Understanding the mechanism of action of the glycosylated bacteriocin glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand
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2019
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Massey University
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Abstract
With the increasing threat posed by antibiotic-resistant bacteria, efforts must be
made to find new antimicrobial agents. One growing area of promise is the bacteriocins, which are a diverse group of antimicrobial peptides produced by bacteria.
This thesis focuses on determining the mechanism of action of one of these peptides,
glycocin F (GccF). GccF is produced by the bacterium Lactobacillus plantarum and
is modified with two N-acetyl glucosamine (GlcNAc) sugar moieties, one located
on an interhelical loop region, and the other at the end of a flexible C-terminal
‘tail’. It has also been shown to exhibit a unique effect on susceptible bacteria,
putting them into a reversible state of hybernation as opposed to outright killing
them. However, little is known about the roles of the structural features of GccF,
how it triggers bacteriostasis in target cells, or even what part(s) of bacterial cells it
targets. This work addresses these questions using three main approaches: studying
the structure-function relationship of different parts of GccF with chemically synthesised analogues; looking at the transcriptional response of a pathogenic bacteria,
Enterococcus faecalis, to GccF; and trying to identify binding partners of GccF and
its respective immunity protein, GccH. The results presented here highlight different
roles of the GlcNAcs attached to GccF, with both the interhelical loop and presence
of GlcNAc on this loop being vital for activity, while the sugar at the C-terminal
position is important, but not crucial for the peptide’s activity. Additionally, a role
of the GlcNAc phosphotransferase system on the mechanism of GccF is strongly
indicated, with evidence from both the transcriptional studies and the protein interaction studies of GccF’s immunity protein. Taken together, the results allow for two
theoretical models of GccF’s mechanism of action to be proposed. These models
presented here should serve to increase the understanding of other glycocin-class
bacteriocins and their mechanisms of action, and possibly contribute towards the
creation of a blueprint for a new class of antimicrobial agents.
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Keywords
Bacteriocins, Peptide antibiotics, Lactobacillus plantarum, Lactobacillus, Genetics