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Fluorescent triple staining
In this study, a triple staining method was used, based on the method described in Mangoni et al. (2004), using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), 4’,6-diamidino-2- phenylindole (DAPI) and fluorescein 5(6)-isothiocyanate (FITC). This triple stain enables the simultaneous viewing of total (stained by DAPI) and viable cells (stained by CTC), as well as cells with modified membrane permeability (stained by FITC). Stationary phase E. coli and B. subtilis cultures were adjusted to a cell density of 64×106 CFU/mL in 10 mM NaP buffer, pH 7.4. A volume of 90 μL of the cell suspension was exposed to 10 μL peptide for 10 minutes at 37°C in a shaking incubator. The final concentrations were 0.77 μM for Os and 1.74 μM for Os-C and concentrations 10x lower (0.07 μM and 0.17 μM). The cells were also exposed to the positive control, Mel, at a concentration of 2.5 μM for 10 minutes at 37°C in a shaking incubator. The bacterial suspensions were subsequently incubated with 900 μL of 5 mM CTC in 10 mM NaP buffer pH 7.4 for 2 hours at 37°C in a shaking incubator. To allow adhesion of cells to the surface of poly-L-lysine coated coverslips, the CTC-bacteria mixture was added to the wells of a 24-well Cellstar polystyrene plate (Greiner Bio-One GmbH, Kremsmünster, Austria) containing the coverslips and incubated at 30°C for 90 min. The coverslips were rinsed with NaP buffer, then a volume of 1 mL of 10 μg/mL DAPI in phosphate buffered saline (PBSa, 0.137 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) was added and incubated at 30°C for 30 min. The coverslips were rinsed with NaP buffer and 1 mL FITC solution (6 μg/mL in 10 mM NaP buffer) added and incubated at 30°C for 45 min. Thereafter the coverslips were rinsed with NaP buffer again, mounted on slides with antifade mounting medium (Sigma-Aldrich, Johannesburg, South Africa) and viewed with the Zeiss LSM 510 Meta Confocal Microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany). The excitation and emission wavelengths used for CTC were 450 nm and 630 nm, for FITC 490 nm and 520 nm and for DAPI 359 nm and 461 nm, respectively. Images of all three dyes were taken separately and overlaid with the Carl Zeiss AIM LSM imaging software into a single image containing the three colour signals.
Gel retardation assay
The DNA of prokaryotic cells is localised in the cytoplasm, has no nuclear membrane or associated histone proteins but exists as a supercoiled structure. Most bacteria also contain separate small pieces of DNA called plasmids (Actor, 2012). If AMPs cross the cell envelope, plasmid/genomic DNA, RNA and/or negatively charged proteins are therefore likely targets. The gel retardation assay was used to determine if peptides bind to plasmid DNA. Ionic interactions between the cationic peptide and plasmid DNA reduce the charge on the plasmid and subsequently the migration in the agarose gel is retarded. To investigate the effect of peptides on DNA, 2.5 μL 10 μg/mL pBR322 vector from E. coli (Sigma-Aldrich, Johannesburg, South Africa) was exposed to 2.5 μL of different concentrations of Os, Os-C and Mel for 1 hour at 37°C. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a positive control for DNA damage, and ddH2O was used as a negative control. A volume of 5 μL of loading solution (40% sucrose and 0.13% bromophenol blue) was added.
The samples were analysed on a 1% agarose gel in TAE buffer (0.8 mM Tris, 0.4 mM glacial acetic acid, 10 mM ethylenediaminetetraacetic acid, pH 8.0). The gel was post stained with a 3X staining solution of GelRed (Biotium, California, USA) as per the manufacturer’s instructions for 1 hour, and imaged with the UVIdoc HD5 gel documentation system (Uvitech, Cambridge, UK). The intensity of three points on each band was measured using the image analysis freeware ImageJ 1.45s (National Institutes of Health, USA), this was done twice. Multiple comparisons were tested by one-way ANOVA followed by the Tukey post hoc test to test for significant difference to the control (GraphPad Prism v6.01, California, USA).
Fluorescent intercalator displacement assay
To confirm results from the SYTOX green membrane permeabilisation assay and the gel retardation assay, the fluorescent intercalator displacement (FID) assay was used. Tse and Boger (2004) described the principle of the assay with ethidium bromide and thiazole orange. A fluorescent dye which intercalates with DNA results in an increased amount of fluorescence upon binding. A test compound which is DNA binding, will displace the intercalator and result in a decrease in fluorescence (Tse and Boger, 2004). The method for the current study was adapted from two previous studies which used ethidium bromide as intercalator (Geall and Blagbrough, 2000, Tse and Boger, 2004). SYTOX green has been shown to bind DNA by intercalation (Thakur and colleagues, 2015), and was chosen for the FID assay in the present study. A volume of 2.5 μL of 10 mg/mL pBR322 vector from E. coli (Sigma-Aldrich, Johannesburg, South Africa) in 0.1 M PBSb (81 mM Na2HPO4.2H2O, 19 mM NaH2PO4.H2O, 0.15 M NaCl, pH 7.4) was incubated with 95 μL of 1 μM SYTOX green in 10 mM NaP buffer in a 96-well polystyrene, flat bottom plate for 15 min at 37°C to allow for equilibration. A volume of 2.5 μL of Mel, Os and Os-C at concentrations of 1 – 50 μM was added and incubated for a further 60 min. The fluorescence intensity was then measured with the FLUOstar OPTIMA plate reader from BMG Labtechnologies (Offenburg, Germany) using an excitation wavelength of 492 nm and an emission wavelength of 520 nm. The fluorescence was expressed as the percentage of the maximum fluorescence of SYTOX green and was corrected for background fluorescence (free SYTOX green in solution) using the formula below. Each peptide concentration was repeated in triplicate.
Chapter 1: Introduction
1.1) Outputs
Chapter 2: Review of the literature
2.1) Immunity
2.2) Antimicrobial peptides
2.3) Background to this study
2.4) Aims
2.5) Objectives
Chapter 3: Mechanism of bacterial killing of Os and Os-C1
3.1) Abstract
3.2) Introduction
3.3) Materials and methods
3.4) Results
3.5) Discussion
3.6) Conclusion
Chapter 4: Effect of Os and Os-C on human blood cells
4.1) Abstract
4.2) Introduction
4.3) Materials and methods
4.4) Results
4.5) Discussion
4.6) Conclusion
Chapter 5: Antioxidant activity of Os and Os-C
5.1) Abstract
5.2) Introduction
5.3) Materials and methods
5.4) Results
5.5) Discussion
5.6) Conclusion
Chapter 6: Concluding discussion
6.1) Limitations and future perspectives
References
