1. The Crud on Cholera:
GPCRs, Immune Complications, & Treatments
Andrew Tung, Ben Magnuson, Jason Soares, and Mark Noble

2. Overview
 Cholera’s impact on GPCR’s
 Symptoms and Mechanism
 Downstream immunological impact
 Inhibition of immune cytokines and effector cells
 Medical treatments
 Oral rehydration and prospective solutions
 Conclusion
 To be revealed

3. Vibrio Cholerae
❏ Bacterium
❏ Secretes protein complex known as Cholera Toxin
❏ Symptoms?
❏ Violent diarrhea
❏ “Rice-water”
❏ Vomiting
❏ Severe dehydration
❏ >100,000 deaths each year
❏ WHO?

4. Cholera Toxin
❏ Has an agonistic effect on G-proteins
❏ What’s an agonist? What’s a G-protein?
❏ You may retake this course next year if you
are unaware of these exquisite biological
phenomenon's…

5. Cholera Mechanism
❏ Monomeric CTA and pentameric CTB
❏ CTB binds surface gangliosides
❏ endocytosis
❏ CTA dissociates and binds Gs
❏ ADP ribosylation
❏ Activation of Adenylyl Cyclase
❏ Sustained cAMP amplification
❏ PKA activation

6. Cholera Mechanism
 Secretion of Cl-, Na+, H2O, and HCO3-
into the intestinal lumen
 Leads to the osmotic imbalance
 Results in deadly diarrhea
 Death as a result of dehydration
 Inhibitory effects on immune system
❏Cytokine production
❏(IL-12, IL-10,TNF-ɑ, IFN-ɣ)

7. Immunological Impact of Cholera Toxin
❏ Does Cholera impact...
❏ Cytokine production?
❏ Interleukin-12, inflammatory cytokine,
stimulates proliferation of effector cells
❏ Effector cell proliferation?
❏ B-cells and T-cells
❏ Protective immunity?
❏ CD8+ T-cells are required components of the innate
and adaptive immune systems

8. Experimental Methods
❏ In vivo mice experimentation
❏ 3 strains of mice
❏ T.gondii parasite only (control)
❏ Cholera Toxin and T.gondii
❏ IL-12 absence and T.gondii
❏ Blood analysis: cytokine and cell counts
❏ Flow cytometry
❏ Live-cell imaging
❏ Photobleaching
❏ Fluorescence

9. Results: Flow Cytometry
❏ Effect of Cholera on effector cell counts
due to IL-12 inhibition
❏ CD4+, CD8+, CD19+
❏ 20-30% reduction in effector cell counts
❏ Dramatic reduction in CD8+ T-cells
compared with other lymphocytes
❏ 5 independent experiments, all of which
had similar results
❏ Very valid
Displays a relatively specific immunological
impact of Cholera Toxin on CD8+ T-Cells
❏ Innate and Adaptive immunity

10. Results: Big Picture
❏ Strain 1 (T.gondii)
❏ Successful immune response
❏ Strain 2 (T.gondii & CT)
❏ Death ~8 days post infection
❏ Strain 3 (T.gondii & no IL-12)
❏ Death ~8 days post infection

11. Conclusion
❏ Cholera inhibits IL-12 production:
❏ Decreased effector cell proliferation
❏ Mainly CD8+ T-cells
❏ Less effective immune response
❏ Similar effect to mice completely lacking IL-12
❏ Detrimental impact on the immune system
❏ Were these valid?
❏ Results were supported with similar findings in a variety of
other experimental literature

12. Medical Experiment for Treatment of Cholera:
❏ Current Treatment: glucose-based oral
rehydration solution
❏ Rehydrates individuals but does not reduce the duration of
diarrhea
❏ Medical Experimentation have been undergone
to find a better treatment

13. Medical Experiment
❏ Oral rehydration solution with high amylose
maize starch (HAMS-ORS) compared with
standard glucose oral rehydration solution
(HO-ORS)
❏ BIG RESEARCH QUESTION: Glucose based or alternative
solution based such as amylose

14. Medical Experiment
 Researchers proposed: oral rehydration solution with
amylose maize starch compared to that of the standard
solution results in shorten duration of diarrhea as well
as recovering the fluid lost

15. Experimental Methods
❏ 50 Adult Males with severe watery diarrhea
❏ 25 given HAMS-ORS
❏ 25 given HO-ORS

16. Results
❏ HAMS-ORS reduced diarrhea duration by 55% compared
to HO-ORS
❏ Patients with HAMS-ORS had a significantly quicker
recovery compared to that of the hypo-osmolar high
glucose solution

17. Conclusion
❏ Oral rehydration solution has saved millions of
children from death via dehydration but this does
not decrease diarrhea
❏ Glucose based solution or amylose solution
❏ Alternative Supplements
❏ Zinc supplements (shown to decrease diarrhea)
❏ Apparent that further medical experimentation is
required to improve the current treatment for
cholera

18. Overall Summary
 Mechanisms of Cholera on G-proteins are well documented
 Downstream Effects upon immune system is validated
through experimentation
 Treatment is targeting symptoms (diarrhea)
 More research is required to target the origin of the Cholera
Toxin on G-proteins

19. References
Atia, A. N., & Buchman, A. L. (2009). Oral Rehydration Solutions in Non-
Cholera Diarrhea: A Review. The American Journal of Gastroenterology
104(10), 2596-2604. doi: 10.1038/ajg.2009.329
Atia, A. N., & Buchman, A. L. (2010). Treatment of cholera-like diarrhoea
with oral rehydration.Annals of Tropical Medicine and Parasitology, 104(6),
465-474. doi: 10.1179/136485910X12786389891164
Dal Molin, F., Zornetta, I., Puhar, A., Tonello, F., Zaccolo, M., & Montecucco,
C. (2008). cAMP imaging of cells treated with pertussis toxin, cholera toxin,
and anthrax edema toxin. Biochemical and Biophysical Research
Communications, 376(2), 429-433. doi:10.1016/j.bbrc.2008.09.012
Das, J. K., Ali, A., Salam, R. A., and Bhutta, Z. A. (2013). Antibiotics for the
treatment of Cholera, Shigella and Cryptosporidium in children. BMC public
health, 13(Suppl 3), S10. doi:10.1186/1471-2458-13-S3-S10
DiLillo, D. J., Yanaba, K., and Tedder, T. F. (2010). B cells are required for
optimal CD4+ and CD8+ T cell tumor immunity: therapeutic B cell depletion
enhances B16 melanoma growth in mice. The Journal of Immunology, 184(7),
4006-4016. doi: 10.4049/jimmunol.0903009.
Dor, Y., Brown, J., Martinez, O., & Melton, D. (2004). Adult pancreatic β-cells
are formed by self-duplication rather than stem-cell differentiation. Nature,
429(6987), 41-46. doi:10.1038/nature02520
Edwards, J. P., and Emens, L. A. (2010). The Multikinase Inhibitor Sorafenib
Reverses the Suppression of IL-12 and Enhancement of IL-10 by PGE 2 in
Murine Macrophages. International immunopharmacology, 10(10), 1220-1228.
doi:10.1016/j.intimp.2010.07.002
Gee, K., Guzzo, C., Mat, C., Nor, F., Ma, W., and Kumar, A. (2009). The IL-12
family of cytokines in infection, inflammation and autoimmune disorders.
Inflammation & Allergy-Drug Targets, 8(1), 40-52. Retrieved from
http://www.researchgate.net/
Ghose, A. C. (2011). Lessons from Cholera & Vibrio cholerae. The Indian
journal of medical research, 133(2), 164. Retrieved from
http://www.ncbi.nlm.nih.gov/pmc/
Gregorio, G. V., Gonzales, M. L. M., Dans, L. F., & Martinez, E. G. (2009).
Polymer‐based oral rehydration solution for treating acute watery diarrhoea.
Cochrane Database of Systematic Reviews, 2, 1-69. doi:
10.1002/14651858.CD006519.pub2
Guettier, J. M., Gautam, D., Scarselli, M., Ruiz de Azua, I., Li, J. H.,
Rosemond, E., Wess, J. (2009). A chemical-genetic approach to study G
protein regulation of beta cell function in vivo. Proceedings Of The National
Academy Of Sciences, 106(45), 19197-19202. doi:10.1073/pnas.0906593106
Inada, A., Nienaber, C., Katsuta, H., Fujitani, Y., Levine, J., Morita, R.,
Bonner-Weir, S. (2008). Carbonic anhydrase II-positive pancreatic cells are
progenitors for both endocrine and exocrine pancreas after birth. Proceedings
Of The National Academy Of Sciences of the United States of America,
105(50), 19915-19919. doi: 10.1073/pnas.0805803105
Jasny, E., Eisenblätter, M., Mätz-Rensing, K., Tenner-Racz, K., Tenbusch, M.,
Schrod, A., and Ignatius, R. (2008). IL-12-impaired and IL-12-secreting
dendritic cells produce IL-23 upon CD154 restimulation. The Journal of
Immunology, 180(10), 6629-6639. doi: 10.4049/jimmunol.180.10.6629
la Sala, A., He, J., Laricchia-Robbio, L., Gorini, S., Iwasaki, A., Braun, M.,
Kelsall, B. (2009). Cholera toxin inhibits IL-12 production and CD8α+ dendritic
cell differentiation by cAMP-mediated inhibition of IRF8 function. The Journal
of experimental medicine, 206(6), 1227-1235. doi: 10.1084/jem.20080912
Lang, P. A., Lang, K. S., Xu, H. C., Grusdat, M., Parish, I. A., Recher, M., and
Ohashi, P. S. (2012). Natural killer cell activation enhances immune pathology
and promotes chronic infection by limiting CD8+ T-cell immunity. Proceedings
of the National Academy of Sciences, 109(4), 1210-1215. doi:
10.1073/pnas.1118834109
Nelson, D. L., & Cox, M. M. (2013). Principles of Biochemistry. New York: W.
H. Freeman and Company
Peyot, M., Gray, J. P., Lamontagne, J., Smith, P. J. S., Holz, G. G., Madiraju,
S. R. M., Heart, E. (2009). Glucagon-Like Peptide-1 Induced Signaling and
Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent
Pancreatic β-Cells. PLoS ONE, 4(7), e6221. doi:
10.1371/journal.pone.0006221
Ramakrishna, B. S., Subramanian, V., Mohan, V., Sebastian, B. K., Young, G.
P., Farthing, M. J., & Binder, H. J. (2008). A Randomized Controlled Trial of
Glucose versus Amylase Resistant Starch Hypo-Osmolar Oral Rehydration
Solution for Adult Acute Dehydrating Diarrhea. PLoS ONE, 3(2), e1587.
doi:10.1371/journal.pone.0001587
Roy, S. K., Hossain, M. J., Khatun, W., Chakraborty, B., Chowdhury, S.,
Begum, A., Chowdhury, R. (2008). Zinc supplementation in children with
cholera in Bangladesh: randomised controlled trial. BMJ, 336(7638), 266-268.
doi: 10.1136/bmj.39416.646250.AE
Serezani, C. H., Ballinger, M. N., Aronoff, D. M., & Peters-Golden, M. (2008).
Cyclic AMP: master regulator of innate immune cell function. American
Journal of Respiratory Cell and Molecular Biology, 39(2), 127-132. doi:
10.1165/rcmb.2008-0091TR
Sonoda, N., Imamura, T., Yoshizaki, T., Babendure, J., Lu, J., & Olefsky, J.
(2008). β-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin
secretion in cultured pancreatic β cells. Proceedings Of The National
Academy Of Sciences of the United States of America, 105(18), 6614-6619.
doi:10.1073/pnas.0710402105
Tognotti, E. (2011). The dawn of medical microbiology: germ hunters and the
discovery of the cause of cholera. Journal of Medical Microbiology, 60(4),
555-558. doi: 10.1099/jmm.0.025700-0
Wilson, D. C., Matthews, S., and Yap, G. S. (2008). IL-12 signaling drives CD8+
T cell IFN-γ production and differentiation of KLRG1+ effector subpopulations
during Toxoplasma gondii infection. The Journal of Immunology, 180(9),
5935-5945. Retrieved from http://