The rise of extensively drug-resistant Mycobacterium tuberculosis (Mtb) has important implications for human health worldwide. However, the development of new antibiotics directed against this organism has been hindered by a lack of knowledge about many of the processes critical to Mtb replication and virulence. While the genes essential for viability of Mtb have been identified, the physiological roles of many of their products are not well understood. Amongst these vital enzymes are homologs of VKOR and DsbA, proteins that our lab has shown can catalyze the formation of disulfide bonds in proteins secreted by E.coli. Disulfide bonds provide stability to many proteins secreted by bacteria. Chief amongst these are proteins crucial to bacterial cell wall integrity, motility, and virulence. Decades of research focused on the Dsb pathway of E.coli conducted by our lab and others has led to the development of many important genetic and biochemical tools that have provided for a thorough understanding of this system. Experiments proposed here will apply those tools to Mycobacteria so as to characterize what appears to be a novel disulfide bond-forming pathway. The essentiality of VKOR and DsbA suggests that the disulfide bond forming system of Mtb is a highly vulnerable component of metabolism that might be exploited as a target for new antibiotics. Outlined here is a high-throughput approach for identifying small molecule inhibitors of MtbVKOR in the model organism Mycobacterium smegmatis. Using M.smegmatis ?vkor strains expressing MtbVKOR or E.coli DsbB, we will look for compounds that specifically inhibit VKOR-mediated growth. Such molecules will be important tools for studying disulfide bond formation in vitro, but may also be developed as new antibiotics.
Mycobacterium tuberculosis is one of the most prolific killers in the world, and antibiotic resistance in this organism is on the rise. Crucial to its success as pathogen is its ability to catalyze the formation of disulfide bonds in secreted proteins. Through experiments outlined in this proposal, we seek to characterize the disulfide bond-forming system of Mycobacteria and to identify small molecule inhibitors of this process that might be developed into effective antibiotics.