Team:Paris Bettencourt/Project/Overview

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  Using <em>Escherichia coli</em> 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 <em>E. coli</em> 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.
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  To defeat tuberculosis, we need new biotechnology. Our work adds 4 new tools to the anti-TB medical armamentarium. <b>Detect</b> - a CRISPR-based biosensor delivered by phage and sequence-specific for antibiotic resistance. <b>Target</b> - an <i>E. coli</i> model hosting an essential mycobacterial metabolic pathway that could simplify drug screening. <b>Infiltrate</b> - an <i>E. coli</i> strain capable of entering infected macrophages and lysing mycobacteria. <b>Sabotage</b> - a non-lytic phage that spreads horizontally in a bacterial population and expresses an siRNA to knock down antibiotic resistance.
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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.
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Diagnosing antibiotic resistance can improve and accelerate treatment. We propose a phage-delivered, CRISPR-based system that cuts specific DNA sequences and detects the presence of resistance genes due to the resulting DNA damage that is reported with a color output.
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We have designed a drug screen to specifically target the mycobacterial protein SirA, using <em>Mycobacterium tuberculosis</em>’ close relative <em>Mycobacterium smegmatis</em>’ synthetic sulfite reduction pathway cloned into an <em>E. coli</em> chassis.  SirA is essential for <em>M. tuberculosis</em> 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.
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<i>M. tuberculosis</i> grows slowly and is hard to study in the lab. We have transferred an essential mycobacterial metabolic pathway to <i>E. coli</i>, where it is easy to screen for targeted small-molecule inhibitors.
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A more efficient treatment of TB should contain a drug which rapidly kills mycobacteria and a delivery system for such drug, which could enable the killing of mycobacteria inside the infected macrophages. In our system the rapid drug is Trehalose Dimycolate Hydrolase (TDMH), while the delivery system is LLO carrying strain of E. coli.
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An effective TB therapy must reach mycobacteria inside lung macrophages. In this system, <i>E. coli</i> express listeriolysin O (LLO) to enter the macrophage cytosol and Trehalose Dimycolate Hydrolase (TDMH) to degrade the pathogen's membrane.
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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.  
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Totally drug-resistant TB (TDR-TB) is an established and growing problem. We have created a phage vector that delivers an siRNA capable of sabotaging drug resistance and restoring sensitivity. By reducing the fitness burden of our construct, we can promote its spread in a population.
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        <a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts: what you need to know about TB.</a> 
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Latest revision as of 00:27, 29 October 2013

FIGHT TUBERCULOSIS WITH MODERN WEAPONS

To defeat tuberculosis, we need new biotechnology. Our work adds 4 new tools to the anti-TB medical armamentarium. Detect - a CRISPR-based biosensor delivered by phage and sequence-specific for antibiotic resistance. Target - an E. coli model hosting an essential mycobacterial metabolic pathway that could simplify drug screening. Infiltrate - an E. coli strain capable of entering infected macrophages and lysing mycobacteria. Sabotage - a non-lytic phage that spreads horizontally in a bacterial population and expresses an siRNA to knock down antibiotic resistance.

Detect

Diagnosing antibiotic resistance can improve and accelerate treatment. We propose a phage-delivered, CRISPR-based system that cuts specific DNA sequences and detects the presence of resistance genes due to the resulting DNA damage that is reported with a color output.

Target

M. tuberculosis grows slowly and is hard to study in the lab. We have transferred an essential mycobacterial metabolic pathway to E. coli, where it is easy to screen for targeted small-molecule inhibitors.

Infiltrate

An effective TB therapy must reach mycobacteria inside lung macrophages. In this system, E. coli express listeriolysin O (LLO) to enter the macrophage cytosol and Trehalose Dimycolate Hydrolase (TDMH) to degrade the pathogen's membrane.

Sabotage

Totally drug-resistant TB (TDR-TB) is an established and growing problem. We have created a phage vector that delivers an siRNA capable of sabotaging drug resistance and restoring sensitivity. By reducing the fitness burden of our construct, we can promote its spread in a population.

TB Facts: what you need to know about TB.
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
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