Team:Paris Bettencourt/Project/Drug Screening

Overview

We have designed a drug screen to specifically target the mycobacterial protein SirA, using M.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. Conventional drug screens involve measuring growth inhibition of batch cultures of M. tuberculosis. However, this method does not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. Whilst a batch-culture model for persistence exists, there are numerous technical difficulties, such as: maintaining the nutrient poor, microaerobic and oxidative conditions required for high-throughput applications. Our model overcomes the problem of the long doubling time of M. tuberculosis and informs us of specific inhibition of the sulfite reduction pathway.

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We achieved this by comparing a drug screen of our E. coli construct against its parent E. coli prior to the deletion of the native sulfite reduction pathway. Any drug candidates that have activity against both the wild-type E. coli and our construct are non-specific inhibitors of E. coli growth. However, any drug candidates that inhibit only the growth of our E. coli construct will have activity specific for our cloned pathway. In order for our construct to be functional, we started with a strain of E. coli which has the sulfite reduction pathway deleted: BL21 (DE3) ΔCysI Δfpr ΔydbK. CysI is responsible for sulfite reduction in E. coli, while fpr and ydbK are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to E. coli in which it is NADPH dependant. To ensure that these two genes do not interfere with our system, we deleted these genes as well. Furthermore, due to the differing cofactors of sulfite reduction between Mycobacteria and Escherichia, we chose to clone two additional genes from Mycobacteria into E. coli; FdxA and FprA. These genes, respectively, are required for production of the ferredoxin cofactor and the NADPH-ferredoxin reductase and are required for SirA to function within E. coli. Upon successful cloning of the three genes into our E. coli deletion strain, we confirmed that all three genes are required for growth on minimum media. We have also characterized the level of induction required by IPTG for effective growth. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in M. tuberculosis and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in E. coli.