Discovery of antifungal compounds produced by Pelargonium sidoides-associated microorganisms

Du Toit, Lauren Anita (2020-04)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Pathogenic fungi cause pervasive diseases with a global impact on human health, agricultural crop yields, food security and natural biodiversity. The incidence of fungal infections continues to rise with increasing rates of drug resistance as a result of the wide- spread use of antifungals in the medical and agricultural sectors. New drug-tolerant pathogenic fungal species also emerge in the wake of global warming. Precariously, only a few new antifungal drugs are in development as these compounds must target fungal cells exclusively, to negate any toxic effects on co-treated mammalian and plant cells. In search of new antifungal drugs, antimicrobial peptides such as lipopeptides (LPs) have received particular attention. In plants, microbial LP producers improve the plant’s overall health by inducing systemic resistance and direct antagonism towards invading pathogens. In this study, tubers of the traditional South African medical plant, Pelargonium sidoides were selected as a potential source of LP-producing microbiota. It has previously been shown that P. sidoides has potent antimicrobial properties. However, the microbial species associated with P. sidoides, and the bioactive compounds they produce, remain mostly unknown. Therefore, this study aimed to characterise these P. sidoides-associated microbes and some of their bioactive compounds, in terms of their structure, haemolytic activity and antifungal activity against a panel of human and plant fungal pathogens. The panel included Candida albicans, Cryptococcus neoformans, Botrytis cinerea and Fusarium oxysporum f. sp. cubense as they are closely related to antibiotic resistant strains which continue to threaten human health, most severely in sub-Saharan Africa, and agriculture, globally. A total of 68 microbial isolates were characterised from wild P. sidoides tubers. Four of these isolates had antifungal activity against the entire panel of fungal pathogens. Using molecular techniques, the isolates were identified as two Penicillium and two Bacillus species. Optimisation of culture conditions for antifungal LP production led to the characterisation of two LPs, fengycin A and iturin AQ, produced by Bacillus sp. YC2. These LPs were characterised using ultra-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Iturin AQ may be a structural variant of iturin A, a well-known antifungal LP produced by many Bacillus species. Extracts containing fengycin A and iturin AQ showed antifungal activity against the yeast cells of C. neoformans and C. albicans and the spore and mycelial cell morphologies of B. cinerea and F. oxysporum. These findings suggest that LP extracts produced by isolate YC2 could be applied to treating these pervasive fungal pathogens in future studies.

AFRIKAANSE OPSOMMING: Middel weerstandige patogeniese swamme veroorsaak steeds deurdringende siektes wat die wêreld se sterftesyfers, die opbrengste van landbougewasse, voedselsekerheid, natuurlike biodiversiteit en verskeie vervaardigingsprosesse beïnvloed. Hierdie infeksies duur voort soos antifungale weerstandigheid voortgaan om te ontwikkel na aanleiding van die wydverspreide toediening van dieselfde antifungale geneesmiddelklasse op dieselfde mediese en landboupatogene. Hierdie patogene is nou goed aangepas om effektiewe behandeling te ontduik. Biomediese navorsing het begin om antimikrobiese peptiede, insluitend antifungale lipopeptiede (LPs) te karakteriseer in die soeke na lukrake antifungale medisinale oplossings. Hierdie amfipatiese verbindings word hoofsaaklik as verdedigende verbindings vervaardig om spesifieke omgewingsantagoniste af te weer. Antagoniste sluit swamspesies in wat meeding om oorlewing op die medisinale plante se oppervlaktes. Dit word voorgestel dat hierdie LP-mikrobiese produsente die plant se gesondheid verbeter deur sistemiese weerstandigheid (ISR) te veroorsaak of deur ‘n bydrae te lewer tot die plant se antimikrobiese eienskappe. In hierdie studie was die Suid-Afrikaanse geranium (Pelargonium sidoides) geselekteer as 'n monster bron vir LP-produserende mikrobes. Hierdie medisinale plant het kragtige antimikrobiese eienskappe. Die mikrobiese spesies wat met P. sidoides geassosieer word, en die bioaktiewe verbindings wat hulle produseer, bly egter ongeïdentifiseer en nie gekenmerk nie. In hierdie studie was 68 suiwer kolonies geïsoleer en gekenmerk deur wilde knolle van P. sidoides. Vier van hierdie mikrobiese isolate het antifungale werking teen Botrytis cinerea, Candida albicans, Cryptococcus neoformans en Fusarium oxysporum f. sp. cubense. Hierdie mikrobes was geselekteer vir molekulêre identifikasie en geïdentifiseer as Penicillium en Bacillus sp. Hulle is ook gekweek vir antimikrobiese LP-produksie en twee LP's, fengycin A en iturin AQ, gekenmerk deur UPLC-MS/MS. Een gekarakteriseerde LP kan 'n strukturele variant van iturien A. wees. Uittreksels wat die twee LP's bevat, het antifungale aktiwiteit teen die geselekteerde patogeniese swamme as gisselle, spore en swam hifes gehad. As dieselfde LP's in toekomstige studies in P. sidoides-uittreksels (EPs 7630) opgespoor word, kan dit bydra tot die antifungale werking van EP's 7630. Hierdie bevindings sal die ekologiese belang van mikrobiese LP's vir plante en hul gepaardgaande mikrobes bevestig, en die potensiaal om meer van hierdie verbindings te gebruik uitlig om deurdringende swamsiektes in toekomstige medisinale en landboukundige toepassings te behandel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108455
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