Tyrocidines, cyclic decapeptides produced by soil bacilli, as potent inhibitors of fungal pathogens

Troskie, Anscha Mari (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The global rise in microbial resistance, ranging from the agricultural industry to the medical sector, has created the urgent need for novel or supplementary antibiotics. Antimicrobial peptides or “nature’s antibiotics” may be the answer to this major problem. In this study a group of antimicrobial peptides, cyclic decapeptides named tyrocidines, produced by the soil bacterium Bacillus aneurinolyticus, was investigated for their antifungal activity, possible mode of antifungal action and potential applications. The study illustrated that the tyrocidines have significant antifungal activity against a range of phytopathogens, including Fusarium solani and Botrytis cinerea, as well as the human pathogen Candida albicans. The activity of the tyrocidines is influenced by the identity of both the target organism and the media environment. Further evidence was obtained in support of the hypothesis that the tyrocidines are extremely sensitive to their environmental conditions and that they tend to self-assemble to form oligomers. The assessment of a small tyrocidine library and analogues, comprised of eight peptides, revealed no overt structure-activity relationships against fungal pathogens, except for the importance of a tyrosine residue. This indicated an important role for the conserved sequence of the tyrocidines, NQYVOLfP, together with the tendency of the tyrocidines to oligomerise into higher-order active structures in their antifungal activity. The tyrocidines were found to be membrane active toward the fungal pathogens. However, supporting evidence was also obtained for additional mode(s) of antifungal action for the tyrocidines which inter alia induces morphological abnormalities in filamentous fungal target cells. Furthermore, the results also indicated that the membrane activity of the tyrocidines may be influenced by additional factors to that of the composition of the target cell membrane, for instance components of the fungal cell wall. This investigation also indicated the significant potential of the tyrocidines to be developed for the commercial sector. The potent activity of the tyrocidines against agronomically important phytopathogens (significantly higher than the commercial fungicide bifonazole) together with their relative salt stability bodes well for their development as bio-fungicides for the agricultural sector. The tyrocidines also exhibited an overt sinergistic effect on the in vitro candidacidal activity of two key antifungal drugs, caspofungin and amphotericin B. Furthermore, tyrocidine A and caspofungin exhibited synergistic activity in vivo which had a significant positive effect on the survival of C. albicans infected Caenorhabditis elegans. Latter results highlighted their potential to serve as candidates for combinatorial treatment in the medical industry.

AFRIKAANSE OPSOMMING: Die globale verskynsel van mikrobiese weerstand, wat strek vanaf die landbou sektor tot in die mediese bedryf, het ’n dringende behoefte vir die ontwikkeling van nuwe antmikrobiese middels geskep. Antimikrobiese peptiede of “die natuur se antibiotika”, kan moontlik die antwoord op hierdie ernstige problem wees. Tydens hierdie studie is ‘n groep sikliese antimikrobiese peptiede, naamlik die tirosidiene wat deur die grondbakterium Bacillus aneurinolyticus geproduseer word, vir hulle antifungiese aktiwiteit, hulle moontlike meganisme(s) van antifungiese werking en hulle potensiёle aanwendings bestudeer. Hierdie studie het getoon dat die tirosidiene uitsonderlike antifungiese aktiwiteit teen ‘n reeks fitopatogene, insluitend Fusarium solani en Botrytis cinerea, asook teen die mens patogeen Candida albicans het. Die aktiwiteit van die tirosidiene is deur beide die identiteit van die teikenorganisme sowel as die mediumomgewing beїnvloed. Daar is ook verdere bewyse verkry wat die hipotese dat tirosidiene uiters sensitief is tot hulle omgewing en dat hulle neig om te oligomeriseer, ondersteun. Die studie van die klein tirosidien-biblioteek, saamgestel uit agt tirosidiene en analoё, het geen ooglopende struktuur-aktiwiteit verwantskappe opgelewer nie, behalwe vir die oёnskynlike invloed van die tirosien-residu. Laasgenoemde het die belangrikheid van die gekonserveerde aminosuurvolgorde van die tirosidiene, NQYVOLfP, asook die neiging van tirosidiene om hoё-orde aktiewe strukture te vorm deur self-verpakking, beklemtoon. Tydens die studie is daar gevind dat die tirosidiene membraan-aktiewiteit toon teenoor fungiese patogene. Daar is egter ook goeie bewyse vir alternatiewe meganisme(s) van antifungiese werking, wat ondermeer tot morfologiese abnormaliteite in filamentagtige fungi-teikenselle lei, vir die tirosidiene verkry. Die resultate het verder ook daarop gewys dat die membraan-aktiwiteit van die tirosidiene ook deur ander faktore, soos deur komponente van die fungiese selwand, en nie net deur die samestelling van die fungiese membraan beїnvloed word nie. Hierdie ondersoek het ook die aansienlike potensiaal van die tirosidiene vir kommersiёle ontwikkeling en gebruik uitgelig. Die merkwaardige aktiwiteit van die tirosidiene teen fitopatogene van agronomiese belang (wat selfs beter as diè van die kommersiёle swamdoder bifonazole was) tesame met die relatiewe sout stabiliteit van die tirosidiene, is belowende tekens om die tirosidiene as bio-swamdoders vir die landbou sektor te ontwikkel. Die tirosidiene het ook ‘n uitgesproke sinergistiese effek op die in vitro candidasidiese aktiwiteit van twee sleutel antifungiese middels, caspofungin en amphotericin B, getoon. Verder is daar in vivo sinergistiese aktiwiteit gewys deur die kombinasie van tirosidien A en caspofungin wat ’n beduidende positiewe effek op die oorlewing van C. albicans geïnfekteerde Caenorhabditis elegans gehad het. Laasgenoemde dui op die potensiaal van die tirosidiene om in die mediese bedryf as kandidate vir kombinasie-behandeling te dien.

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