Date of Award:

5-1-1997

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

Stanley D. Allen

Committee

Mark C. Healey

Committee

Bradley R. Kropp

Committee

William B. Barnett

Abstract

New antifungal agents are needed to combat the increasing incidence of fungal diseases. Strains of Pseudomonas syringae produce cyclic lipodepsinonapeptides (CLPs) such as syringomycin, syringostatin, and syringotoxin, which could be developed as therapeutic antifungal agents. Of these CLPs, syringostatin has been difficult to produce in vitro. Syringostatin production was achieved using syringomycin minimal medium supplemented with arbutin and D-fructose, and a high cell density. Syringostatin A (SS-A) was purified in quantities sufficient to evaluate its antifungal activities. The antifungal activities and erythrocyte toxicities of syringomycin E (SR-E), SS-A, and syringotoxin B (ST-B) were evaluated. All showed fungicidal activities against most of the organisms tested. SR-E and SS-A showed the highest activities and had similar antifungal activities and erythrocyte toxicities. ST-B was less active than SR-E and SS-A against most fungi and was less toxic to erythrocytes. In vivo evaluation of SR-E in a murine model of vaginal candidiasis showed that SR-E 12% was effective and may be more effective than clotrimazole 1%. No SR-E was detected in body tissues and no inflammatory responses were seen by histological examination of vaginal tissues of non-challenged animals treated with SR-E. To discover new strains of P. syringae that produce CLPs, a PCR-based test was developed using oligonucleotide primers specific for the genes syrB and syrD. SyrB primers were specific for the syrB gene. SyrD primers were also able to detect the presence of the syrD gene, but were not specific to this gene. Partial sequence comparisons of syrB-amplified fragments showed that syringostatin and syringotoxin producing strains could be predicted based upon sequence similarities with known CLP producers. Using this PCR-based approach, new isolates were screened for the possession of the syrB and syrD genes. Antifungal compounds from three syrB and syrD positive strains, ESC-10, ESC-11, and M2, were characterized. All three strains produced SR-E. In addition to SR-E, strain M2 produced a novel CLP, M2-NP, with a MH+ molecular weight of 1165 and an amino acid composition of glutamine, homoserine, threonine, arginine, and alanine in a molar ratio of 1:2:3:1:2. M2-NP had 32 to 64 times less antifungal activity than SR-E and was sensitive to alkaline pH. This is the first case of a strain of P. syringae producing more than one class of CLP. In summary, the potential of the CLPs to be useful antifungal agents was shown, and a new P. syringae CLP was isolated and identified.

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