Probiotics in a Gram-Negative Organism

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it is becoming more and more apparent that the old generation of small molecule antibiotics are becoming more and more out of date.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/Tse2 System

Our plan is to turn the Type VI Secretion System (T6SS)/Tse2 from Pseudomonas aeruginosa into a targeted antibiotic suitable for use in E. coli. The T6SS is a 22 protein complex found in many gram-negative species ( including P. aeruginosa, but not E. coli). The T6SS acts much like a spear, physically puncturing the cell membrane of the target cell and providing a channel through which proteins can be secreted. In P. aeruginosa, one of the major substrates of the T6SS is the toxic protein Tse2 (Type Six Excreted 2). Tse2 has been previously shown to be toxic to a wide range of prokaryotic cells, including E. coli( Hood, 2010). Normally, Tse2 forms a complex with Tsi2 ( Type Six Immunity 2), a protein co-expressed with Tse2 that acts as an antitoxin. Before secretion of Tse2, Tsi2 dissociates from Tse2. Tse2 is then secreted into the target cell by the T6SS, killing the target cell.

Regulating the Probiotic

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.

Type VI Secretion