Investigations into the biosynthesis and mode of action of glycocins

Biswas, Subhanip

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Description

Title

Investigations into the biosynthesis and mode of action of glycocins

Author(s)

Biswas, Subhanip

Issue Date

2020-06-08

Director of Research (if dissertation) or Advisor (if thesis)

van der Donk, Wilfred A

Doctoral Committee Chair(s)

van der Donk, Wilfred A

Committee Member(s)

• Hergenrother, Paul J
• Mitchell, Douglas A
• Nair, Satish K

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Glycocins
• sublancin
• S-linked
• heterologous expression

Abstract

Antimicrobial resistance is an emerging global problem that poses a serious threat to our health and economy. The problem is further amplified by the limited number of antimicrobial targets in bacteria that are affected by the current pool of antibiotics. This problem necessitates finding antimicrobial compounds that have new targets or a novel mode of action. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with diverse structures and interesting biological activities. Glycocins (glycosylated bacteriocins) are a family of RiPPs, which are currently characterized by the presence of two helices stabilized by two disulfide bonds. Additionally, the side chains of cysteine or serine residues, which are present on a loop connecting the two helices, are modified with at least one sugar moiety. Sublancin is one of the members of the glycocin family with remarkable stability and antimicrobial activity against multidrug resistant Gram-positive pathogens such as methicillin resistant Staphylococcus aureus (MRSA). Previously, the NMR structure of sublancin was elucidated by our laboratory. In my research, we used this structural information to perform structure activity relationship studies on the solvent exposed residues of sublancin, which is described in Chapter 2. Our studies revealed that two residues at the C-terminus of the peptide are important for activity. Additionally sublancin was determined to be ‘glycoactive’, or in other words , the sugar on sublancin was deemed essential for its bioactivity. Previous studies implicated the involvement of the glucose-phosphotransferase system (glucose PTS) in the sensitivity to sublancin. I further investigated the role of the glucose PTS in the bioactivity of sublancin by overexpressing the genes of the PTS operon. The results presented in Chapter 3 indicated that such strains were hypersensitive and showed decreased resistance frequencies suggesting that sublancin leads to a yet unknown deleterious gain of function of the glucose PTS. In the course of my studies in Chapter 2, I developed a system that enabled the reconstitution of the biosynthesis of sublancin in a heterologous host, Escherichia coli. I realized that the approach can be extended to the rapid discovery of new glycocins that are available in the database via genome mining and that currently cannot be cultured in the laboratory, such as those from pathogens and extremophiles. Indeed, in collaboration with the Zhao laboratory we discovered four new glycocins with novel biological activity and new structures, and these studies are described in Chapter 4. Additionally, the glycosyltransferase SunS shows remarkable chemoselectivity for Cys22 in the core peptide of sublancin. In an effort to understand the specificity, I synthesized some sugar nucleotide derivatives, one of which turned out to be an inhibitor for the enzyme and enabled the crystallization of the full-length protein with the sugar nucleotide. Additionally, the X-ray crystal structure enabled the rational design of a minimal glycosylation motif for the enzyme. These studies are described in Chapter 5. Understanding the biosynthesis and mode of action of these natural products can aid in the development of new antimicrobial compounds.

Graduation Semester

2020-08

Type of Resource

Thesis

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Copyright © 2020 Subhanip Biswas

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