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USING AUTHENTIC SUBSTRATES TO PROBE WALL TEICHOIC ACID ASSEMBLYGale, Robert T January 2018 (has links)
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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Glycosylation of Wall Teichoic Acids in Gram-Positive BacteriaAllison, Sarah 04 1900 (has links)
The biosynthetic enzymes involved in wall teichoic acid biogenesis in Grampositive
bacteria have been the subject of renewed investigation in recent years with the
benefit of modem tools of biochemistry and genetics. Nevertheless, there have been only
limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old
experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage
resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the
wall teichoic acid glycosyltransferase. This study and others have provided only indirect
evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we
showed that deletion of tagE results in the loss of a-glucose at the C-2 position of
glycerol in the poly(glycerol phosphate) polymer backbone. We also report the first
kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using
clean synthetic substrates. We investigated the substrate specificity ofTagE using a wide
variety of acceptor substrates and showed that this enzyme has a strong kinetic preference
for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form.
Further, we showed that the enzyme recognizes its polymeric (and repetitive) substrate
with a sequential kinetic mechanism. This work provides direct evidence that TagE is the
wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for
further studies on the mechanism of wall teichoic acid glycosylation, a largely uncharted
aspect of wall teichoic acid biogenesis. / Thesis / Doctor of Philosophy (PhD)
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