New Antimicrobials to Target Gut Pathogens

Scadden, Jacob (2022) New Antimicrobials to Target Gut Pathogens. Doctoral thesis, University of East Anglia.

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Abstract

Nisin O is an antimicrobial peptide produced by the human gut bacterium Blautia obeum A2-162 which has antimicrobial activity against clinically relevant organisms, such as Clostridium difficile and Clostridium perfringens. The biosynthetic gene cluster of nisin O is unusual when compared to other nisin variants, with four putative structural peptides, two two-component regulatory systems and no leader peptide cleaving protease. This project aimed to investigate the regulation of this cluster and to search for the presence of a protease within the B. obeum A2-162 genome.

Various induction conditions previously used for antimicrobial production in B. obeum A2-162 were assessed, however no antimicrobial activity was observed. A previously designed heterologous nisin O expression system in Lactococcus lactis UKLc10 was optimised for nisin O production. A pan-genome analysis of 2094 Lachnospiraceae genomes was performed and identified six nisin O-like clusters across multiple genera, including Blautia, Dorea and Ruminococcus. Additional novel lantibiotic clusters were identified in Pseudobutryvibrio and Ruminococcus species. The B. obeum A2-162 genome was searched for candidate proteases, of which 10 were identified, with six cloned into inducible plasmids, and used in pre-Nisin O leader peptide cleavage assays. None of the proteases tested were able to cleave the NsoA1-3 leader peptide, however leader peptide cleavage and antimicrobial activity was observed in the presence of trypsin. The two two-component regulatory systems were constitutively expressed and used in a pepI gene reporter assay to assess their interaction with the predicted promoters. It was shown that the PnsoR2K2 provided constitutive expression and that the PnsoA would only interact with the regulatory system from nisin A when induced with nisin A. No activity was seen with any other promoter-regulatory system interaction.

This project has identified a number of novel nisin O-like clusters. However, no candidate protease or nisin O regulatory system interaction could be elucidated.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	03 Jan 2023 11:13
Last Modified:	31 Oct 2024 01:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/90352
DOI:

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