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New and Effective Killer of Gram-Negative Bacteria

Probiotic Delivers a Toxin Specifically to Pathogens

It is becoming increasingly apparent that the utilization of small molecule antibiotics is becoming out-dated and less effective as time passes. Pathogens are continually evolving resistance to currently used antibiotics, and the discovery or modification of antibiotic treatments are slow to match this resistance, resulting in antibiotics that are less effective if not completely futile. Another problem with today's antibiotics is that they do not differentiate between pathogenic and non-pathogenic bacteria - they kill both. There are many common diseases caused by Gram-negative bacteria which inhabit the human gut, such as, but not limited to: cholera (Vibrio cholerae), dysentery (Shigella), and food poisoning (Salmonella Typhimurium). When these diseases are often treated with antibiotics, but in addition to killing pathogens (unless resistance has been obtained), these antibiotics also kill other bacteria naturally found in the gut, causing a diverse range of side effects. Many of the bacteria in the gut are advantageous: they aid the body in digestion, production of vitamins such as vitamin K, and competitively exclude pathogenic invaders. Both problems of resistance and non-specificity could be lessened by an antibacterial agent that selectively kills pathogenic bacteria. This would limit the chance of the development of resistance by limiting exposure, and would drastically decrease damage to the helpful gut flora. The goal of this project is to turn the natural bacterial weapon to kill other bacteria, the Type VI secretion system / toxin injection system, into a probiotic that specifically targets a pathogenic Gram-negative bacteria and is activated only when that specific bacterium is present.

The Type VI Secretion System

T6SS Creates a Channel that Secretes Proteins from Host Cell into Target Cell

The Type VI Secretion System (T6SS) is an injection mechanism found in many Gram-negative bacteria (such as Pseudomonas aeruginosa) [2]. The T6SS acts much like a spear by physically puncturing the cell membrane. After puncturing, the "spear" provides a channel through which proteins, such as toxins, can be inserted into the punctured cell. We wanted to put this system into a model bacterium and use it to target Gram-negative pathogens. The T6SS is physically incapable of puncturing the cell membrane of Gram-positive bacteria, or the cell membrane of eukaryotic cells. This makes the T6SS a perfect candidate for a probiotic because it is unable to harm either human cells or helpful Gram-positive bacteria. This system has been characterized in the lab of [http://depts.washington.edu/micro/faculty/mougous.htm Dr. Joseph Mougous]. E. coli is one bacterial species that does not contain the T6SS. Since E. coli is amenable to genetic changes, is easy to work with in the lab, and is a common Gram-negative bacteria found naturally in the gut, it seemed only logical to move the T6SS into E. coli.

Toxin Antitoxin System

In Pseudomonas aeruginosa, one of the major proteins secreted by the T6SS into target Gram-negative bacteria is the toxic protein Tse2 [1]. Tse2 has been shown to cause cell death when expressed internally in a wide variety of prokaryotic and eukaryotic cells. Normally, Tse2 (yellow in the figure below) forms a complex with Tsi2 (green in the figure below), which is a protein coexpressed with Tse2 that serves as an antitoxin. Cells that express Tse2 and Tsi2 are immune to the toxic effects of Tse2. Before Tse2 is secreted into the target cell, Tsi2 and Tse2 become unbound and Tse2 is secreted into the target cell by the T6SS. The presence of Tse2 in the absence of Tsi2 causes the target cell to die. By controlling the expression of Tse2 and Tsi2 production and having it turn on only when a pathogen is present, we have an antibiotic that is fairly selective only for the pathogen. This helps combat both problems of specificity and resistance.

Schematic of our probiotic system ( adapted from [1] )

References

1. Hood RD, Singh P, Hsu F, Güvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL, Li M, Schwarz S, Wang WY, Merz AJ, Goodlett DR, Mougous JD. [http://www.cell.com/cell-host-microbe/abstract/S1931-3128%2809%2900417-X#Summary A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria]. Cell Host Microbe. 2010 Jan 21;7(1):25-37. PMID: 20114026
2. Filloux, Alain, Hachani, Abderrahman, Bleves, Sophie [http://mic.sgmjournals.org/cgi/content/full/154/6/1570#SEC7 The bacterial type VI secretion machine: yet another player for protein transport across membranes] Microbiology 2008 154: 1570-1583

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-=Probiotics in a Gram-Negative Organism=
+=New and Effective Killer of Gram-Negative Bacteria=
-[[Image: Washington Get away.jpg]]
-As time passes, it is becoming more and more apparent that the old generation of small molecule antibiotics are becoming more and more out of date.  Increasingly, pathogens are evolving resistance  to the currently used antibiotics, but new  antibiotics are being discovered slower than pathogens are evolving antibiotic resistance. This is neccesitating the use of less effective antibiotics, as pathogens are often resistant to stronger, more effective antibiotics. Another problem with small molecule antibiotics is that they indescriminatlhy kill bacteria, wheither or not the bacteria are actually pathenogenic. This causes problems, especially when the antibiotic targets bacterial pathogens in the gut( most of which are gram(-), including ''Vibrio cholerae''(cholera), ''Shigella'' ( dysentary), and ''Salmonella'' ( food poisoning) . Most of the bacteria in the gut is actually helpful, aiding the body in digestion, production of vitamins such as vitamin K, and competitively excluding pathogenic invaders. Both of these problems could be lessened by an antibacterial agent that only kills  bacteria when a pathogen is present. This would limit the chance of the developement of resistance by limiting exposure, and  would limit damage to the helpful gut flora.  The goal of this project is to turn the Tse2/Type VI secretion system  toxin/injection system  into a probiotic anti-gram negative agent that is active only when a specific gram (-) pathogen is present.
-=The T6SS System=
+[[Image:UWT6SSOverview.png|center|600px|thumb|'''Probiotic Delivers a Toxin Specifically to Pathogens''']]
-The Type 6 secretion system is an injection mechagnism found in many gram (-) bacteria, including ''Pseudomonas aeruginosa'', but not ''E. coli''. The T6SS acts much like a spear, physically puncturing the cell membrane and providing a channel through which proteins can be inserted into the punctured cell.  The T6SS is physically incapable of puncturing gram (+) cell walls, or the cell membrane of  eukaryotic cells. Therefore, our probiotic would be unable to harm either human cells, or helpful gram (+) bacteria.
+It is becoming increasingly apparent that the utilization of small molecule antibiotics is becoming out-dated and less effective as time passes.  Pathogens are continually evolving resistance to currently used antibiotics, and the discovery or modification of antibiotic treatments are slow to match this resistance, resulting in antibiotics that are less effective if not completely futile.  Another problem with today's antibiotics is that they do not differentiate between pathogenic and non-pathogenic bacteria - they kill both.  There are many common diseases caused by Gram-negative bacteria which inhabit the human gut, such as, but not limited to: cholera (''Vibrio cholerae''), dysentery (''Shigella''), and food poisoning (''Salmonella Typhimurium'').  When these diseases are often treated with antibiotics, but in addition to killing pathogens (unless resistance has been obtained), these antibiotics also kill other bacteria naturally found in the gut, causing a diverse range of side effects.  Many of the bacteria in the gut are advantageous: they aid the body in digestion, production of vitamins such as vitamin K, and competitively exclude pathogenic invaders.  Both problems of resistance and non-specificity could be lessened by an antibacterial agent that selectively kills pathogenic bacteria.  This would limit the chance of the development of resistance by limiting exposure, and would drastically decrease damage to the helpful gut flora.  The goal of this project is to turn the natural bacterial weapon to kill other bacteria, the Type VI secretion system / toxin injection system, into a probiotic that specifically targets a pathogenic Gram-negative bacteria and is activated only when that specific bacterium is present.
-=Regulating the Probiotic=
+=The Type VI Secretion System=
-One of the goals of this project is to regulate the Tse2/Tsi2 locus so that  the probiotic only kills cells when a pathogen is present. This would require a promoter that is induced by an excreted molecule unique to a specific pathogenic gram-negative species. As a proof of concept, this project uses the LuxR-pLux transcription factor- promoter system from ''Vibrio fischeri''  to regulate expression of the Tse2-Tsi2 locus. ''V. fischeri'' excretes 3OC6HSL, a small cell membrane permeable molecule. 3OC6HSL binds to LuxR, changing the conformation of LuxR, resulting in expression from the pLux promoter. Since ''V. fischeri'' also produces 3OC6HSL,  expression from the pLux promoter is linked to cell density. This is referred to as quorum sensing. Quorum sensing is found in many pathogenic species, making the use of the pLux-LuxR system a good proof-of concept. When our probiotic detects a gram-negative pathogen-specific molecule (  modeled by 3OC6HSL), transcription is induced from an inducible promoter( modeled by pLux). This leads to expression of Tse2 ( a toxic protein) and Tsi2 ( its antitoxin). The Type VI Secretion System then attacks the pathogen, puncturing the cell wall. Tse2 is then secreted into the gram negative pathogen, killing the pathogen.
+[[Image:UWT6SSSchematic.png|300px|right|thumb|'''T6SS Creates a Channel that Secretes Proteins from Host Cell into Target Cell''']]
-==Type VI Secretion==
+The Type VI Secretion System (T6SS) is an injection mechanism found in many Gram-negative bacteria (such as ''Pseudomonas aeruginosa'')[[#References | [2]]].  The T6SS acts much like a spear by physically puncturing the cell membrane.  After puncturing, the "spear" provides a channel through which proteins, such as toxins, can be inserted into the punctured cell.  We wanted to put this system into a model bacterium and use it to target Gram-negative pathogens.  The T6SS is physically incapable of puncturing the cell membrane of Gram-positive bacteria, or the cell membrane of eukaryotic cells.  This makes the T6SS a perfect candidate for a probiotic because it is unable to harm either human cells or helpful Gram-positive bacteria.  This system has been characterized in the lab of [http://depts.washington.edu/micro/faculty/mougous.htm Dr. Joseph Mougous].  ''E. coli'' is one bacterial species that does not contain the T6SS.  Since ''E. coli'' is amenable to genetic changes, is easy to work with in the lab, and is a common Gram-negative bacteria found naturally in the gut, it seemed only logical to move the T6SS into ''E. coli''.
-[[Image:Washington_T6SS_Intro.jpg|805px]]
+<br style="clear: both" />
+=Toxin  Antitoxin System=
+In ''Pseudomonas aeruginosa'', one of the major proteins secreted by the T6SS into target Gram-negative bacteria is the toxic protein Tse2 [[#References | [1]]]. Tse2 has been shown to cause cell death when expressed internally in a wide variety of prokaryotic and eukaryotic cells. Normally, Tse2 (yellow in the figure below) forms a complex with Tsi2 (green in the figure below), which is a protein coexpressed with Tse2 that serves as an antitoxin.  Cells that express Tse2 and Tsi2 are immune to the toxic effects of Tse2.  Before Tse2 is secreted into the target cell, Tsi2 and Tse2 become unbound and Tse2 is secreted into the target cell by the T6SS.  The presence of Tse2 in the absence of Tsi2 causes the target cell to die.  By controlling the expression of Tse2 and Tsi2 production and having it turn on only when a pathogen is present, we have an antibiotic that is fairly selective only for the pathogen.  This helps combat both problems of specificity and resistance.
+<br>
+[[Image:washington_hoodpic.jpg|500px|thumb|center| Schematic of our probiotic system ( adapted from[[#References | [1]]] )]]
+<br>
+==References==
+.  Hood RD, Singh P, Hsu F, Güvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL, Li M, Schwarz S, Wang WY, Merz AJ, Goodlett DR, Mougous JD. [http://www.cell.com/cell-host-microbe/abstract/S1931-3128%2809%2900417-X#Summary A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria]. Cell Host Microbe. 2010 Jan 21;7(1):25-37. PMID: 20114026
+<br>
+. Filloux, Alain, Hachani, Abderrahman, Bleves, Sophie
+[http://mic.sgmjournals.org/cgi/content/full/154/6/1570#SEC7 The bacterial type VI secretion machine: yet another player for protein transport across membranes]
+Microbiology 2008 154: 1570-1583
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