Browsing by Author "Mamhende, Precious Gamuchirai Muchaneta"

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    Investigation of the antifungal activity of tryptophan-rich cyclic peptides
    (Stellenbosch : Stellenbosch University, 2019-03) Mamhende, Precious Gamuchirai Muchaneta; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The global population of immunocompromised individuals has been rising due to the immunosuppressive nature of advanced medical interventions such as chemotherapy for cancer and immunosuppressive conditions such as HIV infection. Concurrent with this is the increasing incidence of life threatening systemic fungal infections as well as the growing resistance towards existing antifungal drugs. As a result, there is an urgent need for the discovery and development of novel drugs of antifungal therapy. Antimicrobial peptides present as promising candidates for this role due to their broad-spectrum antimicrobial activity and the limited potential for the evolution of resistance against them. In this study, the potential of small AMPs to serve as therapeutic agents against fungal infections was investigated by characterising the antifungal activity and mode of action of natural cyclodecapeptides and synthetic RW-hexapeptides against a human fungal pathogen, Aspergillus fumigatus. In addition, biophysical characterisation of their structures was conducted to evaluate the role of structure in antifungal activity. Biophysical characterisation of the secondary structure of cyclodecapeptides confirmed their conserved β-sheet structure. Structural characterisation of cyclic RW-hexapeptides highlighted their secondary structure to be quite stable and to consist of β-sheet and β-turns. On the other hand, the secondary structure of the linear analogues was shown to be flexible, changing from highly irregular (undefined) structures to more defined structures as the polarity of the environment was altered. The cyclodecapeptides and RW-hexapeptides potently inhibited the germination and growth of A. fumigatus spores highlighting the potential of both peptide groups as antifungal agents. However, the cyclodecapeptides exhibited pronounced haemolytic activity which compromised their selectivity for the fungal target. In contrast, the RW-hexapeptides had little to no haemolytic activity which translated to a greater selectivity for the fungal pathogen. A partial characterisation of the mode action employed by the cyclodecapeptides and the RWhexapeptides was done. The cyclodecapeptide, tryptocidine C (WC), rapidly induced the uptake of the membrane impermeable dye, SYTOX Green (SG) in A. fumigatus hyphae. This result confirmed the previously described membranolytic mode of action of the cyclodecapeptides. In contrast, RW-hexapeptides were not as effective at inducing the uptake of SG, indicating that membrane lysis is not their principal mode of action. Thus, the mode of action of RW-hexapeptides remains unknown but ROS could potentially be involved in the antifungal mechanism of action as one of the cyclohexapeptides, cWWW, significantly increased the accumulation of endogenous ROS in A. fumigatus hyphae. Furthermore, the slightly antagonistic interactions between WC and the most active RW-hexapeptide analogues, especially cWWW, in the peptide combination studies could indicate that the two groups of peptides may share a target but act on it differently. While RW-hexapeptides may not be lytic, the membrane could still be their target meaning that the lytic activity of WC prevents them from acting on their target hence the antagonistic interactions. In conclusion, the demonstrated selectivity of the RW-hexapeptides renders them very attractive for potential development as antifungal agents for systemic use.