Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Borrageiro, Christopher Matthew"

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    Formulation of natural cyclodecapeptides for surface sterilisation.
    (Stellenbosch : Stellenbosch University, 2023-03) Borrageiro, Christopher Matthew; Rautenbach, Marina; Van Rensburg, Wilma; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Bacterial adhesion to various surfaces leading to the formation of biofilms is a serious and persisting problem within various industries. Furthermore, once a biofilm reaches its mature stage, its mechanical removal becomes increasingly difficult and expensive. Additional to this rising problem of antimicrobial-resistant bacteria, another concern is further resistance development towards current surface sterilisation and disinfection techniques. This would be attributed to the chemical agents influencing the bacterial microenvironment imposing a constant selective pressure on the bacteria that promote tolerance over time. To circumvent this, the research focus has moved into developing active surfaces/ materials by changing the approach to preventing bacterial adhesion or killing bacterial cells that are in contact with the surface. Antimicrobial peptides (AMPs) serve as viable active ingredients due to their broad range and rapid activity towards various targets, limited resistance and multiple modes of action. A group of AMPs of interest are the tyrocidines (Trcs) and analogues, produced by the soil bacterium Brevibacillus parabrevis. The Trcs have a broad range of activity towards bacterial targets, various pathogenic and filamentous fungi, the malaria parasite Plasmodium falciparum and viruses. Recently it was shown that the peptides have potent activity in various materials and on surfaces towards the bacterial pathogens Listeria monocytogenes and Staphylococcus aureus. As limited solvent and formulation studies have been performed on the Trcs for the treatment of materials and depositing on surfaces, the aim of this study was to investigate the influence varying formulations had on the Trcs surface activity and biophysical properties. The Trcs were formulated in six different solvents, acetonitrile (ACN), ethanol (EtOH), methanol (MeOH), iso-propanol (IPA), tertiary butanol (TBA) and propylene glycol (PG) with or without co-formulants namely zinc chloride (ZnCl2), calcium chloride (CaCl2) or glycerol (Glr). Activity studies revealed that when assessed against both targets (L. monocytogenes and S. aureus), EtOH serves as the best general solvent to be used without any additives compared to the other five solvents, The addition of 1% Glr resulted in significantly lower activity in the more polar solvents when compared to formulation in the solvents alone. However, PG and TBA in formulation with 1% Glr + 100 µ M CaCl2 were found to be the two best formulations. TBA as solvent was marginally better due to a higher activity against both targets compared to PG. How the surface activity links with the formulant influence on the peptide structure was assessed by mapping activity versus chemical parameters, changes in Trp environments (fluorescence) and oligomerisation (ion mobility mass spectrometry). The relative dielectric constant (ε) and relative molar mass (Mr) values for all solvents, with and without 1% Glr, revealed a distinct breakpoint of ε = 65 and relative Mr = 32-33 for activity towards both bacterial targets. This suggests that when the solvent or additives are within the defined Mr range, they are able to disrupt or destabilise inactive oligomers whereas outside of the defined Mr range (Mr < 33) could result in stabilising inactive oligomers and obscuring active structures. Evaluation of the Trp fluorescence versus activity revealed that the Trp environment in solution does not have an overt link with the Trc surface activity. Although Trp may have an important conformational and activity role when the Trcs are organised on the surface, the cationic residues, Orn⁹ and Lys⁹ are known to have major importance in recognising negatively charged cellular targets and in the peptide’s activity. Ion mobility mass spectrometric analysis of Trc oligomerisation indicated 1% Glr as additive resulted in a higher total ion signal for dimers than in the solvent alone. Glr may be acting as a chaotropic agent disrupting larger oligomers by competing for hydrogen bonds and thereby releasing stabile dimers. Amphipathic dimers are regarded as active structures. Therefore, in light of the detection of lower activity in the presence of Glr, this higher dimer signal may not be due to the availability of active amphipathic dimers, but rather inactive non-amphipathic dimers. The addition of CaCl2 showed a lower than expected dimer contribution, but no change in activity. However, 1% Glr + 100 µ M CaCl2 as Trc co- formulants resulted in a substantially increased total ion signal for Trc dimers combined with the highest activity compared to other formulations. There is a weak link between active Trc dimers detected in vacuo, but elucidation of the surface structure-activity relationship is clearly complex and demands an in-depth study of the peptide structure on surfaces, as well as the release of active structures.