Semester

Spring

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Daniel G Panaccione

Abstract

Ergot alkaloids, indole-derived mycotoxins, interact with multiple monoamine neurotransmitter receptors and cause disease in exposed individuals. They have been well studied in the ergot fungus, Claviceps purpurea, and have been reported in some closely related grass endophytes, as well as the distantly related opportunistic human pathogen Aspergillus fumigatus. A. fumigatus, which sporulates prolifically, produces clavines, specifically festuclavine and fumigaclavines A, B, and C in association with asexual spores and the total mass of alkaloids constitutes over 1% of the spore mass. These alkaloids differ from those of most clavicipitaceous fungi, which consist of different clavines and often more complex lysergic acid derivatives. However, the ergot alkaloid pathways of A. fumigatus and ergot fungi are hypothesized to share early biosynthetic steps, diverging at some point after the formation of the intermediate chanoclavine. A homologue of dmaW, a gene encoding dimethylallyltryptophan synthase in Neotyphodium endophytes, was found in the A. fumigatus genome. By gene knockout analysis, dmaW was shown to be required for ergot alkaloid production in A. fumigatus. Comparison of genes clustered around A. fumigatus dmaW to those clustered with dmaW in the ergot fungi revealed potential homologues that could encode proteins controlling early, shared steps in the pathway. Functional analysis via gene knockout of three additional A. fumigatus genes (easA, easE, and easF) rendered mutants with altered alkaloid profiles, demonstrating their involvement in ergot alkaloid biosynthesis. All mutants lacked normal ergot alkaloid production from the latter part of the pathway; complementation with a functional copy of the respective gene, restored normal ergot alkaloid production in each mutant. Analyses of intermediates positioned the products of easF and easE as the second and third enzymatic steps of the pathway. Knockout of easA caused accumulation of multiple intermediates, including chanoclavine; complementation with the C. purpurea easA gene resulted in accumulation of agroclavine, setoclavine, and its isomer isosetoclavine. These data confirm easA involvement post chanoclavine synthesis and more specifically assign its role to reduction of chanoclavine aldehyde, the branch point of the two lineage-specific pathways. These mutants, due to their differing ergot alkaloid profiles, are valuable for testing the role of specific ergot alkaloids in animal pathogenesis and toxicoses. Elucidation of ergot alkaloid biosynthesis, along with the capacity to control the spectrum of alkaloids produced, may be beneficial to agriculture and medicine. Additional studies demonstrated that production of ergot alkaloids was restricted to conidiating cultures. Disruption of brlA, a regulatory gene involved in conidiation, interfered with conidiophore development, as well as ergot alkaloid production. The association of these toxins with sporulation may offer insight into their ecological significance and utility to the fungus.

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