Type Six Secretion System

Using a fosmid to transfer the T6SS genes into E. coli

The T6SS is comprised of 23 genes on several operons. Capturing and moving these genes via standard restriction digest cloning was determined to be impractical. We discovered that the sequence of the P. aeruginosa strain (pao1) we were using was solved using fosmids (essentially large plasmids). We were able to locate these fosmids and were very excited to find that one of the fosmids contained all of the necessary T6SS genes. The fosmid was successfully transfered into E.Coli. It was not clear, however, whether or not the genes would be expressed in E.coli. In order to verify expression of the T6SS genes we performed a western blot using antibodies against Fha1, a critical protein of the secretion system, and a reporter for its activity.

Testing the native Pseudomonas aeriginosa promoter in E. coli

The fosmid was tranfered into E. coli by recombineering, and the cells were pelleted and then a western blot was preformed for Hcp, one of the proteins of the "shaft" of the T6SS. This was done in order to determine if the genes in the T6SS were being expressed from the native P. aeriginosa promoter. The western blot showed no bands (shown below), indicating that E. coli was not transcribing from the native P. aeriginosa promoter. It was decided that the promoter would need to be changed to a bidirectional promoter compatible with E. coli. It was decided that the native promoter would need to be switched to a bidirectional T7 promoter. T7 promoters are well characterized, and known to be highly robust. This makes them relatively easy to work with.

Tse2/Tsi2 Toxin/ Antitoxin System

Regulating the Toxin and Antitoxin

The purpose of the Tse2/Tsi2, toxin/antitoxin, circuit is to regulate the probiotic in a way that allows it to be more effective at killing gram-negative bacteria. If our probiotic system were constantly producing Tse2 and killing gram-negative cells there would be an increased chance of the E.Coli evolving and developing resistance to the tse2. The helpful gut flora would also be adversely affected. It would therefore be preferential to be able to activate the T6SS/Tse2/Tsi2 system only when a pathogen is present. While it is possible to regulate this probiotic system by regulating the expression of the T6SS, the response time of such a system would be limited by the complex 23 protein nature of the T6SS. Consequentially it would be most effective to be able to activate our probiotic by inducing Tse2 expression only in the presence of a pathogen.

Design of our Inducable Tse2/Tsi2 system

Activating Tse2 production when a pathogen is present would require a promoter inducable by some molecular stimulis unique to a specific pathogen. In addition, the expression of Tsi2 would need to be constituitve, or induced by the same stimulis that induces Tse2 expression. 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 membrane permeable molecule( hereafter refered to as HSL). HSL binds to LuxR, changing the conformation of LuxR, which then induces the pLux promoter. Since V. fischeri also produces HSL, 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 HSL), 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. This system could easily be changed to target a wide range of gram (-) pathogens by just changing the regulation of the Tse2/Tsi2 locus.

Diagram of the Tse2/Tsi2 HSL inducible circuit

The Tse2/Tsi2 toxin/antitoxin system has a relativly simple circuit design. Tse2 and Tsi2 are present in one operon ( as in Pseudomonas aeruginosa) regulated by the pLux promoter. The LuxR transcriptional factor is constituitively expressed, as no tetR is present to repress the production of LuxR. When HSl is present, it binds to LuxR, resulting the in the induction of Tse2 and Tsi2 production. The pTet, LuxR, and pLux region of the construct is present in part [http://partsregistry.org/Part:BBa_F2620 F2620], easing the contruction of the circuit.