Bioengineering of nisin to enhance functionality against dairy pathogens

The bacteriocin class of antimicrobial peptides have emerged as a viable alternative to at least partially fill the void created by the end of the golden age of antibiotic discovery. Along with this potential use in a clinical setting, bacteriocins also play an important role as bio-preservatives in the food industry. This thesis focuses on a specific bacteriocin group, the lantibiotics (Lanthionine-containing antibiotics). Their numerous methods of appliance in a food setting and how their gene-encoded nature can be modified to improve on overall bioactivity and functionality are explored here. The use of a lantibiotic (lacticin 3147) producing starter culture to control the Crohn’s disease-linked pathogen Mycobacterium paratuberculosis was assessed in a raw milk cheese. Although lacticin 3147 production did not effectively control the pathogen, the study provided an impetus to employ a variety of PCR-based mutagenesis techniques with a view to the creation of enhanced lantibiotic derivatives. Through the use of these techniques, a number of enhanced derivatives were generated from the ‘hinge’ region of the nisin peptide. Furthermore, a derivative in which the three hinge amino acids were replaced with three alanines represents the first enhanced derivative of nisin to have been designed through a rational process. This derivative also formed the backbone for the creation of an active, trypsin resistant, variant. Through the employment of further mutagenesis methods a derivative was created with potential use as an oral anti-bacterial in the future. Finally a number of lead nisin derivatives were investigated to assess their anti- Streptococcus agalactiae ability, a mastitis associated pathogen. Also a system was developed to facilitate the large scale production of these candidates, or other nisin derivatives, from dairy substrates.

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.