Discovery of a general mechanism for bacterial cell envelope polymer acylation

Abstract

Bacteria frequently decorate their cell envelope polymers with acyl groups that regulate physiology, enhance virulence, and contribute to antibiotic resistance. How cells tackle the challenge of moving activated acyl groups from the cytoplasm—where they are made—onto extracytoplasmic polymers has been a longstanding question. In the work described in this thesis, I uncover a widespread strategy for bacterial cell envelope polymer acylation in which a membrane-bound O-acyltransferase (MBOAT) protein transfers acyl groups from intracellular thioester donors to the side-chain hydroxyl group of an extracytoplasmic tyrosine residue. The acylated tyrosine then serves as a donor for a separate transferase responsible for moving acyl groups to their next destination, usually the cell envelope polymer itself. In the pathway for D-alanylation of lipoteichoic acids, which is the primary focus of this thesis, the key extracytoplasmic tyrosine is located within a highly conserved six-amino acid motif at the C-terminus of a small membrane protein called DltX that holds the MBOAT protein and the other transferase together in a tripartite complex. For other pathways found in diverse bacteria and some archaea, the tyrosine is present in a similar six-amino motif at the C-terminus of the MBOAT protein itself. This work establishes that the function of the vast majority of bacterial MBOAT proteins is to produce acyl-tyrosine intermediates critical for cell envelope polymer modification, setting the stage for function-informed development of inhibitors that target these proteins. In Chapter 1 of this thesis, I introduce bacterial cell envelope synthesis and modification as an important target for antimicrobial therapeutics. I also describe the history of the study of bacterial MBOAT-based cell envelope polymer acylation systems. In Chapter 2, I investigate the identity of the putative unknown intermediate in the long-studied lipoteichoic acid D-alanylation pathway, and I explain how I developed the hypothesis that a tyrosine-containing motif at the C-terminus of the small protein DltX is the key “missing piece” to that pathway’s mechanistic puzzle. I go on to describe how the work on DltX led me to propose that the specific mechanism I discovered is widespread across different polymer acylation pathways in diverse bacteria. Next, in Chapter 3, I collaborate to use in vitro biochemistry and structural biology approaches to provide solid experimental evidence for my proposed mechanism, again focusing primarily on the protein machinery responsible for lipoteichoic acid D-alanylation. Finally, I conclude in Chapter 4 with a discussion of interesting avenues for future exploration related to both the MBOAT-based acyl transfer pathways I explored in my thesis work and also acyl transfer pathways that use a different class of membrane-bound acyltransferases which I became interested in over the course of my studies.