dc.description.abstract |
<p>Although the majority of the population carries <em>Candida spp</em> as normal
components of their microflora, these species are important human pathogens that have
the ability to cause disease under conditions of immunosuppression or altered host
defenses. The spectrum of disease caused by these species ranges from cutaneous infections
of the skin, mouth, esophagus and vagina, to life-threatening systemic disease. Despite
increases in drug resistance, the antifungal armamentarium has changed little over
the past decade. Thus increasing our understanding of the life cycles of these organisms,
not only how they propagate themselves, but also how genetic diversity is created
within the population is of considerable import. Additionally expanding our knowledge
of key signal transduction cascades that are important for cell survival and response
to stress will add in developing new antifungal therapies and strategies. </p><p>This
thesis addresses both of these key areas of fungal pathogenesis. In the first chapter,
we use genome comparisons between parasexual, asexual, and sexual species of pathogenic
<em>Candida</em> as a first approximation to answer the question of whether examining
genome content alone can allow us to understand why species have a particular life
cycle. We start by examining the structure of the mating type locus (<em>MAT</em>)
of two sexual species <em>C. lusitaniae</em> and <em>C. guilliermondii</em>. Interestingly,
both species are missing either one or two (respectively) canonical transcription
factors suggesting that the control of sexual identity and meiosis in these organisms
has been significantly rewired. Mutant analysis of the retained transcription factors
is used to understand how sexual identity and sporulation are controlled in these
strains. Secondly, based on the observation that these species are missing many key
genes involved in mating and meiosis, we use meiotic mapping, SPO11 mutant analysis,
and comparative genome hybridization to demonstrate that these species are indeed
meiotic, but that the meiosis that occurs is occasional unfaithful generating aneuploid
and diploid progeny. </p><p>In the second and third chapters we examine the calcineurin
signaling pathway, which is crucial for mediated tolerance to cellular stresses including
cations, azole antifungals, and passage through the host bloodstream. First, we show
that clinical use of calcineurin inhibitors in combination with azole antifungals
does not result in resistance to the combination, suggesting that if non-immunosuppressive
analogs could be further developed this combinatorial strategy may have great clinical
efficacy. Second, we use previous studies of the calcineurin signaling pathway in
<em>S. cerevisiae</em> to direct a candidate gene approach for elucidating other components
of this pathway in <em>C. albicans</em>. Specifically, we identify homologs of the
<em>RCN1, MID1</em>, and <em>CCH1</em> genes, and use a combination of phenotypic
assays and heterologous expression studies to understand the roles of these proteins
in <em>C. albicans</em>. Although the mutant strains share some phenotypic properties
with calcineurin deletion strains, none completely recapitulate a calcineurin mutant.
</p><p>In the last chapter, we examine the plausibility of targeting the homoserine
dehyrogenase (Hom6) protein in <em>C. albicans</em> and <em>C. glabrata</em> as a
novel antifungal strategy. Studies in <em>S. cerevisiae</em> had demonstrated a synthetic
lethality between hom6 and fpr1, the gene encoding FKBP12 a prolyl-isomerase that
is the binding target of the immunosuppressant FK506. Thiss synthetic lethality was
due to the buildup of a toxic intermediate in the methionine and threonine biosynthetic
pathway as a result of deletion of hom6 and inhibition of FKBP12. We deleted <em>HOM6</em>
from both <em>C. albicans</em> and the more highly drug-resistant species <em>C.
glabrata</em>. Studies suggest that regulation of the threonine and methionine biosynthetic
pathway in <em>C. albicans</em> has been rewired such that the synthetic lethality
between hom6 and FKBP12 inhibition no longer exists. However, in <em>C. glabrata</em>
preliminary analysis suggest that similarly to <em>S. cerevisiae</em> hom6 and inhibition
of FKBP12 can result in cell death.</p>
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