PAF26 is a synthetic hexapeptide that has been shown to be highly effective at killing filamentous fungi whilst showing low toxicity against human and bacterial cells. Unlike membrane permeabilising antimicrobials, PAF26 at low fungicidal concentrations is endocytically internalised by fungal cells and accumulates in the vacuole, which undergoes expansion. At a certain point, PAF26 is transported out of the vacuole into the cytoplasm and this is followed by cell death. Previously, cytosolic free Ca2+ ([Ca2+]cyt) has been shown to exhibit a dose-dependent increase in response to PAF26 but the significance of this was not understood. The main objective of my PhD research was to analyse what role(s) Ca2+ signalling has in the mode-of-action of PAF26 using Neurospora crassa as an experimental model. In the first part of my research, evidence for Ca2+ signalling having a significant role in the PAF26 mode-of-action was obtained by testing the PAF26 sensitivity of deletion mutants defective in different components of their Ca2+ signalling machinery. The Deltacch-1, Deltayvc-1 and Deltanca-2 strains were found to exhibit the greatest resistance against PAF26. Screening of a range of vacuolar fusion mutants provided evidence for PAF26-induced vacuolar expansion involving the fusion of small vacuoles. Evidence was also obtained for PAF26-induced programmed cell death occurring via the pathway involved in HET-C-mediated heterokaryon incompatibility. In the second part of my research, I used live-cell imaging with fluorescently labelled PAF26 to investigate the pattern and kinetics of peptide interaction, internalization and distribution within the cells of the Deltacch-1, Deltayvc-1 and Deltanca-2 PAF26-resistant strains compared with the parental wild type. From these studies I obtained evidence for: CCH-1 being required for endocytic internalization of PAF26; YVC-1 being required for PAF26-induced vacuolar expansion; NCA-2 being involved in PAF26 binding to the cell envelope and subsequent endocytic cell internalization and transport to vacuoles; and cell death not being simply induced by PAF26 making contact with the cytoplasm. In the third part of my research I used the genetically encoded bioluminescent Ca2+ reporter aequorin to investigate differences in the average [Ca2+]cyt responses to PAF26 in cell populations of the Deltacch-1, Deltayvc-1 and Deltanca-2 mutant strains compared to that of the wild type. The Ca2+ signatures of the dose-dependent [Ca2+]cyt increases of each mutant were significantly different to each other and to the wild type, and external Ca2+ was required to initiate the [Ca2+]cyt responses. Evidence was obtained for an as yet unknown mechanism of Ca2+ entry into the fungal cell that was independent of the Ca2+ channels, CCH-1 and YVC-1.In the fourth part of my research, I used the genetically encoded fluorescent Ca2+ reporter GCaMP6 to visualise the Ca2+ signatures within individual cells in response to a low fungicidal concentration (3.5 M) of PAF26. This peptide treatment resulted in [Ca2+]cyt spiking at irregular intervals within all cells but there was considerable heterogeneity in the Ca2+ signatures of individual cells of a germling population. Furthermore, subcellular regions within which transient [Ca2+]cyt increases occurred were spatially correlated with increased membrane fusion events between vacuolar-like structures. In the fifth part of my research, YVC-1 and CCH-1 were labelled with GFP. Whilst YVC-1 was consistently associated with the vacuolar membrane, CCH-1 was harder to visualise. CCH-1 appeared to localise to intracellular membranes in conidia and germlings and to the plasma membrane in mature hyphae. In the final part of my thesis I investigated the hypothesis, based on previous evidence, that PAF26 disrupts pH homeostasis by using live cell imaging with the fluorescent, ratiometric pH dye, carboxy SNARF-1. PAF26 treatment was found to cause acidification of the cytoplasm.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:719321 |
| Date | January 2017 |
| Creators | Alexander, Akira |
| Contributors | Read, Nick |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-ca2-signalling-in-the-modeofaction-of-paf26(72c277a9-66ae-4757-a7d6-458264eb6ed8).html |
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