Probiotics in a Gram-Negative Organism

A Model of Probiotic Therapeutics

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.

Type VI Secretion

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

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. aeruginosafrom 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

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.

Toxin/Antitoxin Inducible Circuit

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.