Characterization of natural antimicrobial peptides adsorbed to different matrices

van Rensburg, Wilma (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.


ENGLISH ABSTRACT: Biofouling is the attachment and biofilm formation that leads to negative repercussions such as persistent post-harvest infections, infections obtained from medical implants and continual surface contamination of food processing plants. Much of the problem lies with the resistance that develops against conventional treatments due to the formation of mature biofilms. Thus the focus has shifted from the removal of biofilms to the prevention of initial attachment of organisms. This entails the use of antimicrobial surfaces that either have an inherent antimicrobial activity, e.g. certain metals, or surfaces that are modified by the attachment of antimicrobial agents. The attachment of antimicrobial agents can either be through covalent bonding or adsorption, depending on the intended use of the surface as well as the mode of action of the antimicrobial agent. Antimicrobial peptides (AMPs) are ubiquitous in nature, tend to have a broad spectrum of activity, are very stable and have been shown to maintain activity when covalently bound to solid surfaces. Tyrocidines (Trcs), antimicrobial peptides produced by Bacillus aneurinolyticus, are cyclodecapeptides with a broad spectrum of activity against Grampositive bacteria, fungi, yeasts and the human malaria parasite, Plasmodium falciparum. The aim of this study was to determine the antimicrobial activity of surfaces treated with a tyrocidine extract, under which conditions the activity remained stable and to look into possible applications of these peptide-treated surfaces. The study focussed on different solid surfaces namely mixed cellulose, polyvinylidene fluoride, polycarbonate, cellulose acetate, cellulose (paper)(CL) and high density cellulose packing material (HDC), as a pilot study to assess the antimicrobial activity of Trc and gramicidin S (GS) treated solid surfaces. Peptide desorption and subsequent analysis by mass spectrometry was used to confirm the presence and integrity of the Trcs adsorbed. Scanning electron microscopy was utilised to show that the adsorbed peptides did not affect the structural integrity of the treated filters. However, it was shown that the adsorbed peptides changed the hydrophobic/hydrophilic character by means of a wettability assay. A cell viability assay and erythrocyte assay were developed from existing methodologies to determine the biological activity of the AMP-functionalised polymeric material. Seven of the AMP treated solid surfaces showed antimicrobial activity when challenged with >105 Micrococcus luteus cells/cm2. Although the polycarbonate filter lost antimicrobial activity at the high cell concentrations, it was shown to have potent antimicrobial activity at lower cell concentrations. Complete inhibition of M. luteus growth was observed for both the gramicidin S and tyrocidine extract treated high density cellulose and cellulose filters. Stability tests showed that the tyrocidines remained adsorbed to cellulose filters and biologically active when exposed multiple water washes, water washes at different temperatures (25°C - 100°C) and pH changes (pH 1-12). The antimicrobial activity was only affected after exposure to the water wash of pH 13 which is possible due to susceptibility of the CL filters to high pH solvents. A preliminary study on the effect of Trcs treated CL filters on the sterilization, germination and effect on tomato seedlings was conducted. It was found that Trcs had no effect on the germination and did not fully sterilise the seeds or environment against fungi. However, it was observed that 5 μg/mL Trcs treated filters promoted root length opposed to the toxic effect seen with filters treated with higher Trc concentrations. It is hypothesised that Trcs prefer to bind to hydrophilic surfaces exposing the hydrophobic residues and the cationic residue of the peptide to interact with the bacterial membrane to elicit its antimicrobial response. The exposed residues contain some of the hydrophobic residues and the cationic Orn9/Lys9, which are crucial to the antimicrobial activity of the peptides. Hydrophobic interaction is particularly important for the haemolytic activity which is currently the only viable method of detection of the adsorbed Trcs. Trcs also have a preference for adsorption onto cellulose and cellulose analogues which points to possible application in protective food wrapping and wood surface protection. Trcs maintains its antimicrobial activity regardless of adsorption to solid surfaces. It can therefore be concluded that Trcs treated solid surfaces hold great potential in preventing the initial bacterial colonization and subsequent biofilm formation. Antimicrobial peptide enriched solid surfaces can thus be developed and tailored to a specific application such as filters, catheters and packaging materials.

AFRIKAANSE OPSOMMING: Biovervuiling is die aanhegting en vorming van biofilms met negatiewe gevolge soos aanhoudende na-oes infeksies, infeksies op mediese inplantings en voortdurende oppervlak besoedeling van voedselverwerkings fabrieke. Die probleem lê grotendeels by die weerstand wat ontwikkel word teen konvensionele behandelings as gevolg van die vorming van volwasse biofilms. Die fokus het gevolglik verskuif vanaf die verwydering van biofilms na die voorkoming van aanvanklike aanhegting van organismes aan oppervlaktes. Dit behels die gebruik van antimikrobiese oppervlaktes wat of 'n inherente antimikrobiese aktiwiteit het, bv. sekere metale óf oppervlaktes wat aangepas is deur die aanhegting van antimikrobiese middels. Die aanhegting van antimikrobiese agente kan of deur kovalente binding óf adsorpsie plaasvind, afhangende van die beoogde gebruik van die oppervlak, sowel as die metode van werking van die antimikrobiese agent. Antimikrobiese peptiede (AMPe) is alomteenwoordig in die natuur, is geneig om 'n breë spektrum van aktiwiteit te hê, is baie stabiel en het getoon dat aktiwiteit in stand gehou word wanneer dit kovalent gebind word op soliede oppervlaktes. Tirosidiene (Trcs), antimikrobiese peptiede wat deur Bacillus aneurinolyticus geproduseer word, is siklodekapeptiede met 'n breë spektrum van aktiwiteit teen Gram-positiewe bakterieë, swamme, giste en die menslike malaria parasiet Plasmodium falciparum. Die doel van hierdie studie was om die antimikrobiese aktiwiteit te bepaal van oppervlaktes wat met 'n tirosidien ekstrak behandel is, te bepaal onder watter omstandighede die aktiwiteit stabiel bly en om te soek na moontlike toepassings van hierdie peptied-behandelde oppervlaktes. Die studie het gefokus op verskillende soliede oppervlaktes naamlik gemengde sellulose, polyvinylidene fluoried, polikarbonaat, sellulose asetaat, sellulose (papier)(CL) en 'n hoë digtheid sellulose verpakkings materiaal (HDC), as 'n loodsstudie om die antimikrobiese aktiwiteit van die Trcs en gramisidien S (GS) behandelde soliede oppervlaktes te ondersoek. Peptied-desorpsie en daaropvolgende ontleding deur massaspektroskopie is gebruik om die teenwoordigheid en integriteit van die geadsorbeerde Trcs te bevestig. Skandering elektronmikroskopie is gebruik om aan te toon dat die geadsorbeerde peptiede geen invloed op die strukturele integriteit van die behandelde filters het nie. Daar is egter getoon dat die geadsorbeerde peptiede die hidrofobiese / hidrofiliese karakter verander. „n Lewensvatbaarheid selgebaseerde toets en eritrosiet toets is ontwikkel uit bestaande metodes om die biologiese aktiwiteit van die AMP-gefunktionaliseerde polimeriese materiaal te bepaal. Sewe van die AMP behandel soliede oppervlaktes het antimikrobiese aktiwiteit getoon wanneer dit met > 105 Micrococcus luteus selle/cm2 gedaag is. Hoewel die polikarbonaat filter antimikrobiese aktiwiteit met hoë sel konsentrasies verloor het, is dit getoon dat dit wel uitgeproke antimikrobiese aktiwiteit het teen laer konsentrasies selle. Volledige inhibisie van M. luteus groei is waargeneem vir beide die hoë digtheid sellulose en sellulose filters wat met GS en tirosidien ekstrak behandel is. Stabiliteit toetse het getoon dat die tirosidiene geadsorbeer en biologies aktief op sellulose filters bly nadat dit blootgestel is aan verskeie water was-stappe, waterwasse by verskillende temperature (25 °C -100 °C) en pH veranderinge (pH 1-12). Die antimikrobiese aktiwiteit was net beïnvloed ná blootstelling aan die water met 'n pH 13, wat moontlik is te danke aan die vatbaarheid van die CL filters by hoë pH oplosmiddels is. 'n Voorlopige studie is gedoen om die uitwerking van Trcs behandelde CL filters op die sterilisasie, ontkieming en tamatiesaailinge te bepaal. Daar is gevind dat Trcs geen effek op die ontkieming het nie, maar dat dit nie volledig die sade en omgewing steriliseer vir fungiese groei nie. Daar is egter waargeneem dat 5 μg/mL Trcs behandelde filters wortel lengte van die saailinge bevorder teenoor die giftige uitwerking soos waargeneem vir die filters wat met hoër konsentrasies Trcs behandel is. Dit word gepostuleer dat Trcs verkies om aan hidrofiliese oppervlaktes te bind wat die van die hidrofobiese aminosure en die kationiese residu van die peptied blootstel om aan die bakteriële membraan te bind om gevolglik antimikrobiese reaksie te ontlok. Die blootgestelde deel bevat sommige van die hidrofobiese residue en positiewe Orn9/Lys9 wat noodsaaklik vir die antimikrobiese aktiwiteit van die peptiede. Die hidrofobiese interaksies is veral belangrik vir die hemolitiese aktiwiteit wat tans die enigste bruikbare metode van opsporing van die geadsorbeerde Trcs is. Trcs het ook 'n tendens vir adsorpsie op sellulose en sellulose analoë wat dui op die moontlike toepassing in beskermende voedselverpakking en die beskerming van houtoppervlaktes. Trcs handhaaf hul antimikrobiese aktiwiteit, ongeag van adsorpsie aan soliede oppervlaktes. Dit kan dus afgelei word dat Trcs-behandelde soliede oppervlaktes die potensiaal het om die aanvanklike kolonisasie van bakterië te voorkom en die daaropvolgende biofilm vorming. Antimikrobiese peptied verrykde soliede oppervlaktes kan dus ontwikkel en aangepas word vir gebruik in spesifieke toepassing soos in filters, kateters en verpakkingsmateriaal.

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