Project Summary Mycobacterium tuberculosis (Mtb) has been the leading cause of infectious disease mortality worldwide for the past few decades with more than 1 million deaths annually. This pathogen has an exceptionally complex cell wall structure, the outer components of which are important for direct interactions with the host, for giving structural support and maintaining a hydrophobic barrier for small molecules and antibiotics. Phthiocerol dimycocerosate (PDIM) is a lipid found in the outer-most layer of the Mtb cell envelope. PDIMs form a permeability barrier, protecting the bacteria from antimicrobial compounds, and are important virulence factors for defense against the host immune system. Paradoxically, despite these important functions, PDIM is dispensable in vitro and its loss confers a growth advantage leading to the selection of spontaneously occurring PDIM negative clones in laboratory cultures. This affects experimental reliability, reproducibly and the interpretation of results. PDIM loss leads to reduced cell wall permeability and virulence attenuation, producing misleading results in infection experiments, drug susceptibility testing and genetic manipulations. To remove PDIM bias from TB research, and to study PDIMs role in pathogenesis and drug tolerance, we need appropriate screening and selection tools. In this grant application we will build on powerful new preliminary results to develop robust PDIM selection, screening and cleanup protocols, determine the propensity of PDIM loss in different Mtb strains and its impact on virulence, and aim to study how PDIM population heterogeneity and propionate metabolism impact infectivity and antibiotic persistence.