Formulation of natural cyclodecapeptides for surface sterilisation.

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dc.contributor.advisorRautenbach, Marinaen_ZA
dc.contributor.advisorVan Rensburg, Wilmaen_ZA
dc.contributor.authorBorrageiro, Christopher Matthewen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Biochemistry.en_ZA
dc.date.accessioned2023-03-03T12:57:45Z
dc.date.accessioned2023-05-18T07:04:05Z
dc.date.available2023-03-03T12:57:45Z
dc.date.available2023-05-18T07:04:05Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Bakteriese adhesie aan verskeie oppervlaktes kan lei tot die vorming van biofilms en is 'n ernstige en volgehoue probleem binne verskeie industrieë. Verder, sodra 'n biofilm sy volwasse stadium bereik, word die meganiese verwydering daarvan al hoe moeiliker en duurder. Bykomend tot hierdie toenemende probleem van antimikrobiese weerstandbiedende bakterieë, is kommer oor verdere potensiële weerstandsontwikkeling teenoor huidige oppervlaksterilisasie en ontsmettingstegnieke. Dit kan toegeskryf word aan die chemiese middels wat die bakteriese mikro-omgewing beïnvloed en 'n konstante selektiewe druk op die bakterieë plaas wat toleransie oor tyd bevorder. Om dit te omseil, het die navorsingsfokus verskuif na die ontwikkeling van aktiewe oppervlaktes/materiale waardeur die benadering verander is om bakteriese adhesie te voorkom of bakteriese selle wat in kontak met die oppervlak is, dood te maak. Antimikrobiese peptiede (AMP's) dien as lewensvatbare aktiewe bestanddele as gevolg van hul wye reeks en vinnige aktiwiteit teenoor verskeie teikens, beperkte weerstand en veelvuldige modusse van aktiwiteit. 'n Groep AMP's van belang is die tirosidiene (Trcs) en analoë wat deur die grondbakterie Brevibacillus parabrevis geproduseer word. Die Trcs het 'n wye reeks aktiwiteit teenoor bakteriese teikens, verskeie patogeniese en filamentagtige swamme, die malariaparasiet Plasmodium flaciparum en virusse. Onlangs is getoon dat die peptiede kragtige aktiwiteit het in verskeie materiale en op oppervlaktes teenoor die bakteriële patogene Listeria monocytogenes en Staphylococcus aureus. Aangesien beperkte oplosmiddel- en formuleringstudies op die Trcs uitgevoer is vir die behandeling van materiale en afsetting op oppervlaktes, was die doel van hierdie studie om die invloed wat verskillende formulerings op die Trc oppervlaksaktiwiteit en biofisiese eienskappe het, te ondersoek. Die Trcs is geformuleer in ses verskillende oplosmiddels, asetonitriel (ACN), etanol (EtOH), metanol (MeOH), isopropanol (IPA), tersiêre butanol (TBA) en propileenglikol (PG) met of sonder ko- formulante naamlik sinkchloried (ZnCl2), kalsiumchloried (CaCl2) of gliserol (Glr). Aktiwiteitstudies het aan die lig gebring dat wanneer dit teen beide teikens (L. monocytogenes en S. aureus) geassesseer word, EtOH dien as die beste algemene oplosmiddel wat gebruik kan word sonder enige ko- formulante in vergelyking met die ander vyf oplosmiddels. Die byvoeging van 1% Glr het ‘n aansienlike laer aktiwiteit tot gevolg gehad vir formulering in die meer polêre oplosmiddels in vergelyking met in die oplosmiddels alleen. Daar is wel gevind dat PG en TBA in formulering met 1% Glr + 100 µ M CaCl2 die twee beste formulerings is. TBA as oplosmiddel was effens beter as gevolg van 'n hoër aktiwiteit teen beide teikens in vergelyking met PG. Hoe die oppervlaksaktiwiteit verband hou met die formulering se invloed op die peptiedstruktuur is geassesseer deur aktiwiteit teenoor chemiese parameters, veranderinge in Trp-omgewings (fluoressensie) en oligomerisasie (ioonmobiliteit-massaspektrometrie) te karteer. Die relatiewe diëlektriese konstante (ε) en relatiewe molêre massa (Mr) waardes vir alle oplosmiddels, met en sonder 1% Glr, het 'n duidelike breekpunt van ε = 65 en relatiewe Mr = 32-33 getoon vir aktiwiteit teenoor beide bakteriese teikens. Dit dui daarop dat wanneer die oplosmiddel of ko-formulante binne die gedefinieerde Mr-reeks is, hulle in staat is om onaktiewe oligomere te ontwrig of te destabiliseer, terwyl buite die gedefinieerde Mr -reeks (Mr <33) kan lei tot stabilisering van onaktiewe oligomere en die afskerming van aktiewe strukture. Evaluering van die Trp-fluoressensie teenoor aktiwiteit het uitgewys dat die Trp-omgewing in oplossing nie 'n duidelike skakeling met die Trc- oppervlaksaktiwiteit het nie. Alhoewel Trp 'n belangrike konformasie- en aktiwiteitsrol kan speel wanneer die Trcs op die oppervlak georganiseer is, is dit bekend dat die kationiese residue, Orn⁹ en Lys⁹ 'n groot belang het in die herkenning van negatiewe gelaaide sellulêre teiken en in die peptied se aktiwiteit. Ioon-mobiliteit massaspektrometriese analises van Trc oligomerisasie het aangedui dat 1% Glr as ko-formulat lei tot 'n hoër totale ioonsein vir dimere as in die oplosmiddel alleen. Dit is ‘n indikasie dat Glr as 'n chaotropiese middel kan optree en groter oligomere ontwrig deur met waterstofbindings te kompeteer en sosoende stabiele dimere vry te stel amfipatiese dimere word as aktiewe strukture beskou. In die lig van laer aktiwiteit waarnemings in die teenwoordigheid van Glr, is hierdie hoër dimeer sein moontlik nie te wyte aan die beskikbaarheid van amfipatiese dimere nie, maar wel onnaktiewe nie-afipatiese dimere. Die byvoeging van CaCl2 het 'n laer as verwagte dimeer bydrae getoon, maar geen verandering in aktiwiteit nie. Die 1% Glr + 100 µM CaCl2 as Trc ko- formulante het egter gelei tot aansienlike verhoogde totale ioonsein vir Trc dimere, gekombineer met die hoogste aktiwiteit in vergelyking met ander formulerings. Daar is slegs 'n swak verband tussen aktiewe Trc dimere wat in vacuo opgespoor word, maar ontrafelling van die oppervlakstruktuur-aktiwiteit-verwantskap is duidelik kompleks en slegs aangespreek word met 'n in-diepte studie van die peptiedstruktuur op oppervlaktes, sowel as die vrysteling van aktiewe strukture.af_ZA
dc.description.versionMastersen_ZA
dc.format.extent174 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/127096
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshPeptides -- Analysisen_ZA
dc.subject.lcshAntimicrobial peptides (AMPs)en_ZA
dc.subject.lcshPeptide antibioticsen_ZA
dc.subject.lcshTyrocidinesen_ZA
dc.subject.lcshPeptides -- Analysisen_ZA
dc.subject.lcshSolvents -- Mechanism of actionen_ZA
dc.subject.lcshSurface sterilisationen_ZA
dc.subject.nameUCTDen_ZA
dc.titleFormulation of natural cyclodecapeptides for surface sterilisation.en_ZA
dc.typeThesisen_ZA
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