Team:Paris Bettencourt/Project/Overview

Using Escherichia coli as a model organism we are developing and testing approaches that could lead to eradication of tuberculosis. A drug screen aimed for a specific mycobacterial metabolic pathway, a phage sensor for detection of a specific antibiotic resistance, a TB-ception E. coli which could invade macrophages and kill mycobacteria, and finally a Trojan horse sRNA which could silence the production of antibiotic resistance proteins thus making antibiotic treatment more effective.

DETECT

We are developing a sensor to target antibiotic resistances in tuberculosis in order to discover which antibiotic resistance a specific strain carries. Our sensor system consists out of a phagemid with a CRISPR/Cas system and LacZ as a reporter under the control of a pREC stress response promoter. When our CRISPR/Cas system binds to the targeted antibiotic resistance gene, a double strand break generated by Cas9 at this specific target site turns on the expression of our reporter as the promoter gets active at stress that results from double strand breaks. Because our system is on a phagemid, the sensor system will be spread all over the population, which will give a clear color output if the target has been detected.

TARGET

We have designed a drug screen to specifically target the mycobacterial protein SirA, using Mycobacterium tuberculosis’ close relative Mycobacterium smegmatis’ synthetic sulfite reduction pathway cloned into an E. coli chassis. SirA is essential for M. tuberculosis persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced. In addition, a homolog within humans has not been found for SirA demonstrating why SirA has become a promising candidate as a drug target. Currently, there are no drug candidates that are known to specifically inhibit SirA.

INFILTRATE

Bacterial vectors offer a biological route to gene and protein delivery to cells such as macrophages. We want to investigate the potential use of E.coli as a bacterial vector to kill M. tuberculosis using two approaches. In the first one we will introduce an expression system containing a gene for a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall and a gene encoding a protein which forms large pores in the phagosomal membrane where mycobacteria are located, thus releasing the target protein into the cytosol. In the second one we will developed a bacteria-based iRNA delivery system for silencing the coronin-1 gene of the macrophages. Coronin-1 is a protein which surrounds the mycobacterial phagosome allowing mycobacteria to survive within them. Silencing of this gene will result in the enhancement of the lysosome/phagosome fusion.

SABOTAGE

We designed a small regulatory RNA (sRNA) that specifically recognizes the Ribosome Binding Site region of resistance genes’ mRNA, thus preventing the binding of the ribosome and subsequent translation into protein. As such silencing will not immediately impair the bacteria’s fitness, the system behaves as a Trojan horse: from the inside it modifies the defense system of bacteria without being noticed preparing for the real attack by an antibiotic that comes from the outside. As we would like such a system to autonomously enter bacteria we choose to use a natural selfish system that carries DNA: bacteriophages. By cloning our sRNA into a phagemid we expect to be able to silently infect bacteria without submitting them to the burden that would normally be due to the production of phages proteins.