"The C. albicans community is currently laying the foundation of understanding how this human pathogen causes infection. C. albicans infections represent a major medical and economic burden for today’s society with an estimated 400,000 blood stream infections worldwide and direct costs exceeding 1$ billion dollar a year in the U.S. alone. Although finding the biological causes of this disease seemed to be beyond our reach in the past, various aspects of the infection have been recently unveiled including its pathology, immunology, histology, and epidemiology. Here we explored the genetic components of this disease by studying the complex host-pathogen dynamics through a series of in vivo, ex vivo and in vitro experiments. By using a pathogen unbiased reverse genetic approach and a host gene candidate strategy we uncovered some of the genes and pathways that are important for pathogenicity and immunity. In particular we explored the complex host-pathogen dynamics using a C. albicans - C. elegans model system and identified four novel putative virulence factors. We focused on Zcf15, a C. albicans transcription factor that has been poorly characterized in the literature and that plays an important role in the pathogen’s ability to resist host generated reactive oxygen species (ROS). By leveraging the power of RNASeq and ChIP-Seq we identified Zcf15 transcriptional targets and DNA binding sites. These studies suggest that Zcf15 plays a critical role in carbon metabolism and that it exerts its ability to protect the pathogen from ROS by controlling the expression of thiol- peroxidases and other detoxifying enzymes. We also showed here that in C. elegans, the host’s ability to counteract the infection relies on the MAPK pathway, evidence that mirrors what has been found by others in mammals and that emphasizes the usefulness of studying C. albicans infections in smaller genetically traceable organisms like C. elegans. The nematode model is also shown here to be a powerful tool not only to study the genetic bases that drive infection and immunity but also to identify new compounds that can be used for therapeutic intervention. This model was instrumental in identifying filastatin, a small molecule that was subsequently found by our collaborators to be capable of reducing virulence in mammals. The antifungal properties of filastatin are currently undertaking further preclinical testing. Overall this thesis shed light on the complex mechanisms of C. albicans pathogenicity and host immunity and identified novel virulence determinants that can be used by the larger community for further biological studies or even drug development. "

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The potential application of weak acids for prevention of germ tube formation, a pathogenic process in <i>Candida albicans</i>, was examined. Previous departmental work revealed a rapid cytoplasmic alkalinization prior to germ tube formation in normal but not hyphal-minus strains, suggesting involvement of cytoplasmic alkalinization in dimorphism. Evidence that benzoate and sorbate can prevent germ tube formation and inhibit growth is given. This is probably due to their ability to shuttle protons across the plasma membrane perturbing intracellular pH control via the ATPase. Benzoate and sorbate could therefore provide an excellent and novel treatment for thrush infections based on a central cellular requirement, that of pH control. In addition they are relatively harmless, cheap and plentiful. The extent to which the azoles affect cellular functions other than ergosterol biosynthesis is unknown. Three separate effects have been identified here which suggest three target sites. High concentrations of azole result in cell death, intermediate concentrations in inhibition of germ tube extension and low concentrations in inhibition of dimorphism. It is already known that fluconazole inhibits cytoplasmic alkalinization and germ tube formation and that azoles inhibit the ATPase. This suggests a relationship between azoles and dimorphism which may be linked through their effect on intracellular pH by direct or indirect interference with ATPase activity. The occurrence of synergism between weak acids and the azoles has been confirmed. This provides the basis for work towards a novel treatment for thrush infections since synergistic relationships enhance effect and delay development of resistance. Growth of <i>Candida albicans</i> hyphae on a solid surface results in the production of a so far unreported coiled morphology. Investigations show that this is probably the result of tip rotation during apical extension which is revealed as a result of adherence to a solid surface. In addition evidence has been obtained of the frequency with which hyphae enter the random pores of Nucleopore membranes. This suggests the possibility (and emphasizes the need for investigation) of contact mediated responses in <i>Candida albicans</i>. Both hyphal coiling and the possibility of contact mediated responses are landmarks in pathogenicity studies of <i>Candida albicans</i>.

In microcosms, <i>Picea sitchensis</i> seedlings were grown aseptically in perlite + vermiculite with NH<SUB>4</SUB>-N or NH<SUB>4</SUB>NO<SUB>3</SUB>-N, with an ectomycorrhizal fungus, <i>Lactarius rufus</i> and/or a saprotrophic fungus <i>Hygrophoropsis aurantiaca</i>. Total and fluorescein diacetate (FDA) active hyphal length, mycorrhizal infection and plant dry weight were measured (8 to 16 weeks). In perlite with NH<SUB>4</SUB>-N, total hyphal length of extramatrical mycelium (EMM) produced by <i>L. rufus</i> was 693 m gdwt perlite or 47 m per mycorrhizal root-tip. This was reduced to 141 m gdwt in the presence of <i>H. aurantiaca</i>. Nutrient solution pH and plant growth were also reduced. In liquid culture, acidification of the medium (H<SUP>+ </SUP> equivalent) per NH<SUB>4</SUB> ion uptake was ≤ 3.8 times greater for <i>H. aurantiaca</i> than <i>L. rufus</i>. Growth (dwt) of <i>H. aurantiaca</i> was inversely related to pH. Growth of <i>L. rufus</i> was unaffected by pH 2.7 to 5.0. In perlite + vermiculite with NH_4-N, <i>L. rufus</i> hyphal length was unaffected by the presence of <i>H. aurantiaca</i>. In this experiment, no mycorrhizas formed in treatments with NH_4NO_3-N and EMM production by <i>L. rufus</i> was greatly reduced compared to treatments with NH_4-N. In a different experiment with NH_4NO_3-N, total hyphal length of EMM for <i>L. rufus</i> grown alone was apparently greatly reduced, (compared with an experiment with NH_4-N) but was increased in the presence of <i>H. aurantiaca</i>. Plant dry weight was also increased in the presence of <i>H. aurantiaca</i> with perlite + vermiculite. Total hyphal length of <i>H. aurantiaca</i> was not affected by the presence of <i>L. rufus</i> in any experiment. FDA-active hyphal length of both fungi decreased or remained constant and was 1% to 3% of total hyphal length at a week 16. Percentage mycorrhizal root-tips was similar between treatments at week 16 but differences occurred at weeks 8, 10 and 12 which suggested an inverse relationship between % infection and pH.

The study of the regulation of chitin synthesis in the pathogen <i>Candida albicans</i> is a challenging field. It not only offers a possibility of a better understanding of the dimorphic transition but also may help in the development of an effective antifungal. The suggestion that the regulation of cell wall synthesis may be closely coupled to the turgor pressure of the fungus has been investigated. The synthesis of chitin by the enzyme chitin synthase was studied under conditions of osmotic stress. Mixed membrane fractions from protoplasts of <i>C.albicans</i> incubated in medium of low osmolality exhibited up to four-fold greater native enzyme activity as compared to protoplasts incubated at high osmolality. This was also the case for preparations from whole cells of <i>C.albicans</i>, <i>Coprinus cinereus</i> and <i>Saccharomyces cerevisiae</i> and also from protoplasts from <i>S.cerevisiae</i>. Trypsin-treated enzyme preparations did not show this regulation to the same degree. The addition of nikkomycin Z, a differential chitin synthase inhibitor, partially restored this regulation. The synthesis of chitin, assessed by following the incorporation of (¹⁴C)-GlcNAc into chitin in the cell wall, was also greater in <i>C.albicans</i> cells incubated in medium of low osmolality. However, the incorporation of (¹⁴C)-GlcNAc into the cell wall of regenerating protoplasts exhibited the opposite effect. This was substantiated further by measuring the fluorescence of regenerating protoplasts following the addition of Calcofluor white. Following the attempted cloning of the <i>C.albicans</i> <i>CHS3 (CSD2</i>) gene, a detailed northern analysis of three chitin synthase genes during growth and dimorphism of <i>C.albicans</i> was performed. <i>CHS1</i> was expressed during both the yeast and hyphal phases of growth while <i>CHS2</i> and <i>CHS3</i> were preferentially expressed in the hyphal form. There was no difference in expression of the chitin synthase genes in invasive and non-invasive clinical isolates of <i>C.albicans</i> and all three genes showed highest levels of mRNA when grown in medium of neutral pH.

The morphology of the fungus Candida albicans was characterised by measurement of cell dimensions with the aid of computerised image analysis. The dimensions were used to calculate a mathematical ratio, the morphology index, which fell in the range 1 to 4.5. Spherical yeast cells gave low Mi values, whilst true hyphal cells gave Mi values greater than 3.2. This study shows that Mi can be used reliably in the place of subjective descriptions of C. albicans morphology. The mean Mi of a cell population varied according to the growth environment and the strain of C. albicans. Mi was found to reveal a continuum of morphologies in C. albicans cells both in vitro and in vivo, with no clear relationship observed between cellular morphology and the pathogenic status of C. albicans. Chemical and cellular analyses were performed on a variety of morphological forms of C. albicans, as determined by Mi. The chitin content of C. albicans cells increased linearly with Mi, although no significant correlation was found between the activity of the polysaccharide degradation enzymes, chitinase and glucanase, and morphology. Autoradiography and analysis of cell wall expansion suggested that the apex of the cell was the main region of expansion, regardless of morphology. General wall expansion was found to be repressed in cells with Mi greater than 2. The rate of overall cell wall expansion increased linearly with Mi. Investigations with "hypha-specific" monoclonal antibodies indicated a correlation between antibody reactivity and Mi, epitope expression appeared to be induced in cells with Mi greater than 3. The linear relationship observed between some properties (polysaccharide composition and overall wall expansion) and Mi suggests that quantitative regulation mechanisms may determine cell morphology.

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