Antibiotic resistance poses a major threat to our healthcare system. Six problem pathogens, the so-called ESKAPE bacteria, are responsible for the majority of drug resistant infections in hospitals. New strategies to treat these infections ar sorely needed. Antibiotics that target peptidoglycan (PG)/cell wall biogenesis are among the most effective drugs for treating bacterial infections, but resistance has emerged to all those currently in clinical use. The proposed work grew out of recent discoveries made using ?-lactams as chemical probes of PG biosynthesis. It is aimed at identifying and validating new targets in the pathways for cell wall assembly in methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). MRSA is the most virulent of the ESKAPE pathogens, and E. coli, an important pathogen in its own right, is the model system for PG biogenesis in all pathogenic Gram-negative rods. Our first two aims are focused on validating a new target for inhibitors that resensitize MRSA to ?-lactams. MRSA have acquired a PG transpeptidase called PBP2A that promotes ?-lactam resistance. We discovered that PBP2A function is dependent on the activity of a glycosyltransferase, TarS, that attaches ?-O-GlcNAc residues to wall teichoic acids (WTAs), an additional cell wall polymer important for cell division in S. aureus. This suggests that the pathways of PG and WTA synthesis are somehow interconnected. We will use a combination of genetic and chemical approaches to uncover the mechanistic basis for these connections so that we can exploit them as targets to combat ?-lactam resistance in MRSA. We will also explore TarS itself as a drug target by monitoring the effect of small molecule ?-lactam potentiators on its activity and solving its structure with and without bound inhibitors. Our second set of aims focus on understanding the function of PG synthesizing machines and validating them as antibiotic targets. Given their importance as potential drug targets, surprisingly little is known about the mechanism of PG assembly by these machines. This has primarily been due to a limited availability of genetic assays to dissect their function. Taking advantage of the genetic tractability of the E. coli system, we developed the first positive selection against the activity of a PG assembly machine, the highly conserved Rod system needed for cell elongation. We used this selection to identify small molecule antagonists of Rod function and propose to determine their specific targets and mode of action. We will also use our selection to genetically interrogate the structure of the multi-protein Rod complex and identify amino acid residues critical for the function of each component. The combined chemical genetic analysis will help us identify and validate aspects of Rod system function amenable to targeting by novel therapeutics. Because the PG and WTA synthesis machineries we will study are highly conserved, our findings in MRSA and E. coli will be broadly relevant to our understanding of cell wall polymer biogenesis in other microorganisms and should significantly impact and inform efforts to generate therapies against MRSA and Gram-negative ESKAPE pathogens.

Public Health Relevance

New strategies to treat antibiotic resistant bacterial infections are sorely needed. This project combines small molecules and genetic methods to identify and validate new antibiotic targets in the pathway for assembly of the bacterial cell wall. The proposed work may lead to new therapies against methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative ESKAPE pathogens.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZAI1-LG-M (J1))
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Huntley, Clayton C
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Harvard University
Schools of Medicine
United States
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Rohs, Patricia D A; Buss, Jackson; Sim, Sue I et al. (2018) A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery. PLoS Genet 14:e1007726
Vickery, Christopher R; Wood, B McKay; Morris, Heidi G et al. (2018) Reconstitution of Staphylococcus aureus Lipoteichoic Acid Synthase Activity Identifies Congo Red as a Selective Inhibitor. J Am Chem Soc 140:876-879
Wood, B McKay; Santa Maria Jr, John P; Matano, Leigh M et al. (2018) A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation. J Biol Chem 293:17985-17996
Sham, Lok-To; Zheng, Sanduo; Yakhnina, Anastasiya A et al. (2018) Loss of specificity variants of WzxC suggest that substrate recognition is coupled with transporter opening in MOP-family flippases. Mol Microbiol 109:633-641
Rajagopal, Mithila; Walker, Suzanne (2017) Envelope Structures of Gram-Positive Bacteria. Curr Top Microbiol Immunol 404:1-44
Chamakura, Karthik R; Sham, Lok-To; Davis, Rebecca M et al. (2017) A viral protein antibiotic inhibits lipid II flippase activity. Nat Microbiol 2:1480-1484
Schaefer, Kaitlin; Matano, Leigh M; Qiao, Yuan et al. (2017) In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins. Nat Chem Biol 13:396-401
Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G (2017) The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli. PLoS Genet 13:e1006934
Meeske, Alexander J; Riley, Eammon P; Robins, William P et al. (2016) SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537:634-638
Lee, Wonsik; Schaefer, Kaitlin; Qiao, Yuan et al. (2016) The Mechanism of Action of Lysobactin. J Am Chem Soc 138:100-3

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