Mechanistic Characterization of Acyltransferases that Modify the Bacterial Cell Envelope

Abstract

Bacteria are surrounded by a complex macromolecular structure called the cell envelope, which is essential for survival. The cell envelope structure varies depending on the bacterial species; however, there are conserved features present in all cell envelopes, like peptidoglycan. Because of its conservation and essentiality, peptidoglycan has long been the target for antibiotics, such as beta-lactams. Unfortunately, bacteria have developed resistance to these antibiotics, and their broad-spectrum activity is considered detrimental to commensal bacteria. With this in mind, it is critical to explore new antibiotic targets within cell envelope biosynthesis. Furthermore, it would be beneficial for these new drugs to have more specific activity, which could be achieved by targeting features that are unique to specific bacterial clades. This work explores the potential of cell envelope-modifying acyltransferases as new targets for antibiotic development. We focus on understanding the basic mechanism of these acyltransferases to lay the groundwork for future drug development campaigns. In this thesis, we determined the mechanism for teichoic acid D-alanylation in Gram-positive bacteria. We showed that the small membrane protein DltX uses a conserved C-terminal hexapeptide motif to help shuttle D-alanine across the membrane. This occurs through a cascade of covalent intermediates that promote thermodynamically favorable transfer. Additionally, we found that cell envelope modifying pathways that lack a DltX homolog use a similar hexapeptide motif; however, it is fused directly to a polytopic membrane protein akin to DltB. We then used cryoEM to structurally characterize the DltXBCD complex to further probe the mechanism. We developed a robust pipeline to determine this structure and applied it to a disulfide mutant that traps DltX in a DltB-bound state. Finally, we turned our attention to an acetyltransferase, TmaT, important for mycomembrane biogenesis in Mycobacteriales. We found that this acetyltransferase interacts with an arabinosyltransferase, AftD, involved in arabinogalactan biosynthesis. We also discovered that the acetyltransferase and the arabinosyltransferase regulate each other’s activities. This work led to a model where the TmaT/AftD complex serves to coordinate cell envelope assembly in Mycobacteriales. Taken together, this work uncovered fundamental mechanistic information about a range of cell envelope-modifying acyltransferases that will serve as a basis for future antibiotic development efforts.