Production and characterisation of analogues of the antimicrobial tyrocidine peptides with modified aromatic amino acid residues
Thesis (MSc)--Stellenbosch University, 2018.
ENGLISH ABSTRACT: With the approach of a post-antibiotic era in which the current arsenal of antibiotic compounds can no longer combat common bacterial infections, the search for novel compounds to fight against antimicrobial resistance is more important than ever. A group of forgotten antimicrobial peptides, termed the tyrocidines, have re-emerged as promising candidates for further development in clinical and industrial settings. These are basic, cyclic decapeptides, with the sequence cyclo(D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5- L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-Orn9-L-Leu10), and are naturally synthesized, together with the neutral, linear pentadecapeptides, the gramicidins, by the soil bacterium Brevibacillus parabrevis. A multitude of structurally similar tyrocidines has been found in extracts of this organism. This arises due to the non-ribosomal, enzymatic, synthesis of these peptides in which certain domains within this multi-domain enzyme system have the ability to incorporate more than one kind of aromatic amino acid. The specific structural variations are at residue positions 3, 4 and 7 in the cyclic decapeptide. These structural variations produce tyrocidines with unique structure-activity relationships, imbuing them with activity against a wide variety of Grampositive bacteria and fungi. Activity against the malarial parasite, Plasmodium falciparum, has also been observed. This study aims to make use of the variability seen for incorporation of different aromatic amino acid residues, specifically of phenylalanine and tryptophan, to determine if non-natural derivatives of these amino acids can be incorporated, to biosynthesize novel tyrocidine, tryptocidine and phenycidine analogues (collectively termed the Trcs), with unique structureactivity relationships. The initial objective of this study was to characterise different available strains of the producer organism, to determine if there was a difference in the production profiles of Trcs by the producer organism. A clear difference was found between the Br. parabrevis 5618, 362, 10068 and 8185 strains in terms of their production profiles, with the 5618 showing a marked improvement in Trc analogues rich in tryptophan. In addition to strain-determined differences in production profiles, differences in production as a result of altered nutrient conditions, in the form of nitrogen, sulphate and carbon sources, added on top of a base media, were also investigated. This was initially done by growth rate analysis of the producer organisms under different conditions, and then followed up by small scale culture to determine the effect on production of Trcs. Altered productions in terms of biomass and extract mass were found, and to a lesser degree the Trc production profile. Urea appeared to have the most marked effect on Trc production, particularly for the 5618 strain. The next objective was to determine if the selected phenylalanine analogues 4-methylphenylalanine (4-MeF), 2-flouro-phenylalanine (2-FF) and 4-trifluoro-phenylalanine (4-CF3F) and tryptophan analogues 5-methyl-tryptohan (5-MeW) and 1-methyl-trpyophan (1-MeW) could be incorporated into the Trcs. This investigation also used initial growth rate analysis of the strains 5618, 362 and 8185 under different supplementation concentrations of the non-natural and natural amino acids. With the exception of 1-MeW and CF3F, most amino acids appeared well tolerated by the strains tested, with a decrease in growth only being seen by most of the amino acids at concentrations of 22 mM. Following this, small scale cultures were done under selected conditions to test for their incorporation using the 5618 strain. Extensive incorporation was found for all the analogues, with the exception of 4-CF3F as determined by novel masses found using ESMS analysis. Finally, medium-scale cultures were done using supplementation with 2-FF and 5-MeW to produce enough peptide for subsequent extraction, purification and analysis. Again, extensive incorporation of these analogues was found, with 5-MeW substituted Trc analogues having a large hydrophobic shift in retention time. A tyrocidine A analogue incorporating three 2-FF residues was found to be the predominate peptide in the 2-FF culture, while a tryptocidine B analogue incorporating either one or two 5-MeW was found on the 5-MeW culture. Subsequent semi-preparative reverse-phase HPLC was done in an attempt to purify individual non-natural analogues, however, difficulties in separation owing to the large variety of Trc analogues produced with overlapping retention times only allowed for enrichment of certain analogues. The enriched analogues were tested against Microccocus luteus. The methylated tryptocidine B analogues appeared to have similar growth and metabolic inhibition activities to the natural analogues, while the fluorinated tyrocidine A analogues appeared to be more active compared to the natural analogue, being a more effective inhibitor of bacterial metabolism. The production of novel tyrocidines with unique structure-activity relationships was shown to be possible by supplementation of the growth media of the producer organism with non-natural aromatic amino acids. This provides a rapid, affordable, and scalable method to produce novel antimicrobial peptides.
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