Production, characterisation and activity of selected and novel antibiotic peptides from soil bacteria
| dc.contributor.advisor | Rautenbach, Marina | en_ZA |
| dc.contributor.author | Laubscher, Wikus Ernst | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Biochemistry. | en_ZA |
| dc.date.accessioned | 2016-12-22T14:22:30Z | |
| dc.date.available | 2019-12-31T03:00:12Z | |
| dc.date.issued | 2016-12 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2016. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: The ever increasing development of pathogen resistance towards conventional antibiotics has necessitated the search for novel antimicrobial molecules. It has been suggested that antimicrobial peptides will form the foundation of a new generation of antibiotics. These small natural antibiotics possess rapid killing mechanisms against a broad spectrum of pathogens. They often disrupt multiple cellular targets resulting in decreased risk of resistance, making them ideal candidates as novel antimicrobials. Selection of a screening source is challenging as antimicrobial peptides are ubiquitously produced by most organisms. However, soil bacteria have historically been shown to produce a large variety of clinically significant antibiotics, including antimicrobial peptides. Furthermore, the soil biome possesses a vast bacterial diversity that has been to date largely unexplored, making it an ideal resource for the discovery of novel antimicrobial peptides. The overall objective of this study was therefore to isolate, identify and characterise novel antimicrobial peptides from soil bacteria. A commercial soil additive containing a lower bacterial diversity than natural soil environments was first used to validate a method for novel antimicrobial peptide discovery. With the goal of discovering novel antimicrobial peptides, this method included the isolation, identification and characterisation of antimicrobial producing bacteria and their active components. Only two of the isolates from the soil additive, denoted LB.4 and LB.5, were selected for further purification and characterisation of their antimicrobial compounds. The LB.4 and LB.5 isolates were determined to be strains of Brevibacillus laterosporus and Bacillus licheniformis respectively. In this dissertation they are referred to as Br. laterosporus LB.4 and B. licheniformis LB.5. The antimicrobial compound produced by B. licheniformis LB.5 was determined to be the well–known antimicrobial peptide, bacitracin A. It was found that Br. laterosporus LB.4 produces two potentially novel antimicrobial peptides termed LB.4-1223 and LB.4-1273. Although the amino acid composition was shown to be: F, L/I, M, N, P, V and Y, the amino acid sequences remain to be determined and their novelty could therefore not be confirmed. Brevibacillus parabrevis was used as a positive control as it is known to produce antimicrobial peptides from the tyrocidine group. The biological activity and mode of action data of the isolated peptides were therefore compared to that of tryptocidine C, a characterised analogue from the tyrocidine group of peptides. Antimicrobial dose response analysis revealed that LB.4-1223, LB.4-1273 and tyrocidine C possess antimicrobial and haemolytic activity, while bacitracin A only showed potent antimicrobial activity. Biophysical studies indicated that both bacitracin A and tryptocidine C disorientated lipid bilayers, however, only tryptocidine C resulted in the formation of transmembrane pores. This was the first study to show pore formation by tryptocidine C. It is known that bacitracin elicits its antimicrobial activity by inhibiting peptidoglycan synthesis, but this study provided the first insight into the interactions between bacitracin A and cellular membranes. However, whether these interactions results in microbial inhibition is still unknown. Screening of an environmental soil sample yielded three isolates with significant antimicrobial activity and suitable low molecular mass spectra to suggest antimicrobial peptide production. A literature study suggested that one of the isolates produces peptides from the bogorol group of antimicrobial peptides, while the other two isolates produce previously uncharacterised compounds. These compounds will be investigated in future studies. This dissertation describes an antimicrobial discovery and characterisation study that led to the discovery of novel antimicrobial peptides/compounds. Furthermore, it was shown that the laborious, time consuming nature of traditional microbiological screening methodology demands that a more effective, higher-throughput methodology be developed to meet the demand for novel antibiotics discovery. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Die onbeheerde toename in patogeen-weerstand teenoor konvensionele antibiotika het die soektog na nuwe antibiotiese molekules genoodsaak. Daar word gespekuleer dat antimikrobiese peptiede die basis van ʼn nuwe generasie antibiotika sal vorm. Hierdie klein, natuurlik geproduseerde antibiotika besit vinnige uitwissingsmeganismes teen a breë spektrum patogene. Antimikrobiese peptiede onderdruk dikwels veelvuldige sellulêre teikens wat lei tot ʼn verlaagde weerstandigheidsrisiko, wat hul dus ideale kandidate maak vir nuwe antimikrobiese terapieë. Meeste organismes produseer antimikrobiese peptiede wat die keuse van siftingsmateriaal bemoeilik. Histories is dit wel duidelik dat bakterieë afkomstig van grond ʼn groot verskeidenheid klinies-belangrike antibiotika produseer, insluitend antimikrobiese peptiede. Die grond bioom besit ook ʼn enorme bakteriële diversiteit wat nog grootliks onverkend is en is dus ʼn ideale hulpbron in die soektog vir nuwe antimikrobiese peptiede. Daarom is die hoofdoel van hierdie studie om nuwe antimikrobiese peptiede afkomende van grond bakterieë te isoleer, identifiseer en te karakteriseer. Eerstens is ʼn kommersiële grondbymiddel, wat ʼn laer verskeidenheid van bakteriële spesies in vergelyking met natuurlike grond omgewings bevat, gebruik om ʼn geldige metodiek te ontwikkel vir die ontdekking van nuwe antimikrobiese peptiede. Isolasie, identifikasie en karakterisering van antimikrobiese bakterieë en hul antimikrobiese aktiewe molekules is ingesluit in hierdie metodiek met die doel van nuwe antimikrobiese peptied ontdekking. Net twee van die grondbymiddel-isolate, genoem LB.4 en LB.5, is geselekteer vir verdere suiwering en karakterisering van hul antimikrobiese komponente. Die LB.4 en LB.5 isolate was as Brevibacillus laterosporus en Bacillus licheniformis geïdentifiseer en word na Br. laterosporus LB.4 en B. licheniformis LB.5 verwys in hierdie skripsie. Daar is beplaal dat die antimikrobiese molekule wat deur B. licheniformis LB.5 geproduseer word die wel-bekende antimikrobiese peptied basitrasien A is. Daar is verder gevind dat twee, moontlike nuwe, antimikrobiese peptiede geproduseer word deur Br. laterosporus LB.4, genoem LB-1223 en LB-1273. Die peptiede se aminosuursamestelling was vasgestel as F, L/I, M, N, P, V en Y, tog kon die uniekheid van die nuwe peptiede nog nie bevestig word nie weens die onbekende aminosuurvolgorde, wat eers in toekomstige studies ondersoek sal word. Brevibacillus parabrevis is bekend vir die produksie van die antimikrobiese peptiede van die tirosidien groep. Inligting van biologiese aktiwiteit en meganisme van werking studies op die geïsoleerde peptide is vergelyk met die gekarakteriseerde tirosidien analoog, triptosidien C. Antimikrobiese dosisrespons analise het bevestig dat LB.4-1223, LB.4-1273 en triptosidien C beide antimikrobiese en hemolitiese aktiwiteit besit, terwyl basitrasien A het egter net kragtige antimikrobiese aktiwiteit getoon het. ʼn Biofisiese ondersoek het aangedui dat beide basitrasien A en triptosidien C dubbellaag membrane disoriënteer, maar dat net triptosidien C transmembraanporieë vorm. Dit was die eerste bewys dat triptosidien C porieë in lipiedmembrane vorm. Dit word algemeen aanvaar dat basitrasien se hoofmeganisme van werking die inhibisie van peptidoglikaansintese behels. In hierdie studie is die eerste insigte verskaf tot die interaksie van basitrasien A met dubbellaagmembrane. Dis nog onbekend of hierdie membraan interaksie wel ʼn rol in mikrobiese inhibisie het. In die ondersoek op ʼn omgewingsgrondmonster is net drie isolate gevind met betekenisvolle antimikrobiese aktiwiteit en lae molekulêre massa spektra wat dui op moontlike antimikrobiese peptiede produksie. Uit literatuurstudies het dit geblyk dat een van die isolate peptiede vanuit die bogorol groep van antimikrobiese peptiede produseer, terwyl die oorblywende isolate ongekarakteriseerde verbindings/peptide produseer. Die bogenoemde verbindings/peptiede sal verder ondersoek word in toekomstige studies. Hierdie tesis beskryf ’n antimikrobiese ontdekkings- en karakteriseringstudie wat gelei het tot die ontdekking van nuwe antimikrobiese verbindings/peptiede. Verder beklemtoon die studie die moeisame, tydrowende aard van tradisionele mikrobiese siftingsmetodologie en sodoende die noodsaak vir nuwe, hoër-deurset metodologie om aan die aanvraag na nuwe antibiotka te voldoen. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.embargo.terms | 2019-12-31 | |
| dc.format.extent | xvii, [161] leaves : illustrations (some color) | |
| dc.identifier.uri | http://hdl.handle.net/10019.1/100378 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Peptide antibiotics | en_ZA |
| dc.subject | Peptides -- Therapeutic use | en_ZA |
| dc.subject | Microbial peptides | en_ZA |
| dc.subject | Soil microbiology | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.subject | Anti-infective agents | en_ZA |
| dc.subject | Bacitracin | en_ZA |
| dc.subject | Tryptocidine C | en_ZA |
| dc.title | Production, characterisation and activity of selected and novel antibiotic peptides from soil bacteria | en_ZA |
| dc.type | Thesis | en_ZA |