Project Summary. Until recently, sphingolipids within the microbial environment have been attributed to host association, as the conventional wisdom was that bacteria did not contain their own biosynthetic pathways to produce them. Recently, members of our team uncovered a new bacterial sphingolipid pathway involving an acyl carrier protein similar to that of fatty acid synthase (FAS). This new acyl carrier protein, AcpSP, appears to play a role distinct from its FAS progenitor, AcpP, whose primary purpose lies in de novo fatty acid synthesis. Remarkably, AcpSP shares less than 30% sequence identity to AcpP, suggesting a unique activity and interaction landscape than that of AcpP. We have identified AcpSP and serine palmitoyltransferase (SPT) within in many pathogenic bacteria, including Neisseria gonorrhoeae, Escherichia coli (studied herein), Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa, indicating that this pathway may be involved in pathogenesis. In this program, our team applies a suite of chemical and structural biology tools originally developed for the study of AcpP in order to explore the interaction of AcpSP with its partner enzymes. Using a combination of chemical probes and structure-based methods, we will characterize the interface between AcpSP and SPT, the first key step in sphingolipid biosynthesis. To further explore the cryptic interactions between sphingolipid biosynthesis and FAS, we will deploy a systems-wide structural analysis to detail AcpSP interactions with de novo FAS enzymes. This program provides an essential step toward understanding bacterial sphingolipid biosynthesis and its role in pathogenesis and will likely provide important new targets for future drug discovery.