Applications of Biotechnology in Third World Problems:Using Transgenic Goats to Induce Cholera Immunity: Applications of Biotechnology in Third World Problems: Using Transgenic Goats to Induce Cholera Immunity Amy Wang 2007


Cholera: Cholera Also known as Asiatic cholera or epidemic cholera Spreads through exposure to contaminated water or waste Caused by cholera toxin released by the bacterium Vibrio cholerae Results in severe dehydration and diarrhea Common in developing countries due to lack of water treatment and sanitation services Figure 1: Vibrio cholerae bacterium


Symptoms of Cholera: Symptoms of Cholera Extreme diarrhea causes the rapid loss of protein-free fluid Fluid contains electrolytes, bicarbonates, ions Loss of fluid leads to severe dehydration and shock Loss of potassium ions creates increased risk of cardiac complications and circulatory failure Untreated cholera has a 50-60% mortality rate


Current Treatment: Current Treatment Mild to moderate forms of cholera can be treated by replacement of fluid and ions through oral rehydration solution (ORS) More severe forms of the disease require intravenous infusion of fluids and antibiotic therapy Figure 2: Patient being treated for cholera in Suchitepequez, Guatemala


Cholera Toxin: Cholera Toxin Enterotoxin from Vibrio cholerae Made up of 2 major protomers The heavy (H) or A subunit The B protomer, made up of 5 light (L) or B subunits Figure 3: Electron micrograph of Vibrio cholerae


B protomer: B protomer Attaches the cholera toxin to intestinal epithelial cells Allows the A subunit to proceed and begin the production of cyclic AMP


A subunit: A subunit The A subunit is split into A1 and A2 A1 is a mono (ADP-Ribose) transferase that activates the production of cyclic AMP Increased levels of cyclic AMP results in excessive amounts of fluid and electrolytes to be released from intestinal crypt cells, causing severe dehydration and diarrhea


Slide8: Figure 4: Cycle of cholera toxin


Question: Question Is it possible to create immunity against cholera through the use of transgenic goat milk?


Immunity: Immunity Body recognizes specific proteins on the surface of the pathogen When the pathogen enters the body, the immune system creates antibodies to fight the bacteria and prevent infection Immunity allows the body to have a constant source of antibodies in case the pathogen re-enters the body, resulting in faster defense and recovery


How this applies: How this applies Antibodies react and attach to specific proteins on the bacteria Isolating the antigen-antibody pair in the bloodstream of an immunized body makes it possible to identify the protein the antibody attaches to Isolating the sequence can be done through a technique known as phage display


Phage Display: Phage Display Selection technique to find a peptide sequence that reacts with a specific protein Used to create physical link between peptide sequences and the DNA encoding each sequence Allows identification of peptide ligands for different target molecules


Phage Display: Phage Display Peptide-molecule identification done though an in vitro selection process known as panning Incubate a library of different phage-displayed peptides on a plate coated with the target molecule Wash away the unbound phages Elute and amplify the specific phage that bound to the molecule Repeat the cycle to concentrate the amount of favorable binding sequences


How this applies: How this applies Phage display isolates the amino acid sequence that triggers the creation of cholera antibodies The amino acid sequence can then be genetically engineered into the DNA of dairy goats This allows for the expression of the amino acid sequence in the goats’ milk Once the sequence is in the milk, the person who drinks it will be able to develop an immunity to the protein that coats the Vibrio cholerae bacteria


Transgenic Goats: Transgenic Goats Animal that carries a foreign gene (transgene) intentionally inserted into its genome Fibroblasts grown in tissue culture are treated with a vector containing A neomycin-resistance gene which isolates the cells that adopted the vector Promoter sites from the beta-lactoglobulin gene which promote hormone-driven gene expression in milk-producing cells Binding sites for ribosomes for translation of the mRNAs


Transgenic Goats: Transgenic Goats A peptide sequence for a common protein in goat’s milk may be added to the gene in order to make the amino acid sequence stable Transformed cells are then combined with enucleated goat eggs and injected into the uterus of a female goat The embryos develop into transgenic goats who produce milk containing the protein expressed by the transgene


How this applies: How this applies The use of transgenic goats would be a feasible way for locals to receive the necessary protein needed for immunization Hormonally induced lactating cycles would allow the goats to provide a regular supply of milk Fig. 6: Transgenic goats


Additional Points: Additional Points A realistic application of this method would be to provide a transgenic “milk goat” to local villages Remember, the antibody for the cholera bacteria is not being provided in the milk. Instead, an antigen is being expressed in the milk, triggering an immunity to the bacteria


Bibliography: Bibliography Akker, F. Van Den . "Structure and Function of Cholera Toxin and Related Enterotoxins." Bacterial Protein Toxins. 2000. Feldstein, Paul. "Re: COSMOS Final Project Question." E-mail to Amy Wang. 27 July 2007. K. Holmes, Randall. "Cholera Toxin and Related Enterotoxins of Gram-Negative Bacteria." Handbook of Natural Toxins Volume 8. 1st ed. 1995. Kimball, John W.. "Transgenic Animals." Kimball's General Biology Text. 2006. 31 Jul 2007 . Mekalanos, John J, W. R. Romig. "Purification of Cholera Toxin and Its Subunits: New Methods." Infection and Immunity 20(1978): 552-558. New England BioLabs. "Rapid Screening of Peptide Ligands with a Phage Display Peptide Library." Ph.D.-12™ Phage Display Peptide Library Kit Jan 2006: 2-4. O'Brien, Philippa M. Antibody phage display methods and protocols. Totowa, N.J.: Humana Press, 2002. Scharfen, Erika Catharine. Utilization of human lysozyme transgenic goat milk in cheesemaking effects on bacterial profile and yield. Davis, CA: 2006. Todar, Kenneth. "Vibrio cholerae and Asiatic Cholera." Todar's Online Textbook of Bacteriology. 2005. University of Wisconsin-Madison Department of Bacteriology . 24 Jul 2007 .


Picture Credits: Picture Credits Figure 1: http://www.britannica.com/eb/art-74724/Vibrio-cholerae-the- bacterium-that-causes-cholera-shown-in-a?articleTypeId=1 Figure 2: http://www.paho.org/English/DPI/100/100feature07_photos.htm Figure 3: http://www.mwra.state.ma.us/germs/cholera.htm Figure 4: http://www.ebi.ac.uk/interpro/potm/2005_9/Page2.htm Figure 5: New England BioLabs. "Rapid Screening of Peptide Ligands with a Phage Display Peptide Library." Ph.D.-12™ Phage Display Peptide Library Kit Jan 2006: 2-4. Figure 6: Sam O’Neill Nexia Biotechnologies http://www.carleton.ca/catalyst/2003/s2.html