Some of the most successful antibiotics in the clinic inhibit bacterial cell wall assembly. However, the continued emergence of multidrug-resistant bacteria is rapidly eroding the efficacy of these drugs - like all other antibiotics - and is a significant threat to human health. New treatment options are urgently needed that hinder bacterial viability, growth, and/or pathogenesis. Wall teichoic acids (WTAs) are integral components of Gram-positive bacterial cell walls and are critical to physiology and pathogenesis. WTA assembly is a bona fide drug target in several pathogens like Staphylococcus aureus. Many whole-cell screens have been performed to find small molecule inhibitors of this process. These efforts require a deep understanding of WTA assembly to help identify, characterize, and exploit high potential targets and therapeutic strategies. Challenges involved in the isolation of authentic substrates have hampered detailed biochemical study of WTA biosynthesis. Many features of the process remain uncharacterized. In this thesis, I detail a novel chemoenzymatic method to prepare authentic WTA substrates. I show that these materials can be used to study WTA biosynthetic enzymes in vitro and to probe enigmatic features of WTA assembly. With authentic WTA substrates, I reconstituted the final step of B. subtilis WTA synthesis in vitro, which involves ligation of WTAs to peptidoglycan. This process eluded biochemical characterization for decades. I investigated the catalytic requirements and substrate preferences for the LytR-CpsA-Psr family of enzymes that mediate this glycopolymer transfer reaction. Further, I discuss the attractiveness of these enzymes as antibiotic drug targets. Authentic WTA substrates will likely continue to be useful tools in understanding molecular features of WTA assembly and serve to assist drug discovery and development efforts targeting this process. / Thesis / Doctor of Philosophy (PhD)

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The peptidoglycan of many Gram-positive bacteria is densely functionalized with anionic glycopolymers called wall teichoic acids (WTAs). Recent studies have shown that these polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of Gram-positive bacterial physiology. Furthermore, in pathogens they are important in host infection and play key roles in antibiotic resistance. In many cases, precise mechanisms for WTA involvement in these processes have not been established. In order to better understand the roles of WTAs in the biology of the human pathogen Staphylococcus aureus, we sought to identify their interactions with other cellular pathways. By employing a transposon screen, we found that lipoteichoic acid (LTA) synthesis, D-alanylation of teichoic acids, cell wall stress sensors, CAAX-like proteases, and peptidoglycan biosynthesis were all synthetically lethal with depletion of WTAs in Staphylococcus aureus . Further investigations revealed that several genes required when WTAs were depleted were not essential when LTAs were removed. Unexpectedly, TA D-alanylation, became essential in the absence of WTAs, but not LTAs. Examination of terminal phenotypes following WTA depletion revealed that strains lacking LTA D-alanine esters died from envelope rupture during ongoing cell division whereas strains lacking LTAs were unable to form Z rings, stopped dividing, and had altered PG biosynthesis. Finally, we designed and implemented parallel, pathway-specific chemical screens to identify inhibitors that specifically kill mutants deficient in WTAs or D-alanylation of TAs. In addition to elucidating new interactions between cell envelope pathways, and establishing distinct roles LTAs and WTAs in the cell envelope of S. aureus, these experiments provide a list of potential targets and a strategy for identifying inhibitors for these targets, in compound combinations as therapeutics against antibiotic-resistant S. aureus infections.