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Electronic version does not contain associated previously published material Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA. |
