The Reconstitution of Mature Cell Wall Polymer and the Roles of LCP Ligases

Abstract

The Gram-positive cell wall is a massive macromolecule composed primarily of two polymers: peptidoglycan (PG) and cell wall glycopolymers. Wall teichoic acid (WTA) is the most abundant cell wall glycopolymer across Gram-positive bacterial species and typically essential for survival. WTA plays important roles in bacterial pathogenesis and is required for beta-lactam resistance in methicillin-resistant Staphylococcus aureus (S. aureus). Detailed characterizations of every step of WTA biosynthesis have been reported, except the final one: the transfer of WTA to peptidoglycan. Here, we develop chemoenzymatic tools to obtain well-defined WTA and peptidoglycan precursors and use these molecules to establish that LCP proteins are the missing cell wall glycopolymer ligases. In a series of chemical-genetic experiments we then reveal that one of three LCP proteins in S. aureus plays a dominant role in WTA biosynthesis. Next, we use these WTA precursors to obtain high-resolution crystal structures of an LCP domain bound to a WTA substrate. With the structural and biochemical information learned from these studies we propose an unusual mechanism for the transfer of WTA to peptidoglycan. Lastly, we prepare discrete, complex PG substrates to address the question of when WTA modifications occur in relation to PG biosynthesis. We provide a definitive conclusion that WTA modifications must take place prior to PG crosslinking. In summary, this work reports biochemical, structural, and genetic characterization of the LCP enzymes, develops novel chemical tools to interrogate their activity, and uses these methods to answer important questions about the roles of LCP enzymes in cells. We hope these studies will provide the basis for characterizing and developing inhibitors against LCP proteins as a new antibacterial target.