Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: The implication of biologically active peptides from different organisms on one another in
complex ecological communities is largely unknown at this stage. The elucidation of the
nature of this influence may have practical implications in terms of organism resistance and
the conservation of an optimal agricultural environment. This study was aimed to elucidate
the effect of antimicrobial peptides from different co-habitational organisms, on each other,
both in terms of bioactivity and interaction. The two peptides investigated were gramicidin S,
a decapeptide from Bacillus brevis, and surfactin, a heptalipopeptide from Bacillus subtilis.
Preliminary studies were also done on iturin A and synthetic analogues of iturin A and iturin
C, both octalipopeptides from Bacillus subtilis.
Analytical antimicrobial assay systems were used to study the effect of surfactin on the
antibiotic action of gramicidin S towards three different target cells namely, a Gram-positive
bacterium (Micrococcus luteus), a Gram-negative bacterium, (Escherichia coli) and a fungus
(Penicillium corylophilium). The investigation of the antifungal activity was hampered by the
insensitivity and subjectivity of the majority of antifungal assays and necessitated the
development of two new testing methodologies.
The investigation showed that surf actin had an antagonistic effect on the antimicrobial
activity of gramicidin S against all three of the target cells. This antagonism is dose-dependent
at concentrations lower than required for surfactin to exert biological activity. Electrospray
mass spectrometry (ESMS) showed the formation of surfactin-gramicidin S complexes in 1:1
and 2: 1 ratios with enhanced complex formation in an apolar environment. Dissociation
experiments indicated that the peptide complexes were slightly less stable than the peptides
alone. The presence of NaCI up to 80 mM had little effect on the stability of preformed
complexes. Incubating surfactin with NaCI and CaCh before titration with gramicidin S also
did not affect complex formation. Furthermore, results from the pre-incubation studies with
CaCh indicated that surfactin-gramicidin S complexes might be formed through the
displacement of the metal ion. The mechanism of this displacement is unlikely to be direct
competition but rather the result of conformational' changes induced by peptide-peptide
interaction/interactions. A likely point of interaction the p-tums in the peptide ring.
Linear iturin A2 and iturin C analogues were synthesised (8-Beta and 8-Betac) with solid
phase peptide synthesis and purified using self-assembly and high performance liquid chromatography. The products of the syntheses wete analysed by ESMS and found to be
correct. The products, together with commercially obtained iturin A, were used in biological
assays and it was found that iturin A antagonises the antibiotic activity of gramicidin S but the
linear analogues had no effect. Complex formation between iturin A and gramicidin S was
observed using ESMS but no complexes were detected for the analogues, which reinforces the
hypothesis that antagonism is related to the formation of inactive complexes.
In general, the formation of peptide-gramicidin S complexes may indicate that a defence
mechanism may be present in which toxic peptides of the competitor organism are inactivated
by peptides from co-habiting organisms. / AFRIKAANSE OPSOMMING: Die invloed wat biologiese aktiewe verbindings van verskillende mikro-organismes in
komplekse ekologiese omgewings op mekaar het, is onbekend. Die ontrafeling van die rol
mag verskeie vrae ten opsigte van weerstandbiedenheid en ontwrigting van ekologiese
landbou-omgewings beantwoord. Die doel van hierdie studie was om die invloed wat
antimikrobiese peptiede, afkomstig van verskillende ko-habiterende organismes, op mekaar
het te ondersoek- beide in terme van biologiese aktiwiteit en interaksie. Die twee peptiede
wat ondersoek is, was gramisidien S, 'n dekapeptied geproduseer deur Bacillus brevis en
surfaktien, 'n heptalipopeptied, geproduseer deur Bacillus subtilis. Voorlopige ondersoeke is
ook uitgevoer op iturin A, en sintetiese iturin A en iturin C analoë, beide oktalipopeptiede van
B. subtilis.
Analitiese antimikrobiese toetsstelsels is gebruik om die effek van surfaktien op die
biologiese aktiwiteit van gramisidien S te bepaal. Drie teikenselle is gebruik nl. 'n Grampositiewe
bakterium (Micrococcus luteus), 'n Gram-negatiewe bakterium (Escherichia coli)
en 'n fungus (Penicillium corylophilium) Die gebrek aan sensitiwiteit van bestaande
antifungiese toetsstelsels het die ontwikkelling van twee nuwe toetstelsels genoodsaak.
Die ondersoek het aangetoon dat surfaktien 'n antagonistiese effek op gramisidien S se
antimikrobiese werking teen al drie teikenselle het. Die antagonisme is waarneembaar by
surfaktien konsentrasies veel laer as wat nodig is vir biologiese aktiwiteit. Elektrosproeimassaspektrometrie
(ESMS) van surfaktien en gramisidien S mengsels het aangedui dat
komplekse in 'n 1:1 en 2: 1 stoichiometrie voorkom. Die vorming van peptiedkomplekse word
ook deur 'n nie-polêre omgewing bevorder. Die stabiliteit van die peptiedkomplekse is ook
geëvalueer met dissosiasie eksperimente en daar is gevind dat die komplekse minder stabiel is
as die peptiede alleen - dit is 'n aanduiding van kompleksdissosiasie. Die teenwoordigheid
van NaCI tot en met 80 mM het 'n minimale invloed op die stabiliteit van voorafgevormde
peptiedkomplekse gehad. Inkubasie van surfaktien met NaCl en CaCh voor titrasie met
gramisidien S, het ook nie die vorming van peptiedkomplekse beïnvloed nie en die studies het
aangetoon dat die komplekse moontlik gevorm word deur die verplasing van die alkaliemetaalioon.
Dit is onwaarskynlik dat die meganisme van ioon-verplasing direkte kompetisie
is, maar eerder as gevolg van interaksie in een van die p-draaie. Liniêre iturin A2 en liniêre iturin Canaloë (8-Beta en 8-Betac) is gesintetiseer met behulp van
soliede fase peptiedsintese en gesuiwer deur middel van "self-assembly" en "high
performance liquid chromatography (HPLC)". Volgens die ESMS analise is die korrekte
produkte verkry. Die analoë en kommersieel beskikbare iturin A is aan biologiese toetsing
onderwerp en daar is gevind dat iturin A, maar nie die analoë nie, die antibiotiese effek van
gramisidien S ophef. Die vorming van iturin en gramisidien S komplekse, wat met ESMS
waargeneem is, versterk die teorie dat opheffing van aktiwiteit verband hou met die vorming
van inaktiewe komplekse. Verder, die analoë het nie komplekse met gramisidien S gevorm
me.
Dit blyk vanuit hierdie studies dat die vorming van peptied komplekse moontlik deel kan
uitmaak van 'n tipe verdedigingsmeganisme waar toksiese peptiede van kompeterende
organismes, deur peptiede van ko-habiterende organismes, geïnaktiveer word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50379 |
| Date | 03 1900 |
| Creators | Vlok, Nicolaas Mare |
| Contributors | Rautenbach, M., Snoep, J., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 1 v. (various pagings) : ill. |
| Rights | Stellenbosch University |
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