
O1 and O139 cholera bacteria strains secrete a toxin that contains Cholera Toxin Subunit B (CTB)
CTB binds to Ganglioside GM1 on the cell membranes of enterocytes and the toxin is internalized
The A1 domain of the toxin’s A subunit is cleaved and travels to the cytosol
A1 binds and activates Gsa, locking it in its GTP form
The GTP form of Gsa stimulates adenylate cyclase (AC), which produces cyclic AMP (cAMP)
Notes:
Cholera secretes a toxin (protein complex) with a pentameric (subunits) subunit, called cholera toxin subunit B (CTB). The other subunit of the cholera toxin is called subunit A
CTB binds to a glycolipid found on the cell membrane of intestinal cells called ganglioside GM1, triggers a signal transduction pathway.
The binding of the B subunit to GM1 induces endocytosis of the toxin
Once inside the cell, the A1 domain of the toxin’s A subunit is cleaved, and thus activated. The activated A1 domain travels to the cytosol, where it binds a G protein called Gsa. A1 locks Gsa in its GTP form when it binds it, and the GTP form of Gsa is a stimulant for adenylate cyclase, which produces cyclic AMP, a secondary messenger.
Whereas activation of cAMP is usually regulated to maintain homeostasis, this constituive expression of cAMP by a constantly active adenylate cyclase causes an abnormal amount of chloride ions, to be released from the cell when cAMP binds to the cystic fibrosis transmembrane regulator on intestinal cell membranes.
As systemic levels of chloride ions rise in the extracellular space, sodium and water ions leave the cell also in order to restore internal and external balance of charge. Thus, abnormal amounts of water and ions leaves the intestine, which causes infected individuals to suffer from constant watery diarrhea.

5. Mechanism
cAMP binds to a transmembrane protein called Cystic Fibrosis Transmembrane Regulator, which releases chloride ions into the extracellular space
Efflux of chloride ions triggers efflux of sodium and water as well, as cell tries to restore a balance in charge
Efflux of abnormal amounts of water and ions from the intestine induces watery diarrhea (which causes dehydration and a decrease in blood volume), cramps (due to electrolyte imbalance), hypovolemic shock (due to decreased blood volume), and vomiting in the infected individual

6. Treatment Oral Rehydration Therapy (ORT) Antibiotics:
Timely administration of oral rehydration salts can reduce the fatality rate to less than 1%
For severe dehydration and hypovolemic shock, intravenous fluids (ex. Ringer’s lactate) and electrolytes are required
Antibiotics:
Recommended as an adjunctive therapy: doxycycline or tetracycline for adults (target the 30S subunit of ribosome), azithromycin for children and pregnant women (target the 50S subunit)
The choice of drug depends on regional antibiotic sensitivity of the bacteria
Since infection with the cholera toxin causes large volumes of fluid loss, the first-line treatment is oral rehydration therapy.
Administer oral rehydration salts to replace lost fluid within 3 to 4 hours of initial presentation to prevent severe dehydration
Solutions consist of salts, glucose and water. Sodium-glucose cotransporter proteins in the small intestine transport a 2:1 ratio of sodium and glucose from the solution across the epithelium, and water follows to maintain an osmotic equilibrium
Does not change the duration of the disease
Less than 1% fatality if ORT is administered early at the onset of symptoms
IV:
Patients who experience severe dehydration or at risk of hypovolemic shock (low vascular volume do to 20% fluid loss) need IV fluids
200 mL- 350 mL/ kg administered within first 24 hours
Standard commercial one is Ringer’s Lactate because it contains both potassium and sodium bicarbonate (both of which are lost)
Antibiotics:
Recommended for severely dehydrated, hospitalized or for patients with hypovolemic shock in endemic areas
Fluid loss assessed based on the volume of fluid depletion that occurs as a percentage of the total body weight.
World Health Organization categorizes fluid loss of more than 10% as severe
In Endemic regions, cholera cots are used to measure the volume of stool and the fluid that is being (hole in the cot allows stool to be collected in bucket that has been calibrated)- rapid fluid loss is typically 10 to 20 mL/kg/hour
Tetraycycline and doxycycline both inhibit protein synthesis by targeting the 30S subunit of the bacterial ribosome and inhibiting translation- essentially preventing growth of bacteria
Antibiotics can reduce stool by up to 92%, decrease duration of diarrhea by ½

7. Treatment Zinc Treatment Oral Vaccines
Recommended for use in cases of pediatric diarrhea
Helps reduce the duration of diarrhea and volume of stool
Reduces cAMP concentration
Oral Vaccines
Bivalent killed whole-cell vaccine (Sanchol) - contains serotypes of V. cholerae O1 and O139 without the toxin B subunit
WC/rBS cholera vaccine (Dukoral) - contains serotypes of V. cholerae O1 and recombinant cholera toxin B subunit
Zinc:
Zinc supplementation works by reducing cyclic AMP concentrations and promoting the absorption of ions (cholera bacteria increases cAMP which causes choride ion secretion).
Reduces duration of diarrhea by
Vaccines:
Vaccines are recommended by the World Health Organization for people residing in endemic regions
Two doses are given to adults, three doses to children within a 7-14 day span
Provides protection for up to 6 months after two doses are given- provides 78-87% protection against the bacteria

8. Prevention Drink and use safe water Wash your hands often
safe water: water that is bottled, has been boiled, or has been treated with a chlorine product
Wash your hands often
Cook your food well and keep it covered
Clean up safely
Consider getting vaccinated (Sanchol, Dukoral)
1. Since cholera is contracted from contaminated water and food, the biggest precaution you can take is to drink and use safe water which is water that is bottled with an unbroken seal, or boiled, or has been treated with a chemical disinfectant such as chlorine.
2. Another thing is to cook your food well and keep it covered while it’s cooking and eat it while it’s hot (don’t let it cool down or sit around).
3. You also need to be cleaning up safely especially in places where lots of water is being used so the kitchen, the bathroom, the laundry room.
4. And there is also an oral vaccine for cholera (Sanchol, Dukoral) which also has been shown to be effective in preventing cholera as Ramsha already mentioned.

9. Prevention Public Health Agency of Canada (PHAC) Recommendations
Eat raw vegetables only if you have prepared them yourself
Avoid salads or raw foods
Eat foods only when they are served hot and cooked well
Ask for drinks without ice
Boil your water, use a chemical disinfectant, or treat your water with an ultraviolet light
Brush your teeth with safe water
Cholera is very uncommon in Canada but the public health agency of Canada has some recommendations for anyone travelling to countries where there isn’t the best sanitation such as sub-Saharan Africa, Southeast Asia, or Yemen as Malak mentioned. Public health advises people to:
only eat raw vegetables if you have prepared them yourself - you should peel + give them a thorough wash with safe drinking water
that also means you should avoid salads or raw foods made with fresh produce
try to eat your food hot and avoid even slightly undercooked foods
another recommendation is to ask for drinks without ice bc the water used in the ice cubes may be contaminated
you could also boil your own water or use a chemical disinfectant to ensure the water you use is safe

10. Summary
Description: Infectious disease caused by ingestion of contaminated food containing bacterium Vibrio cholerae
2 strains of Vibrio cholerae causes outbreak - O1 and O139
Symptoms (if any) include: vomiting, leg cramps and watery diarrhea
Mechanism:
The B subunit of the cholera toxin binds to GM1 on intestinal cell membranes, causing toxin to be internalized
Once in the cell, the A1 domain of the toxin is cleaved and binds to the Gsa G-protein in the cytosol, locking it in its GTP-form
The GTP-form of Gsa activates adenylate cyclase, which produces cAMP. cAMP binds to Cystic Fibrosis Transmembrane Factor (CFTR), which exports chloride ions into the extracellular space.
The efflux of chloride ions triggers the efflux of sodium and water loss, which produces the symptoms of cholera (if any exist) in the infected individual.
Treatment: Oral Rehydration Therapy is the first-line treatment, antibiotics (doxycycline or tetracycline for adults, azithromycin for children and pregnant women) can be used as an adjunctive therapy
Prevention: use safe water (boiled or water treated with a chemical disinfectant), cook your food well and eat it hot, wash your hands often, consider vaccination (Sanchol and Dukoral)

11. References
Centers for Disease Control and Prevention. (2018). Cholera - Vibrio cholerae Infection. Retrieved from
Cholera: Diseases and Conditions. (2018). Ontario Ministry of Health and Long-Term Care. Retrieved from
Ganglioside GM1. (2018). U.S. National Library of Medicine. Retrieved from
Kitaoka, M., et al. (2011). Antibiotic resistance mechanisms of Vibrio cholerae. Journal of Medical Microbiology. 60, doi: /jmm
LaRocque, R., Harris, JB. (2018). Cholera: Clinical features, diagnosis, treatment, and prevention. UpToDate. Retrieved from
Lauer, S., Goldstein, B., Nolan, R., Nolan, J. (2002). Analysis of Cholera Toxin−Ganglioside Interactions by Flow Cytometry. Biochemistry. 41(6): doi: /bi

12. References
Lyons, K. (2017). Yemen’s cholera outbreak now the worst in history as millionth case looms. The Guardian. Retrieved from worst-in-history-1-million-cases-by-end-of-year
McDowall, J. (2018). Cholera Toxin. InterPro. Retrieved from
Qadir, MI., et al. (2013). Zinc: Role in the management of diarrhea and cholera. World Journal of Clinical Cases. 1(4), doi: /wjcc.v1.i4.140
Reuters. (2018). Yemen cholera outbreak accelerates to cases per week: WHO. Retrieved from