Mycobacterium tuberculosis is a tough bug to kill. The sterilization of a M. tuberculosis infection ina patient with active tuberculosis requires a minimum of 6 months of treatment with 3-4 drugs takendaily. This tenacity is unparalleled in any other human bacterial pathogen. M. tuberculosis hasevolved strategies to survive in the face of drugs and the host immune responses. The currentdogma in the field suggests that survival is the result of the bacteria's ability to enter into a drugtolerant state, where it is non-replicative and dormant, but clearly viable. If drugs are stopped earlyor the immune system is compromised, dormant bacteria can revive and go on to active disease.As part of the program project, we are dedicating our time and resources to find the 'Achilles' heel'of the dormant bacteria using mycobacterial genetics. For this proposal, we have generated a highthroughput methodology of specialized transduction, which can be used to disrupt any nonessentialgene. In addition, we have used conditional expression and specialized transduction todevelop CESTET, a method to prove essentiality and to study a mycobacterial cell's fate upondepletion of the gene product. In vitro and in vivo assays have been developed to screen foressential genes and persistence factors. The group has a proven track record of successfullysolving structures having solved 70% of the Mtb structures in the PDB, and we will build on thissuccess to solve the structures of the new essential or persistence targets. Using the structuralinformation, virtual ligand screens will be performed to identify new inhibitors. Thus, the goal of thisProject is to validate targets, solve their three dimensional structures, and propose novel inhibitorsfor further drug development to radically shorten the time to treat TB.
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