Optimised bacterial production and characterisation of natural antimicrobial peptides with potential application in agriculture

Thesis (PhD)--Stellenbosch University, 2016ENGLISH ABSTRACT : The loss incurred due to microbial spoilage is a great concern to the agricultural and food industries. Microbial pathogens have typically been controlled through the use of chemical control agents and antibiotics. However, combination of emerging microbial resistance together with consumer opposition and regulation toward these chemical agents has instigated the need for so called green-biocides which have a reduced ecological impact together with lower potential for resistance development. The tyrocidines and their analogues, cyclic decapeptides produced by the soil bacterium Bacillus aneurinolyticus, show potential to serve as green-biocides. Potent antimicrobial activity toward bacterial and fungal pathogens which affect both the food and agricultural industries is achieved by different cyclodecapeptide analogues. The objective of this study was the development and evaluation of the future potential of the cyclodecapeptides produced by B. aneurinolyticus to serve as green-biocides. In this thesis an overview is given in Chapter 1 of the challenges facing agricultural production due to microbial spoilage and resistance, as well as the potential of antimicrobial peptides, specifically the tyrocidines and analogues to address this problem. The optimal conditions allowing for future large scale production of target-specific subsets of these cyclodecapeptides and economical downstream purification of the peptide mixtures and single peptides is covered in Chapters 2-4. The produced cyclodecapeptide mixtures/formulations were investigated to elucidate some parameters which may allow for optimisation to target different agricultural and/or food related pathogens/problem organisms (Chapters 5 and 6). Initial formulation parameters were evaluated together with the relative safety of their application within an agricultural setting by determining their in vivo toxicity toward honey bees (Chapter 7). A Summary of the findings in each of the different chapters is presented together with proposed future work (Chapter 8). The experimental chapters of this thesis were written as independent units so as to enable future publication. In some chapters repetitions were unavoidable, every attempt was made to keep this to a minimum. The first goal of this study was the increased natural production of these cyclodecapeptides to enabling their tailored application to target different pathogens in an agricultural environment. In order to achieve the goal of this study the following objectives were set: Manipulation of the natural cyclodecapeptide profile of B. aneurinolyticus to produce defined peptide subsets and single peptides. (Chapter 2). Construction of a computational model of cyclodecapeptide production by B. aneurinolyticus (Chapter 3). Optimise production and purification methodology of the tyrocidines and their analogues for future large scale production (Chapter 4). The second goal was to characterise the peptides in order to facilitate formulation of the peptides for in vivo applications. In order to achieve the goal of this study the following objectives were set: Investigate the structure and oligomerisation relationships of the major cyclodecapeptide analogues produced in the different subsets (Chapter 5). Correlation of the oligomerisation characteristics of co-produced cyclodecapeptides with their antimicrobial activity toward two representative target organisms (Chapter 6). Formulation of the natural cyclodecapeptide extracts and investigation of their in vivo toxicity toward honey bees as representative non-target species (Chapter 7)

Synergistic Activity of the Tyrocidines, Antimicrobial Cyclodecapeptides from Bacillus aneurinolyticus, with Amphotericin B and Caspofungin against Candida albicans Biofilms

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment