A proteomic investigation into the bacteriostatic mechanism of glycocin F, secreted by Lactiplantibacillus plantarum KW30 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand
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Date
2021
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Massey University
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Abstract
Antimicrobial resistance to clinical antibiotics has increased significantly over the past decade and continues to pose a serious public health concern worldwide. Due to this rise in antimicrobial resistance, clinical health professionals and scientific researchers alike have been eager to explore alternative options in place of traditional treatments (e.g. antibiotics). Bacteriocins are antimicrobial polypeptides secreted by bacteria. The therapeutic potential of these diverse natural products is largely unexplored and, as such, they constitute a novel source of potentially viable treatment options in the fight against resistant strains of pathogenic bacteria. At the most basic level, bacteriocins can be divided into two groups, those that are modified and those that are not. Glycocins fall into the former group as they are post-translationally modified by one or more monosaccharide moieties. These monosaccharides can be O-linked to the hydroxyl group of serine or threonine residues, or S-linked to the sidechain thiol group of cysteine residues. Glycocin F (GccF) is a doubly glycosylated bacteriocin secreted by the lactic acid bacterium Lactiplantibacillus plantarum (Lb. plantarum) KW30 that inhibits the growth of its bacterial targets. Although it is known that GccF is bacteriostatic, the exact mechanism by which it inhibits cell growth within two minutes, at nanomolar (nM) concentrations, remains unknown. Previous bacterial genomics, nuclear magnetic resonance (NMR) spectroscopy, total chemical synthesis and analysis of synthetic peptides, and transcriptomic studies have provided the framework for this present research. Collectively, these studies led to the proposal that GccF binds to the transmembrane domain of a specific N-acetylglucosamine (GlcNAc) phosphotransferase system (PTS) transporter on the surface of targeted bacterial cells, causing the amplification of a signal that results in the rapid onset of bacteriostasis. This project used proteomic methods to investigate changes that occurred in the proteome of susceptible cells when treated with GccF. Cultures were sampled immediately before, and then at specific timepoints after the addition of GccF. Sampled cells were separated into membrane and cytosol fractions, then analysed for changes in their proteomes. Changes in the abundances of specific target cell proteins were linked to biochemical pathways that may be affected by treatment with GccF, providing clues to its mechanism of action. The results showed clear changes in the abundance of proteins involved in cell wall metabolism and protein translation in GccF-treated Lb. plantarum ATCC 8014 cells.
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Māori Masters Thesis