Engineering novel lantibiotics to target gut pathogens

Gherghisan-Filip , Cristina (2016) Engineering novel lantibiotics to target gut pathogens. Doctoral thesis, University of East Anglia.

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Infections with multi-drug resistant bacteria are an urgent health security threat and bacteriocins such as the subclass of lantibiotics represent a promising alternative source to traditional antibiotics. The gut intestinal tract ecosystem has a complex set of bacteria with a huge bacteriogenic potential and it can be explored as a source for new lantibiotic peptides. Using a genome mining approach, a clos gene cluster that has the potential to encode for novel lantibiotics was previously discovered in a strain of Blautia isolated from the human gut. This clos operon may encode two nisin-like peptides. Several nisin derived clos-like sequences were generated, however they did not have improved properties when tested under GI tract conditions. Since we lack the molecular tools to manipulate the original Blautia obeum A2-162 gut bacterium that harbours the clos operon, the entire clos cluster was cloned in Lactococcus lactis where it was possible to obtain heterologous expression of the Clos peptides under the control of the nisin inducible NisRK regulatory system. The production of preClosA was demonstrated using an antibody raised against the Clos leader. The biological activity of the Clos peptide was confirmed after treatment with trypsin where the Clos leader was presumably cleaved, releasing the biologically active and mature Clos peptide that was highly active against two clinically important pathogens, Clostridium perfringens and Clostridium difficile as well as against L. lactis MG1614 sensitive indicator strains. Using immunoprecipitation, we obtained pure Clos peptides in solution and after trypsin treatment and alkylation of the cysteine residues, the post-translational modifications and the structure of the preClosA peptides were confirmed. Some of the anticipated dehydrations and ring formations (Rings D and E) were also identified using LC-MS.
The approach taken in this PhD project confirms that genome mining can be used to identify more gut bacteria with potential to produce novel lantibiotics.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 4971 not found.
Date Deposited: 23 Jun 2017 12:51
Last Modified: 30 Jun 2018 00:38


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