Difference between revisions of "Team:UMass-Dartmouth/ourproject"
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<em>Helicobacter pylori</em>, a Gram-negative bacterium, is classified as a class I carcinogen. An <em>H. pylori</em> infection causes gastric duodenal ulcers, gastric lymphoma, and chronic gastritis, which is a precursor to gastric carcinoma. <em>H. pylori</em> infects about half of the human population, with high prevalence in developing countries. <em>H. pylori</em>-induced gastric inflammation usually does not cause symptoms in the infected person, meaning the prevention of gastric ulcers is virtually impossible. The current method of treating an infection of <em>H. pylori</em> is with the use of antibiotics, including Protein Pump Inhibitor (PPI), amoxicillin, clarithromycin, and metronidazole2. The rising complication with using antibiotics, however, is the propagation of antibiotic resistant bacteria, as well as the eradication of predominantly beneficial gut bacteria. In the absence of antibiotic therapy, <em>H. pylori</em> can live in the stomach for decades, or even an entire lifetime. What is most notable about <em>H. pylori</em> is its ability to secrete products that are linked with its ability to evade the host's innate immune system1. There is a great need for a new approach of eliminating <em>H. pylori</em> from the body. <br><br> | <em>Helicobacter pylori</em>, a Gram-negative bacterium, is classified as a class I carcinogen. An <em>H. pylori</em> infection causes gastric duodenal ulcers, gastric lymphoma, and chronic gastritis, which is a precursor to gastric carcinoma. <em>H. pylori</em> infects about half of the human population, with high prevalence in developing countries. <em>H. pylori</em>-induced gastric inflammation usually does not cause symptoms in the infected person, meaning the prevention of gastric ulcers is virtually impossible. The current method of treating an infection of <em>H. pylori</em> is with the use of antibiotics, including Protein Pump Inhibitor (PPI), amoxicillin, clarithromycin, and metronidazole2. The rising complication with using antibiotics, however, is the propagation of antibiotic resistant bacteria, as well as the eradication of predominantly beneficial gut bacteria. In the absence of antibiotic therapy, <em>H. pylori</em> can live in the stomach for decades, or even an entire lifetime. What is most notable about <em>H. pylori</em> is its ability to secrete products that are linked with its ability to evade the host's innate immune system1. There is a great need for a new approach of eliminating <em>H. pylori</em> from the body. <br><br> | ||
Urease is an extracellular enzyme, produced by <em>H. pylori</em>, which allows the microenvironment surrounding <em>H. pylori</em> to be buffered against the harsh acidity of the mammalian stomach via the cleavage of urea to ammonia and carbon dioxide by urease.3 Synthetic peptides have been produced in studies utilizing phage display, which demonstrate a non-competitive interaction with urease by a unique 6mer peptide. It has been suggested that this particular peptide (YDFYWW) interacts with urease via hydrophilic/hydrophobic interactions, causing a spatial modification to the enzyme’s catalytic core, thus inhibiting urea cleavage.4 Given this information, and the requirement of <em>H. pylori</em> to have the ability to buffer its microenvironment via urea cleavage by urease, we hypothesize that an environment physiologically similar to the mammalian stomach and appropriately saturated with this particular 6mer peptide could potentially inhibit the growth of <em>H. pylori</em>, or any other non-acidophile, as the pH of the environment would be sufficient to inhibit growth.<br><br> | Urease is an extracellular enzyme, produced by <em>H. pylori</em>, which allows the microenvironment surrounding <em>H. pylori</em> to be buffered against the harsh acidity of the mammalian stomach via the cleavage of urea to ammonia and carbon dioxide by urease.3 Synthetic peptides have been produced in studies utilizing phage display, which demonstrate a non-competitive interaction with urease by a unique 6mer peptide. It has been suggested that this particular peptide (YDFYWW) interacts with urease via hydrophilic/hydrophobic interactions, causing a spatial modification to the enzyme’s catalytic core, thus inhibiting urea cleavage.4 Given this information, and the requirement of <em>H. pylori</em> to have the ability to buffer its microenvironment via urea cleavage by urease, we hypothesize that an environment physiologically similar to the mammalian stomach and appropriately saturated with this particular 6mer peptide could potentially inhibit the growth of <em>H. pylori</em>, or any other non-acidophile, as the pH of the environment would be sufficient to inhibit growth.<br><br> | ||
| − | Additionally, studies into the regulation of urease production and acid-tolerance of <em>H. pylori</em> have shown that, in acidic environments, a promoter region directly upstream of the <em>ureA</em> and <em>ureB</em> genes, encoding the <em>ureA</em> and <em>ureB</em> subunits of urease, is regulated by the well-studied ArsR regulatory system, which is present in Escherichia coli.5 The utilization of a pH promoter, | + | Additionally, studies into the regulation of urease production and acid-tolerance of <em>H. pylori</em> have shown that, in acidic environments, a promoter region directly upstream of the <em>ureA</em> and <em>ureB</em> genes, encoding the <em>ureA</em> and <em>ureB</em> subunits of urease, is regulated by the well-studied ArsR regulatory system, which is present in <em>Escherichia coli</em>.<sup><em>5</sup></em> The utilization of a pH promoter, P<sub><em>ureA</sub></em>, in a biological system designed to minimize antibiotic “noise”, in the form of molecules intended to inhibit microbiological growth, appears to be desirable.<br><br> |
Therefore, it is our intention to design a biological system to regulate production of a 6mer peptide in host organism <em>E. coli</em>, and to produce the 6mer when in an acidic environment, as regulated naturally by P<sub><em>ureA</sub></em> in <em>H. pylori</em> as apart of its innate metabolic regulatory machinery. Additionally, we will couple the 6mer peptide to a Twin-Arginine Translocation tag sequence, in order for the 6mer to be recognized by the innate TAT peptide secretion mechanism in <em>E. coli</em>. With a construct containing these genetic components, and additional subsets of these components coupled with standard laboratory reporter genes, we will be able to test and quantify the efficacy of this novel, organism-specific, anatomically specific anti-H. pylori biological treatment system.<br><br><br> | Therefore, it is our intention to design a biological system to regulate production of a 6mer peptide in host organism <em>E. coli</em>, and to produce the 6mer when in an acidic environment, as regulated naturally by P<sub><em>ureA</sub></em> in <em>H. pylori</em> as apart of its innate metabolic regulatory machinery. Additionally, we will couple the 6mer peptide to a Twin-Arginine Translocation tag sequence, in order for the 6mer to be recognized by the innate TAT peptide secretion mechanism in <em>E. coli</em>. With a construct containing these genetic components, and additional subsets of these components coupled with standard laboratory reporter genes, we will be able to test and quantify the efficacy of this novel, organism-specific, anatomically specific anti-H. pylori biological treatment system.<br><br><br> | ||
References<br> | References<br> | ||
| − | 1. Scott Algood H, Cover T. | + | 1. Scott Algood H, Cover T. “<em>Helicobacter pylori</em> Persistence: an Overview of Interactions between <em>H. pylori</em> and Host Immune Defenses.” Clinical Microbial Review. 2006; 19(4): 597-613. <br> |
| − | 2. Zou Q, Wei W. “Phage Therapy: Promising For H. pylori Infection.” Clinical Microbiology. 2013; 2: 112. <br> | + | 2. Zou Q, Wei W. “Phage Therapy: Promising For <em>H. pylori</em> Infection.” Clinical Microbiology. 2013; 2: 112. <br> |
| − | 3. Blanke S, Ye D. Alternative Mechanisms of Protein Release. In: Mobley H, Mendz G, Hazell S, editors. Helicobacter pylori: Physiology and Genetics Washington DC: ASM Press; 2001. Chapter 20.<br> | + | 3. Blanke S, Ye D. Alternative Mechanisms of Protein Release. In: Mobley H, Mendz G, Hazell S, editors.<em> Helicobacter pylori</em>: Physiology and Genetics Washington DC: ASM Press; 2001. Chapter 20.<br> |
| − | 4. Houimel M, Mach J, Corthesy-Theulaz I, Corthesy B, Fisch I. New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries. Eur J Biochem. 1999; 262:774-780.<br> | + | 4. Houimel M, Mach J, Corthesy-Theulaz I, Corthesy B, Fisch I. New inhibitors of <em>Helicobacter pylori</em> urease holoenzyme selected from phage-displayed peptide libraries. Eur J Biochem. 1999; 262:774-780.<br> |
| − | 5. Pflock M, Kennard S, Delany I, Scarlato V, Beier D. Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of Helicobacter pylori. Infect Immun. 2005; 73(10): 6437-6445.<br> | + | 5. Pflock M, Kennard S, Delany I, Scarlato V, Beier D. Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of <em>Helicobacter pylori</em>. Infect Immun. 2005; 73(10): 6437-6445.<br> |
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