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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]]
-=Use of Engineered Bacteria as Probiotic Therapeutics=
-'''Change into section about gram negative bacteria, state importance of combating gram negative pathogens ( short list) '''
-Probiotics are becoming more and more common.  The basic idea is to purposely inoculate a body with "useful" bacteria that will prevent the growth of pathogenic bacteria, usually by competitive inhibition.  But what if we could make this better?
-We propose to create a probiotic system that could work actively against dangerous enteric bacteria.  This system would consist of harmless gut flora that, when it comes in contact with pathogens, targets the pathogenic organism and injects it with a toxin which is harmless to the host but deadly to the invader.
+[[Image:UWT6SSOverview.png|center|600px|thumb|'''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=
+[[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''.
-'''Include Diagram of T6SS'''
-Type VI Secretion is a recently discovered secretion system that has now been found in most Gram-negative bacterial genomes. Its hallmarks are two proteins, Hcp and VgrG, which form a "tube and spike" structure which is homologous to phage tail spikes. Through secretion and assembly of these two proteins, effectors produced in the cytoplasm of the donor cell can be transported directly into the cytoplasm or periplasm of the recipient.
-The ''Pseudomonas aeruginosa'' genome includes a Type VI Secretion System (T6SS) which has been shown to target prokaryotic cells with a toxin (Hood, 2010). This toxin is not targeted to eukaryotic cells, and has been shown to kill Gram-negative organisms in a cell contact-dependent manner. Using this natural system, transported into a harmless enteric organism, we believe will allow for a new kind of defense against pathogenic enteric bacteria.
-==Tse2/ Tsi2 toxin/antitoxin system==
+<br style="clear: both" />
-'''include simple diagram explaining Tse2=death, Tse2+Tsi2= survival'''
-One of the major proteins excreted into prokaryotic cells  by the T6SS in ''P. aeruginosa''  is the protein Tse2 (Type six exported 2). Tse2 is a protein that is toxic to a wide variety of gram negative bacteria. In  the ''P. aeruginosa'' genome, Tse2 is coexpressed on the same operon with Tsi2. Tsi2 is a short protein that binds with Tse2 until excretion, and acts as an antitoxin, preventing ''P. aeruginosa''from suffering any ill effects due to Tse2 production. By regulating when Tse2 is produced, one could regulate when a T6SS strain of bacteria acts as an antibacterial agent. This would allow for a genetically engineered T6SS containing bacterial strain to be an antibacterial agent much more precisely targeted than traditional chemical antibiotics.
-==Using Type VI Secretion as an Antibacterial Agent==
+=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>
-The T6SS we want to use has several difficulties in its current form. First, it is found in P. aeruginosa which can be pathogenic, as well as not being an enteric bacteria. As our model is anti-enteric, the native system would be unfeasible.
-Secondly, T6SS is highly regulated at both the transcriptional and post-transcriptional level. We want our toxin delivery system to be expressed in either a constitutive or easily inducible manner.
+==References==
-==Tse2/Tsi2 Toxin/Antitoxin Inducible Circuit==
+.  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
-One of the major proteins exported into prokaryotic cells by the T6SS of is the  toxin Tse2 ( type six excreted 2). Tse2 is toxic to a wide range of gram-negative bacteria. In ''P. aeruginosa'', Tse2 is coexpressed on the same operon with Tsi2. Tsi2 binds to Tse2 until Tse2 is excreted by the T6SS, and acts as an antitoxin.  Inducing transcription of the Tse2/Tsi2 operon initiated the ability of the T6SS to cause cell death ( puncturing of the cellular membrane with the T6SS does not cause cell death). By inducing Tse2 production only when a pathogen is present,  T6SS engineered bacteria would be a more finely targeted antibacterial agent than traditional chemical antibiotics.
+<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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