Team:UT-Dallas/temp

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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><br><h2>Introduction</H2><p style="display:block">
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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><i>“We have witnessed in our days the birth of a new pestilence, which, in the short space of fourteen years, has desolated the fairest portions of the globe, and swept off at least fifty MILLIONS of our race. It has mastered every variety of climate, surmounted every natural barrier, conquered every people. It has not, like the simoon, blasted life and then passed away; the cholera, like the small-pox or plague, takes root in the soil which has once possessed.”
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We envision a new paradigm for treating infections of the human gastrointestinal tract through exploitation of engineered probiotics that produce anti-microbials with high specificity for pathogens. The anti-microbials we are exploring do not utilize a one-fit all therapy mold, but target unique features specific to organisms at the genetic level. Towards this aim, we have utilized a general-purpose system that will be delivered to pathogenic bacteria from an engineered bacterial species found in the GI tract (Escherichia coli), which will cleave pathogenic genes with single nucleotide resolution. To achieve specific genome targeting, we will utilize the CRISPR/Cas9 system with gRNA engineered to recognize genes from infectious bacteria that contribute to pathogenicity in humans. Our CRISPR/Cas9 system will be delivered from the engineered E. coli to infectious bacteria using bacterial specific phages, minimizing any side-effects to native microbiota and human-host cells. As a proof-of-principle for our engineered probiotic, we are starting by targeting Vibrio cholerae, however we hope to expand the system to other pathogens of the GI tract.  
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(The London and Paris Observer, 11/27/1831)</i><br><h2>Introduction</H2><p style="display:block">
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Cholera is a gastrointestinal disease caused by a toxin released by aquatic bacterium Vibrio cholerae. Symptoms of cholera are acute watery diarrhea and vomiting. In severe cases, patients may show signs of severe dehydration, such as sunken eyes, decreased skin turgor, muscle cramping, and decreased blood pressure. If left untreated, severe cases can result in death within a few hours. There are two serotypes of V. cholerae responsible for the global cholera pandemics: O1 and O139. O1 has two biotypes - classical and El Tor. Non-O1 and non-O139 V. cholerae infections can cause similar but with milder symptoms (1). There have been seven recorded cholera pandemics beginning in 1816 and many more outbreaks in that time. Today, cholera infects approximately 3-5 million people every year and between 100,000-120,000 of these cases are fatal (1). Although cases of cholera are rare in developed countries, it continues to be a public health concern in regions with underdeveloped water treatment practices because of its relatively high death rate and persistence in the environment.  
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<br><h2>Probiotics</H2><br><p style="display:block">
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<br><h2>Mechanism of Infection</H2><br><p style="display:block">
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We used E. coli as our chassis for our experiments in the lab, but we envision that our system would be used in a common probiotic strain of bacteria, such as Lactobacillus acidophilus. Our probiotic system can be used prophylactically to persons in a region experiencing a cholera outbreak, or it can be administered to someone already exposed to V. cholerae to quickly deliver therapeutic phage directly to the site of the infection. We believe our system would be particularly useful to military personnel or aid workers stationed in a cholera outbreak zone where they would be frequently exposed to V. cholerae. Although an oral cholera vaccine exists, its efficacy is relatively low: 52-62% in healthy adults and as low as 38% for the highest risk age group - children under 5 years of age (1).
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Although most V. cholerae ingested dies in the acidic stomach, the survivors can quickly colonize the small intestine. The symptoms associated with V. cholerae infection are primarily caused by the cholera toxin, but there are several genes that contribute to the colonization and pathogenicity of V. cholerae. Many of the genes associated with the virulence of V. cholerae are located on a pathogenicity island believed to originate from phage (2).  We chose several genes identified for their role in pathogenicity and colonization to target for our project. <br>
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Once complete, our system will be able to remain as a stable population in a person’s gut until the event of a V. cholerae infection. Detection of V. cholerae will then activate production of the phage delivery system, which will package the gRNA and Cas9 targeting system into a phage coat, exit the probiotic and transfer the system into V. cholerae present in the gut (transmission of a heterologous DNA message via phage was demonstrated by Ortiz and Endy in 2012 and featured in Waterloo’s 2013 iGEM project (2)). Once inside V. cholerae, the targeting system will bind and cleave sites complementary to the gRNA that correspond to selected pathogenicity genes. We proposed using Cas9/gRNA to target and kill pathogens as opposed to traditional phage therapy using a targeted lytic phage because it allowed us to differentiate and kill harmful pathogens of a strain that has both harmful and harmless serotypes. The gRNA in our system, while specific to V. cholerae, can easily be altered with PCR to target unique regions in other gastrointestinal pathogens.
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<u>Cholera Toxin:</u> <br>
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The cholera toxin is an oligomeric protein made up 6 subunits: 1 copy of subunit A (ctxA) and 5 copies of subunit B (ctxB). The 5 ctxB subunits form a ring that directly binds to the GM1 ganglioside receptors on the surface of intestinal epithelial cells, causing the cholera toxin to be endocytosed. Once inside, the disfulfide bridges in the toxin are reduced, freeing the A subunit which then activates G proteins inside the cell,  causing constitutive production of cAMP. This activates chloride ion channels, causing efflux of chloride ions, followed by water, Na+, and K+ due to osmotic and electrical gradients. This produces the characteristic watery diarrhea and rapid loss of water and electrolytes (3).  
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<u>Transcriptional regulator toxT:</u> <br>
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ToxT is a transcriptional regulator that indirectly activates the cholera toxin operon and the toxin coregulated pilus operon (4). ToxT is activated when ToxR recruits TcpP to the upstream promoter.
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<u>Toxin Coregulated Pilus:</u> <br>
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The toxin coregulated pili are bundles of fimbriae extending from the poles of V. cholerae that are coregulated with the cholera toxin. They have been shown to play an important role in V. cholerae colonization of the gut (5). Their exact cell surface interactions are unknown.
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<u>Accessory Colonization Factor:</u> <br>
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  The exact mechanism of the accessory colonization factor proteins are unknown, but they have been shown to enhance colonization of V. cholerae in the gut in infant mice (6).  
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<h3>Cholera and the beginnings of phage therapy:</h3> <br>
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In the late 1800s, English chemist Ernest Hanbury Hankin traveled to India to study the cholera outbreak. He observed bactericidal activity in the water taken from the Ganges river when applied to cultures of V. cholerae. In his notes, he described the agent responsible as being able to pass through a fine porcelain filter but became inactive when boiled. He also noted that people living in regions that obtained their water from the Ganges river were not experiencing a cholera outbreak (7) </p><br><br>
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<br><h2>References</H2><br><p style="display:block">
<br><h2>References</H2><br><p style="display:block">
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1. Sinclair D., Abba K., Zaman K., Qadri F., Graves P.M., Oral vaccines for preventing cholera. Cochrane Database Syst Rev. 2011 Mar 16;(3):CD008603.<br>
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1. World Health Organization. (2014). Cholera (Fact sheet No. 107). Retrieved from http://www.who.int/mediacentre/factsheets/fs107/en/ <br>
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2. Ortiz M.E., Endy D. Engineered cell-cell communication via DNA messaging. J Biol Eng. 2012 Sep 7;6(1):16.</p><br><br>
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2. Rajanna, C., Wang, J., Zhang, D., Xu, Z., Ali, A., Hou, Y.-M., and Karaolis, D. K. R. The Vibrio Pathogenicity Island of Epidemic Vibrio cholerae Forms Precise Extrachromosomal Circular Excision Products. J. Bacteriol. December 2003 185:23 6893-6901 <br>
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3.  Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417. <br>
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4. Krukonis, E.S., Yu, R.R., Dirita, V.J. The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol. 2000 Oct;38(1):67-84 <br>
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5. Herrington, D.A., Hall, R.H., Losonsky, G., Mekalanos, J.J., Taylor, R.K., and Levine, M.M., Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med Oct 1 1988 168:1487-1492 <br>
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6. Peterson, K.M., Mekalanos, J.J., Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization. Infect Immun. 1988 Nov;56(11):2822-9. <br>
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7. Abedon, S.T., Thomas-Abedon, C., Thomas, A., Mazure, H. Bacteriophage prehistory: Is or is not Hankin, 1896, a phage reference? Bacteriophage. 2011 May-Jun; 1(3): 174–178
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Revision as of 00:14, 18 October 2014

“We have witnessed in our days the birth of a new pestilence, which, in the short space of fourteen years, has desolated the fairest portions of the globe, and swept off at least fifty MILLIONS of our race. It has mastered every variety of climate, surmounted every natural barrier, conquered every people. It has not, like the simoon, blasted life and then passed away; the cholera, like the small-pox or plague, takes root in the soil which has once possessed.” (The London and Paris Observer, 11/27/1831)

Introduction

Cholera is a gastrointestinal disease caused by a toxin released by aquatic bacterium Vibrio cholerae. Symptoms of cholera are acute watery diarrhea and vomiting. In severe cases, patients may show signs of severe dehydration, such as sunken eyes, decreased skin turgor, muscle cramping, and decreased blood pressure. If left untreated, severe cases can result in death within a few hours. There are two serotypes of V. cholerae responsible for the global cholera pandemics: O1 and O139. O1 has two biotypes - classical and El Tor. Non-O1 and non-O139 V. cholerae infections can cause similar but with milder symptoms (1). There have been seven recorded cholera pandemics beginning in 1816 and many more outbreaks in that time. Today, cholera infects approximately 3-5 million people every year and between 100,000-120,000 of these cases are fatal (1). Although cases of cholera are rare in developed countries, it continues to be a public health concern in regions with underdeveloped water treatment practices because of its relatively high death rate and persistence in the environment.




Mechanism of Infection


Although most V. cholerae ingested dies in the acidic stomach, the survivors can quickly colonize the small intestine. The symptoms associated with V. cholerae infection are primarily caused by the cholera toxin, but there are several genes that contribute to the colonization and pathogenicity of V. cholerae. Many of the genes associated with the virulence of V. cholerae are located on a pathogenicity island believed to originate from phage (2). We chose several genes identified for their role in pathogenicity and colonization to target for our project.

Cholera Toxin:
The cholera toxin is an oligomeric protein made up 6 subunits: 1 copy of subunit A (ctxA) and 5 copies of subunit B (ctxB). The 5 ctxB subunits form a ring that directly binds to the GM1 ganglioside receptors on the surface of intestinal epithelial cells, causing the cholera toxin to be endocytosed. Once inside, the disfulfide bridges in the toxin are reduced, freeing the A subunit which then activates G proteins inside the cell, causing constitutive production of cAMP. This activates chloride ion channels, causing efflux of chloride ions, followed by water, Na+, and K+ due to osmotic and electrical gradients. This produces the characteristic watery diarrhea and rapid loss of water and electrolytes (3).

Transcriptional regulator toxT:
ToxT is a transcriptional regulator that indirectly activates the cholera toxin operon and the toxin coregulated pilus operon (4). ToxT is activated when ToxR recruits TcpP to the upstream promoter.

Toxin Coregulated Pilus:
The toxin coregulated pili are bundles of fimbriae extending from the poles of V. cholerae that are coregulated with the cholera toxin. They have been shown to play an important role in V. cholerae colonization of the gut (5). Their exact cell surface interactions are unknown.

Accessory Colonization Factor:
The exact mechanism of the accessory colonization factor proteins are unknown, but they have been shown to enhance colonization of V. cholerae in the gut in infant mice (6).

Cholera and the beginnings of phage therapy:


In the late 1800s, English chemist Ernest Hanbury Hankin traveled to India to study the cholera outbreak. He observed bactericidal activity in the water taken from the Ganges river when applied to cultures of V. cholerae. In his notes, he described the agent responsible as being able to pass through a fine porcelain filter but became inactive when boiled. He also noted that people living in regions that obtained their water from the Ganges river were not experiencing a cholera outbreak (7)




References


1. World Health Organization. (2014). Cholera (Fact sheet No. 107). Retrieved from http://www.who.int/mediacentre/factsheets/fs107/en/
2. Rajanna, C., Wang, J., Zhang, D., Xu, Z., Ali, A., Hou, Y.-M., and Karaolis, D. K. R. The Vibrio Pathogenicity Island of Epidemic Vibrio cholerae Forms Precise Extrachromosomal Circular Excision Products. J. Bacteriol. December 2003 185:23 6893-6901
3. Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417.
4. Krukonis, E.S., Yu, R.R., Dirita, V.J. The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol. 2000 Oct;38(1):67-84
5. Herrington, D.A., Hall, R.H., Losonsky, G., Mekalanos, J.J., Taylor, R.K., and Levine, M.M., Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med Oct 1 1988 168:1487-1492
6. Peterson, K.M., Mekalanos, J.J., Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization. Infect Immun. 1988 Nov;56(11):2822-9.
7. Abedon, S.T., Thomas-Abedon, C., Thomas, A., Mazure, H. Bacteriophage prehistory: Is or is not Hankin, 1896, a phage reference? Bacteriophage. 2011 May-Jun; 1(3): 174–178



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