Thesis (Ph. D.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Rick P. Morrison. Includes bibliographical references (leaves 207-235).

published_or_final_version / Oral Biology / Doctoral / Doctor of Philosophy

No description available.

The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.

Three overlapping genomic clones covering 29.0 kilobases of cotton DNA were found to encompass a cluster of two presumptive osmotin genes (OSMI and OSMII) and two osmotin pseudogenes (OSMIII and OSMIV). A segment of 16,007 basepairs of genomic DNA was sequenced from the overlapping genomic clones (GenBank Accessions AY303690 and AF304007). The two cotton osmotin genes were found to have open reading frames of 729 basepairs without any introns, and would encode presumptive osmotin preproteins of 242 amino acids. The open reading frames of the genes are identical in sequence to two corresponding cDNA clones (GenBank Accessions AF192271 and AY301283). The two cDNA inserts are almost full-length, since one lacks codons for the four N-terminal amino acids, and the other cDNA insert lacks the coding region for the 34 N-terminal amino acids. The cotton osmotin preproteins can be identified as PR5 proteins from their similarities to the deduced amino acid sequences of other plant osmotin PR5 preproteins. The preproteins would have N-terminal signal sequences of 24 amino acids, and the mature 24 kilodalton isoforms would likely be targeted for extracellular secretion. Prospective promoter elements, including two ethylene response elements, implicated as being positive regulatory elements in the expression of a number of PR-proteins, occur in the 5'-flanking regions. The mature osmotin proteins accumulate in cotton plants treated with the inducers ethephon and hydrogen peroxide. Thus, the two cotton osmotin genes encode osmotin proteins. The coding regions of the two genes have been expressed and isolated as fusion polypeptides in a bacterial expression system. Binary constructs containing the open reading frames of the two osmotin genes under the control of the 35S CaMV promoter have been generated for eventual production of transgenic Arabidopsis and cotton plants for potential constitutive expression of the osmotin proteins for increased resistance against fungal pathogens.

The significance of fungal infections has been grossly underestimated. Only a few drugs are clinically available to treat life-threatening fungal infections, and resistance against these drugs is rising. Antifungal peptides (AFPs) are being actively explored as novel pharmaceuticals. PAF26 is a de novo designed hexapeptide possessing N-terminal cationic and C-terminal hydrophobic regions. Previously the roles of each of these motifs in the antifungal mode-of-action of PAF26 have indicated that it involves three stages: interaction with the plasma membrane, internalisation, and cell killing. The overall aim of my project was to obtain further insights into its mode-of-action and develop more active antifungal derivatives of PAF26. Three experimental fungal systems were used in this study: the model Neurospora crassa, the human pathogen Aspergillus fumigatus and the plant/human pathogen Fusarium oxysporum. The first objective of the study was to evaluate the impact of different fluorescent labels on the intracellular localisation and antifungal properties of PAF26. For this purpose a library of PAF26 labelled with 13 different fluorophores was synthesised. This library contained PAF26 conjugates of broad chemical and spectral diversity. These fluorescent PAF26 conjugates were analysed by live-cell imaging and tested for their antifungal activities. The different fluorescent labels were found to have significant impacts both on intracellular localisation and antifungal activities. TMR, carboxyfluorescein, NBD and DMN were found to be the best labels for live-cell imaging because they had the least influence on the intracellular localisation and antifungal activity of PAF26. The second objective was to identify target proteins of PAF26 in N. crassa cells. A large number of proteins were identified as binding to PAF26 from a protein pull-down and mass spectroscopy analysis using TMR- and fluorescein-labelled PAF26. One of these proteins was the highly abundant plasma membrane ATPase PMA-1. An in-silico analysis showed that PMA-1 is likely to be a major target protein of PAF26. The final objective was to develop novel antifungals based on PAF26 with improved activities and stability. Novel cyclic derivatives of PAF26 were designed in-silico against PMA-1. These peptides were synthesised and tested against N. crassa, A. fumigatus and F. oxysporum and were found to have higher activities (at the sub-micromolar level) and greater stability than the linear PAF26. Overall this study has provided novel mechanistic insights into the mode-of-action of PAF26 and discovered novel highly active antifungal peptides with clinical potential as therapeutics.

The n-butanol extract of aerial parts of Cordyline australis demonstrated antifungal activity. n-Butanol and chloroform extracts of dried or fresh leaves of C. australis afforded a steroidal glycoside, which was identified as 5α-spirost-25(27)-en-3β-ol 3-O{O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside}, saponin 1. This spirostanol glycoside showed strong antifungal activity towards Trichophyton mentagrophytes and some aspecific activity and cytotoxicity against MRC5 cell. The chloroform extract of fresh leaves of C. australis yielded a second new spirostanol glycoside which was identified as 5α-spirost-25(27)-ene-1β,3β-diol 1-{O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside}, saponin 2. The n-butanol extracts of senescent leaves of C. australis afforded a third new spirostanol glycoside that was identified as 5α-spirost-25(27)-ene-1β,3β-diol 1-{O-β-D- fucopyranoside, saponin 3. A mixture of two isomeric flavonoid glycosides was isolated from dried leaves of C. australis and shown to be a ca 1:1 mixture of isorhamnetin-3-O-{O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside}, 4 and isorhamnetin-3-O-{O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside}, 5. Three other known steroidal glycosides, β-sitosterol glucoside, 6, prosapogenin A of dioscin, 7, and trillin, 8 were also isolated from the leaves of C. australis. The n-butanol extract of dried stems of C. australis afforded (25S)-5α-spirostane-1β,3α-diol 1-{O-β-D-glucopyranoside}, 9. This spirostanol glycoside showed moderate cytotoxicity against Herpes simplex type I virus (ATCC VR733) and Polio Virus Type I (Pfiser vaccine strain).

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: IAA; S. cerevisiae; quorum sensing. Includes bibliographical references (p.57-59).

Graduation date: 2000

Human systemic fungal infections are increasing, and causing high morbidity and mortality. Treatment is challenging because fungi share many metabolic pathways with mammals. Current antifungals are losing effectiveness due to drug resistance. In immunocompromised patients Aspergillus fumigatus causes systemic aspergillosis, the most important airborne fungal disease. Mortality from aspergillosis exceeds 50% even with aggressive treatment. We need novel antifungal drug targets. Fungal cell wall components are promising targets for antifungal therapy as they are essential for fungi and absent from humans. The sugar galactofuranose (Galf) is a 5-memberd ring form of galactose that is found in the cell walls of many fungi, but not in mammals. I used molecular biology and microscopy techniques to characterize Galf biosynthesis enzymes in the model species A. nidulans. I studied three enzymes that catalyze sequential steps in Galf biosynthesis: UgmA, UgtA and UgeA. UDP-galactopyranose mutase (UgmA) creates UDP-galactofuranose (UDP-Galf) from UDP galactopyranose (UDP-Galp) in the cytoplasm. The UDP-Galf transporter (UgtA) moves UDP Galf into membrane bound organelles for incorporation into cell wall compartments. Upstream of UgmA, UDP-glucose/galactose epimerase (UgeA) interconverts UDP-glucose into UDP-Galp, the UgmA substrate. Neither UgmA nor UgtA has a human counterpart; UgeA is in the Leloir galactose metabolism pathway that found in many organisms from bacteria to humans. None of UgeA, UgmA and UgtA is essential for viability of A. nidulans, but deleting any one of them substantially reduces colony growth and sporulation (Figure i). Wild type and Galf defective strains (ugeA∆, ugmA∆ and ugtA∆) were quantified for colony growth, cell morphometry, spore formation and germination, as well as wall architecture. The abundance of these proteins was regulated using the alcA promoter. Galf content was assessed by immunolocalization in the Galf defective strains, showing that those strains lacked immunodetectable Galf. Gene products were localized with fluorescent protein tags; both UgmA and UgeA were cytoplasmic, whereas UgtA was Golgi localized. Wall surfaces were imaged and force-probed using transmission electron microscopy and atomic force microscopy. Overall, Galf deletion strains had aberrant wall maturation, and poorly consolidated surfaces. Our results indicate that Galf is necessary for abundant sporulation, wild type growth and full maturation of Aspergillus cell wall. Galf deletion strains were assessed for sensitivity to antifungal agents in clinical use. They were significantly more sensitive to caspofungin and amphotericin B that target cell wall synthesis and cell membrane chemistry, respectively. Thus, anti-Galf drugs (once created) may be useful in combination with existing antifungal drugs. In summary, Galf biosynthesis pathway appears to be promising as an antifungal drug development target.