Project 2 - Metabolic determinants of barrier function in rifampin-sensitive and -resistant Mtb Project Leader: Kyu Rhee Co-investigator: Valerie Mizrahi Collaborating investigators: Jeremy Rock (Core D), D. Branch Moody (Core B) ABSTRACT The overarching goal of this project is to elucidate specific cell envelope structure-activity relationships mediating barrier function. A key and quite literal barrier to safer and simpler drugs and treatments for TB is its highly unusual cell envelope. Improved knowledge of the barrier function of the Mtb envelope thus represents a potential blueprint to developing better safer drugs. Unlike most bacteria, the Mycobacterium tuberculosis (Mtb) envelope consists in a unique multilayered structure whose physico-chemical properties are widely believed to mediate an intrinsic mechanism of antibiotic resistance. Using newly developed whole organism chemical profiling and genome scale CRISPRi technologies, we challenge the longstanding view of Mtb cell wall as a static and impermeant chemical barrier. Preliminary studies indicate that the Mtb cell envelope is chemically selective, and its barrier activity is not a simple product of its bulk physico-chemical properties. These data further show that both its composition and barrier function are dynamically regulated. This project specifically focuses on Mtb barrier function through the therapeutic lens of the frontline TB drug rifampicin. We focus on rifampicin because of its unique treatment shortening and sterilizing activities against drug-sensitive TB, and its defining role in the biology of drug-resistant TB. In addition, because rifampicin has a single, well understood target of action, the b subunit of RNA polymerase, which is located in the cytosol, it serves as a potential model that could elucidate general principles that apply to any drug with cytosolic targets.