Team:Paris Bettencourt/Project/Target

   We have designed a drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA, using M.Smegmatis’ synthetic sulfite reduction pathway cloned into an E. coli chassi. 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. We plan to achieve 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.

   We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth was restored with the mycobacteria pathway resulting in a f value of 0.9105.

   We designed a synthetic M.smegmatis derived sulfite reduction pathway containing SirA; the sulfite reductase, and two supporting genes that are required for its function in E.coli; FdxA and FprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The sequence used as a template was taken from literature in which these genes were previously expressed in E. coli for purification and in vitro characterization of the enzyme kinetics with restriction sites removed and then codon optimized for expression in E. coli. These genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genes before being transformed into our knock out mutant strains of E. coli.