Investigation of complexation and antimicrobial activity of gramicidin S in the presence of lipopeptides from Bacillus subtilis

Vlok, Nicolaas Mare (2005-03)

Thesis (PhD)--Stellenbosch University, 2005.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/50379
This item appears in the following collections: