PROJECT SUMMARY: Mycobacterium tuberculosis (Mtb) is the leading cause of death worldwide due to a single pathogen. Mtb is exceptionally reliant on specialized lipids, yet little is known about how lipids and lipid- linked glycans transit across the membranes and aqueous periplasm of mycobacteria. My objective is to reveal how Mtb controls the localization and effects of important lipidic antigens, virulence factors, and cell envelope components. I found that one of the primary mycobacterial lipid transport systems, Mce4, is not restricted to exogenous lipid import as previously thought, but maintains steady-state levels of hundreds of native mycobacterial lipids. I will define how periplasm-spanning Mce channels mediate directional lipid transport in Mtb, using unbiased lipidomic methods, a new reverse micelle separation technique, and collisional mass spectrometry. This will push the field beyond previous studies of selected exogenous lipids, and will identify the roles that Mce channels play in native lipid transport within laboratory and clinical strains of Mtb. I also found that the putative transport lipid mannosyl phosphomycoketide (MPM) is covalently linked to one or more Mtb glycans, and that the gene pks12—which is required for the synthesis of MPM—is important for glycan localization. This suggests that MPM is at the center of an undiscovered lipoglycan transport mechanism. I will uncover this mechanism through chromatography and mass spectrometry-based identification of MPM-linked glycans. Through the same methods along with electron microscopy and glycan labeling, I will determine the effects of pks12 on exported Mtb glycans. I will finish by determining the role that pks12 and glycans play in a growth defect I discovered, thereby providing a mechanistic basis for the likely in vivo essentiality of pks12 in human disease. This proposed investigation of understudied Mtb lipid and lipoglycan transport will address a critical knowledge gap that hinders dev