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- All Subjects: Fungi--Hyphae.
- Creators: Roberson, Robert W.
- Member of: ASU Electronic Theses and Dissertations
Description
In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection is ingestion of contaminated meat or dairy product originating from cattle or vegetables contaminated with bovine manure. Since cattle are the main reservoir for human infection with E. coli O157:H7, the reduction of intestinal colonization by these bacteria in cattle is the best approach to prevent human infections. Intimin is an outer membrane protein of E. coli O157:H7 that plays an important role in adhesion of the bacteria to the host cell. Hence, I proposed to express intimin protein in tomato plants to use it as a vaccine candidate to reduce or prevent intestinal colonization of cattle with E. coli O157:H7. I expressed His-tagged intimin protein in tomato plants and tested the purified plant-derived intimin as a vaccine candidate in animal trials. I demonstrated that mice immunized intranasally with purified tomato-derived intimin produced intimin-specific serum IgG1and IgG2a, as well as mucosal IgA. I further demonstrated that mice immunized with intimin significantly reduced time of the E. coli O157:H7 shedding in their feces after the challenge with these bacteria, as compared to unimmunized mice. Shiga toxin is the major virulence factor that contributes to HUS. Since Shiga toxin B subunit has an important role in the attachment of the toxin to its receptor, I fused intimin to Shiga toxin B subunit to create multivalent subunit vaccine and tested the effects upon immunization of mice with the B subunit when combined with intimin. His-tagged intimin, Shiga toxin B subunit, and Shiga toxin-intimin fusion proteins were expressed in E. coli and purified. I demonstrated that this multivalent fusion protein vaccine candidate elicited intimin- and Shiga toxin B-specific IgG1, IgG2a, and IgA antibodies in mice. I also showed a reduction in the duration of the bacterial shedding after the challenge compared to the control sham-immunized groups.
ContributorsTopal, Emel (Author) / Mason, Hugh S. (Thesis advisor) / Bingham, Scott E. (Committee member) / Mor, Tsafrir (Committee member) / Roberson, Robert W. (Committee member) / Arizona State University (Publisher)
Created2010
Description
The distinguishing feature of the filamentous fungi is the hyphae - tube-like microscopic cells that exhibit polarized growth via apical extension and allow the fungus to interact with its environment. Fungi elongate at the hyphal apex, through the localized construction of new plasma membrane and cell wall through the exocytosis of secretory vesicles. One population of these vesicles have been identified as chitosomes, containing chitin synthase isoenzymes, which are responsible for the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin, the primary fibrillar component of the fungal cell wall. The chitosomes, in addition to other vesicles, can be observed aggregating in the hyphal tip in most filamentous fungi. In the Ascomycota and Basidiomycota, this collection of vesicles exhibits discrete organization and has been termed a Spitzenkörper. Although accumulations of vesicles can be observed in the hyphal tip of many growing filamentous fungi, some debate continues as to what precisely defines a Spitzenkörper. This study reports the details of three separate projects: first, to document the effects of deleting a single chitin synthase, CHS-1 and CHS-6 in Neurospora crassa with regards to hyphal ultrastructure, cytoplasmic organization, and growth in comparison to the wild-type. Given the importance of chitin synthesis in fungal cell growth, deletion of a critical chitin synthase presumably impacts cell wall structure, fungal growth and cytoplasmic organization. Second, an examination of the ultrastructure of four zygomycetous fungi - Coemansia reversa, Mortierella verticillata, Mucor indicus, and Gilbertella persicaria has been conducted. Utilization of cryofixation and freeze-substitution techniques for electron microscopy has produced improved preservation of cytoplasmic ultrastructure, particularly at the hyphal apex, allowing detailed analysis of vesicle size, contents, and organization. Lastly, hyphal tip organization was reviewed in a broad range of fungi. Previous studies had either focused on a few select fungi or representative groups. Vesicle organization, composition and size do appear to vary among the classes of fungi, but some trends, like the vesicle crescent in the zygomycetous fungi have been documented.
ContributorsFisher, Karen Elizabeth (Author) / Roberson, Robert W. (Thesis advisor) / Chandler, Douglas (Committee member) / Riquelme, Meritxell (Committee member) / Stutz, Jeam (Committee member) / Wojciechowski, Martin (Committee member) / Arizona State University (Publisher)
Created2015