Systemic candidiasis is most commonly studied in animal models, particularly the murine intravenous (IV) challenge model, where infection with a virulent Candida albicans strain leads to increasing fungal kidney burdens and increasing pro-inflammatory cytokines in the kidneys. Based upon the finding that early renal levels of the chemokine KC correlate with infection outcome, a new in vitro model, utilising the murine renal M-1 cortical collecting duct epithelial cell line, was developed to evaluate virulence of C. albicans isolates and mutants, in attempts to reduce the number of mice used in C. albicans virulence studies, addressing the 3Rs. The epithelial cells were shown to respond only to live fungal cells, unlike immune cells, responding more robustly to hyphae rather than to cells growing as yeasts. We also demonstrate that non-albicans Candida species, which are attenuated in the mouse IV challenge model, are unable to elicit chemokine responses from mouse kidney epithelial cells, despite increasing the inoculums used. Renal epithelial cell responses observed in the new model reflect early events in the mouse model, with chemokines KC and MIP-2 produced in response to virulent C. albicans strains or mutants. This chemokine production correlates with C. albicans damage to epithelial cells. Some involvement of TLR4 signalling was demonstrated as blocking of TLR4 signalling reduced epithelial KC production, and it was demonstrated that the renal epithelial cells respond strongly to more complex glycan molecules. Using this new in vitro model we have confirmed that renal epithelial cells are able to discriminate between virulent and attenuated strains of C. albicans, allowing this model to be used as an initial screen for altered virulence and for investigating how renal epithelial cells detect the presence of pathogenic fungi.

Thesis (Ph. D.)--Oregon State University, 2003. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

One of the major aims of this thesis was to identify novel regulators that drive C. albicans adaptation during growth under different nutrient and temperature conditions. The classical stress response cascades have been previously characterised under standardized, but physiologically irrelevant growth conditions (YPD at 30°C). In this study these pathways and other regulators were examined under more physiologically relevant inputs because metabolic plasticity and thermo-tolerance have been shown to affect stress adaptation (Arguelles et al., 1999; Brown et al., 2014; Cowen, 2009; Diezmann et al., 2014). In this study, we characterized 18.5% of the functional C. albicans ORFeome under 144 different stress conditions by employing a standardized system of robotic screening (Chapter 3). These screens highlighted extensive carbon and temperature-conditional regulators in C. albicans. We identified carbon-conditional contributions of the transcriptional regulators Sfp1 and Rtg3 to stress adaptation in this pathogenic fungus (Chapter 4). Sfp1 was found to regulate the expression of key stress regulators during growth on glucose, whereas Rtg3 induced the expression of these stress genes during growth on lactate. Our screens also revealed a distinct set of transcription factors, Hap43, Swi4, Sfp1, Cap1 and Zcf31, that control regulators of cell wall integrity and that promote antifungal drug resistance in a temperature dependent and yet Hsp90- independent manner. The screens also provided new information about a relatively obscure group of transcriptional regulators in C. albicans; the zinc cluster proteins with focus on Zcf3 and Zcf18 which we further pursued with RNA-sequencing to establish them as modulators of cell cycle, stress resistance and virulence in C. albicans. Lastly, our screens reveal a network of regulators that are homologous to human oncogenes and control fungal growth via modulation of TOR signaling. In conclusion, this thesis has revealed many novel targets for possible antifungal drug development and highlighted the extensive and intricate cross-talk between stress response modules facilitated by physiologically relevant nutrient sources and ambient temperatures.

Graduation date: 2006

Victoria blight of oats is caused by the fungus Cochhobolus victoriae. This fungus is pathogenic due to its ability to produce the host-selective toxin victorin. Previously, a 100-kD protein that binds victorin in vivo only in susceptible genotypes was identified as the P protein of the glycine decarboxylase complex (GDC). Victorin is a potent in vivo inhibitor of GDC. Leaf slices pretreated with victorin displayed an effective Victorin inhibited the concentration for 50% inhibition (EC������) of 81 ��M for GDA. glycine-bicarbonate exchange reaction in vitro with an EC������ of 23 ��M. We also identified a 15-kD mitochondrial protein in susceptible and resistant genotypes that hound victorin. Amino acid sequence analysis indicated this protein is the H protein component of the GDC. Thus, victorin specifically binds to two components of the GDC. Victorin had no detectable effect on GDC in isolated mitochondria, apparently due to the inability of isolated mitochondria to import victorin. The interaction of victorin with the GDC may be central to victorin's mode of action. Supporting this observation is the finding that CO��� gives partial protection against victorin. Elevated CO��� is known to ameliorate the effect of GDC inhibition. Victorin treated plants incubated in the light develop more severe symptoms than dark-incubated plants. Victorin appears to induce a plant-wide signal transduction cascade, resulting in diverse effects. Victorin induces specific proteolytic cleavage of the Rubisco large subunit (LSU). Leaf slices incubated with victorin for 4 hours in the dark accumulate a form of LSU which is cleaved after the N-terminal lysine 14. LSU cleavage in leaf slices is prevented by the protease inhibitors E-64 and calpeptin. LaCl��� prevents this cleavage of LSU and LaCl��� also confers complete protection against victorin at the whole plant level. Victorin also causes lipid peroxidation as measured by MDA accumulation. DNA laddering is seen in leaves after 3 hr treatment with toxin. The ethylene inhibitors AOA and STS give significant protection against victorin at the whole plant level, and also prevent LSU cleavage. / Graduation date: 1998