Date of Award:

2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Advisor/Chair:

Jon Y. Takemoto

Abstract

Fungicide discovery is relatively neglected when compared to the investment in the development of antibacterial, antiviral, and anti-cancer therapeutics. Due to extensive use of currently available fungicides in agriculture and medicine, resistance is emerging among plant and animal pathogenic fungi. This necessitates the search for novel antifungal agents that are effective and less toxic and that do not promote resistance.

FG08 and K20 are novel aminoglycoside analogs synthesized from kanamycin B and A, respectively. The antimicrobial properties of these analogs were tested in vitro against a wide range of agriculturally and clinically important fungal pathogens. Both compounds showed broad-spectrum antifungal properties, but they did not inhibit bacteria such as Escherichia coli and Staphylococcus aureus. The hemolytic activities and cytotoxicities of FG08 and K20 were also evaluated. They showed no toxicity or lowered toxicity against animal cells at their antifungal minimum inhibitory concentrations (MICs).

The fungicidal mechanisms of action of FG08 and K20 were examined using intact cells of Saccharomyces cerevisiae, Cryptococcus neoformans, hyphae of Fusarium graminearum. FG08 and K20 caused SYTOX Green dye uptake and potassium efflux by intact cells, indicating that they increase plasma membrane permeability. FG08 and K20 also caused leakage of pre-loaded calcein from small unilamellar vesicles (SUVs) composed of lipids that mimic the lipid composition of fungal membranes, further suggesting increased membrane permeability as their mechanism of action.

The synergistic interactions of K20 with six azoles (such as itraconazole, and fluconazole) were investigated against a wide array of fungal pathogens. The in vitro results revealed strong synergy between K20 and azoles against plant and human pathogenic fungi. Their synergies were furthered confirmed by time kill curves and disk diffusion methods.

In conclusion, FG08 and K20 are broad-spectrum antifungal agents that do not inhibit bacteria. At their antifungal MICs, they are not toxic to animal cells, but they inhibit fungi by interacting with the fungal plasma membrane, leading to pore formation. These novel aminoglycoside analogs appear attractive for applications as fungicides in agriculture and medicine.

Included in

Biology Commons

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