Synthesis and utilization of N-acetyl muramic acid derivatives as tools for probing information about peptidoglycan structure, biogenesis and immunological response

All bacteria contain an essential glycopeptide polymer known as peptidoglycan (PG). PG is important in determining cell shape, serving as a scaffold for other envelope structures, and protecting bacteria from osmotic lysis. Its biosynthesis is the main target for widely used antibiotics, such as vancomycin and penicillin, that function by inhibiting various steps of its production. This structure is also important for guiding interactions with the human innate immune system. Innate immune cells, such as macrophages, sense and respond to fragments produced by bacteria, especially fragments produced by PG breakdown. Previous work from the Grimes lab led to the development of N-acetyl muramic acid (NAM) based probes that can be metabolically incorporated into bacterial PG and allow for visualization of this polymer. In this thesis, improved methodologies were developed to significantly increase and enhance the established PG labeling strategies. This endeavor was fueled by innovative chemical syntheses of novel NAM chemical probes, which demanded rigorous yet scalable routes allowing for late-stage modifications to the carbohydrate core. This was demonstrated through active participation in the commercialization of the probes testing the reproducibility of these syntheses. In addition to streamlining the synthesis of the probes, I also considered the metabolic processing of these probes by the bacteria. Leveraging genetic engineering and novel enzymes from the human oral pathogen, Tannerella forsythia, (N-acetylmuramate/N-acetylglucosamine kinase AmgK, and N-acetylmuramate alpha-1-phosphate uridylyltransferase, MurU) I developed bacterial strains and protocols that facilitated more efficient utilization of NAM probes. Finally, I collaborated with the Fox lab at UD to develop a new bioorthogonal NAM probe. This probe capitalized on the tetrazine-trans-cyclooctene ligation reaction, currently the fastest known bioorthogonal reaction, via the synthesis and development of a minimal tetrazine NAM. I designed downstream workflows that utilized high resolution microscopy, high resolution mass spectrometry, and flow cytometry to quantify and visualize the labeling of PG both in isolated bacterial cultures and in the presence of human macrophages. Furthermore, my thesis work demanded that I work closely with microbiologists to validate known biological processes and unveil novel pathways. My main collaborations included Prof. Ashu Sharma (SUNY Buffalo), Prof. Sloan Siegrist (UMass Amherst), and Prof. Natacha Ruiz (The Ohio State University). This was accomplished with the creation of protocols that are thorough and transferable as well as providing in-depth, species-specific troubleshooting. These advancements will change the way that scientists can view live bacterial systems, as traditional PG visualization methods struggle to match the rapid rate of cell growth and PG turnover.

Grantor dc:publisher

University of Delaware

Year dc:date.issued

2024

Author dc:creator

• Hyland, Stephen N.

• Peptidoglycan
• Human oral pathogen
• Human macrophages
• Innate immune cells

DOI

https://doi.org/10.58088/b3v7-kx75

OAI identifier oai:identifier

oai:udspace.udel.edu:19716/35463