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Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028)
Dissertation (MSc (Biochemistry))--University of Pretoria, 2020.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2020
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| _version_ | 1867613610835968000 |
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| access_status_str | Open Access |
| author2 | Gaspar, A.R.M. (Anabella Regina Marques) |
| author_browse | Gaspar, A.R.M. (Anabella Regina Marques) |
| author_facet | Gaspar, A.R.M. (Anabella Regina Marques) |
| collection | Thesis |
| dc_rights_str_mv | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc (Biochemistry))--University of Pretoria, 2020. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/75104 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:38:53.515Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/75104 Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) Gaspar, A.R.M. (Anabella Regina Marques) u04667124@hotmail.co.za Bester, Megan J. Taute, Helena Moller, Dalton Sharl Antimicrobial peptides Drug discovery Biotherapeutics UCTD Dissertation (MSc (Biochemistry))--University of Pretoria, 2020. Candida albicans is a fungus found in the normal biota of humans, but in immuno-compromised individuals, C. albicans forms complex biofilms on the surface of medical prosthetics, skin, oral cavities, the urinary tract, and other epithelial cell layers. Biofilms and the development of drug resistance has limited treatment options. Antimicrobial peptides (AMPs) are increasingly becoming attractive therapeutic agents for the treatment of these infections due to their multifunctional properties, multiple cellular targets, and the lower incidence of resistance development. Previous studies have shown that Os, an AMP derived from the tick defensin OsDef2, has antifungal activity against C. albicans. Preliminary antifungal mode of action studies indicated that Os induces the formation of reactive oxygen species although not a primary mode of killing. Os causes membrane permeabilization, which is inhibited by an excess of free laminarin and mannan. Furthermore, Os was shown to bind plasmid DNA but was inactive in high salt conditions. The aim of this study was to further explore the mode of action of Os in planktonic C. albicans (ATCC 90028) cells. A modified microbroth dilution assay was developed to allow rapid screening of salt sensitive AMPs such as Os. With this method the IC50 of the positive control, amphotericin B (AmpB), and Os were determined as 0.547 ± 0.056 μM and 1.163 ± 0.116 μM, respectively. The effects of AmpB and Os on cellular morphology were evaluated using scanning electron microscopy and transmission electron microscopy at their respective IC25, IC50 and IC75 values. When comparing the effects of Os with AmpB on the cell wall and membrane, Os had more severe and nonspecific effects. Os induced the formation of pits on the cell surface and pores in the cell membrane, as well as increased budding scars. Using isothermal titration calorimetry, no interaction between Os and the fungal cell wall components, mannan and laminarin, could be detected. Factors such as the lack of tryptophan and aspartate residues as well as β-sheet secondary structures may account for the lack of interaction. However, with the modified microbroth dilution assay in the presence of excess of mannan or laminarin (20 mg/mL), reduced activity from Os was observed. The formation of soluble macro-complexes between Os and the cell wall components at high concentrations may account for reduced activity. The ability of Os to cause membrane depolarization was evaluated with bis-(1,3-dibutylbarbituric acid) trimethine oxonol. The control, melittin, caused a linear increase in depolarization with a significant increase at 0.63 μM, while Os caused a sigmoidal increase in depolarization with a significant increase at 2.5 μM. Therefore, membrane depolarization occurs following membrane permeabilization which occurs at 2 μM. Finally, the localisation of 0.5 μM and 6.4 μM (IC25, IC75) 5-FAM-Os, and concurrently the effect on vacuoles loaded with CellTracker Blue-CMAC, was determined with flow cytometry and confocal laser scanning microscopy (CLSM). Findings were that Os, at a concentration below its IC50, binds to the cell membrane, then translocates and binds DNA. At a concentration above its IC50, Os accumulates in the cytoplasm and causes destruction of membranes, including that of vacuoles, leading to cell death. In conclusion, this study shows that Os is a membrane acting AMP that can be further developed for clinical application as an antifungal drug. NRF Biochemistry MSc (Biochemistry) Unrestricted 2020-07-09T08:48:15Z 2020-07-09T08:48:15Z 2020-09 2020-07 Dissertation Moller, DS 2020, Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028), MSc (Biochemistry) Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/75104> S2020 http://hdl.handle.net/2263/75104 en © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Antimicrobial peptides Drug discovery Biotherapeutics UCTD Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title | Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title_full | Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title_fullStr | Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title_full_unstemmed | Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title_short | Antifungal mode of action studies of an antimicrobial peptide, Os, in planktonic Candida albicans (ATCC 90028) |
| title_sort | antifungal mode of action studies of an antimicrobial peptide os in planktonic candida albicans atcc 90028 |
| topic | Antimicrobial peptides Drug discovery Biotherapeutics UCTD |
| url | http://hdl.handle.net/2263/75104 |