Browsing by Author "Liang, Hai"
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Item Coupling Novel Probes with Molecular Localization Microscopy Reveals Cell Wall Homeostatic Mechanisms in Staphylococcus aureus(ACS Chemical Biology, 2022-11-22) Lund, Victoria A.; Gangotra, Haneesh; Zhao, Zhen; Sutton, Joshua A. F.; Wacnik, Katarzyna; DeMeester, Kristen; Liang, Hai; Santiago, Cintia; Grimes, Catherine Leimkuhler; Jones, Simon; Foster, Simon J.Bacterial cell wall peptidoglycan is essential for viability, and its synthesis is targeted by antibiotics, including penicillin. To determine how peptidoglycan homeostasis controls cell architecture, growth, and division, we have developed novel labeling approaches. These are compatible with super-resolution fluorescence microscopy to examine peptidoglycan synthesis, hydrolysis, and the localization of the enzymes required for its biosynthesis (penicillin binding proteins (PBPs)). Synthesis of a cephalosporin-based fluorescent probe revealed a pattern of PBPs at the septum during division, supporting a model of dispersed peptidoglycan synthesis. Metabolic and hydroxylamine-based probes respectively enabled the synthesis of glycan strands and associated reducing termini of the peptidoglycan to be mapped. Foci and arcs of reducing termini appear as a result of both synthesis of glycan strands and glucosaminidase activity of the major peptidoglycan hydrolase, SagB. Our studies provide molecular level details of how essential peptidoglycan dynamics are controlled during growth and division.Item Metabolic labeling of the carbohydrate core in bacterial peptidoglycan via the Pseudomonas putida cell wall recycling enzymes AmgK and MurU and its applications(University of Delaware, 2018) Liang, HaiBacterial cells are surrounded by a polymer called peptidoglycan (PG), which is a core component of bacterial cell wall. PG assists in bacterial cell division, maintains the strength of cell structure and protects the cell from changes in osmotic pressure and small molecule insults. PG is composed of two conserved carbohydrates N-acetyl-glucosamine (NAG, or GlcNAc) and N-acetyl-muramic acid (NAM, or MurNAc), and peptides chains containing D-amino acids. ☐ PG is also an essential polymer for human life, serving as an antibiotic target and also a recognition element for the human innate immune system. It has been proposed that the production of specific bacterial cell wall fragments and the proper innate immune recognition is critical for human to differentiate and manage bacterial species from human microbiome. Misrecognition of bacterial PG fragments is proposed to be the cause of a variety of diseases, including Crohn’s disease, arthritis and asthma. ☐ In order to develop a novel methodology for labeling on the PG at the conserved NAM residue to study the innate immune recognition of PG fragments, here I demonstrated the power of connecting the bacterial PG recycling program via enzymes AmgK and MurU, to the biosynthetic pathway to ultimately metabolically label bacterial PG with bioorthogonal handles. Using the synthetic modifiable NAM carbohydrate derivatives, these probes were installed into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole bacterial cells are labeled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This method also provides a powerful tool for research on the bacterial cell division on the carbohydrate level. These studies will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.