Project Summary / Abstract Mtb utilizes host-derived lipids to promote pathogenesis and this is a defining feature of this intracellular pathogen. During infection Mtb imports and metabolizes host lipids to support pathogenesis by producing: i) energy, ii) central metabolic intermediates, or iii) polyketide virulence lipids. While the metabolic pathways in Mtb that degrade or process lipids are complex and contain redundant enzymes, the bacterial Mce lipid transporters appear to be specific for dedicated lipid substrates. Aim 1 of this work proposes to employ genetic and biochemical approaches to identify and characterize novel gene/proteins required for fatty acid import in Mtb. While it is understood that Mce1 imports fatty acids, the substrate specificity of this transporter is unknown. Therefore, we intend to define substrates and the biochemical basis of Mce1 substrate specificity. Our preliminary studies indicate that Mtb transports fatty acid precursors of immune signaling lipids via Mce1 and we include here studies to evaluate if scavenging of this immune lipid precursors by Mtb impacts the immune response. Aim 2 proposes to identify and characterize protein subunits that are shared by all the Mce transporters and are required for lipid import in Mtb. We have determined that LucA is required for Mce1- and Mce4-mediated transport and LucA stabilizes these transporter complexes. These studies seek to characterize the basis for this transporter stabilization. Similarly, MceG is required for Mce1- and Mce4-mediated transport and we intend to understand how MceG stabilizes and interacts with Mce1. We will use a genetic approach to silence LucA and MceG in Mtb within chronically infected mice and quantify bacterial fitness to determine the therapeutic potential of drugs that potentially block these proteins.