UVM Theses and Dissertations
Format:
Print
Author:
Midkiff, John Frank
Dept./Program:
Microbiology and Molecular Genetics
Year:
2013
Degree:
MS
Abstract:
Candida albiearis is a commensal organism that is also a opportunistic pathogen, causing disease primarily in immunocompromised individuals. C. albieans is a pleomorphic fungus capable of assuming a range of morphologies in response to environmental signals, primarily the budded, pseudohyphal, and the hyphal states. The proper transitioning between the budded and the hyphal states, also known as the budded-tohyphal transition, is essential for the formation of mature biofilms on biotic and abiotic surfaces and for virulence.
The budded-to-hyphal transition is stimulated by diverse environmental signals and stresses, such as the presence ofserum, amino acids, CO₂, glucose and nitrogen starvation, and these signals are transmitted a several parallel, yet intercommunicating signaling pathways. Each of the pathways are responsible for the detection and transmission of a subset of the total signals and activate specific transcription factors that then generate a hyphal-specific response. The two primary morphogenic pathways are the CekI-mediated MAPK and the cAMP/PKA pathways, and these pathways activate the transcription factors Cph1 and Efg1, respectively. Deletion or mutation of genes in these two pathways may cause a loss of filamentation, hyperfilamentation, or have no morphological phenotype, depending upon or refractory to the environmental conditions in which the cells are grown.
Previously, our lab identified 16 small molecule inhibitors of the budded-to-hyphal transition that were able to block filamentation under inducing condition (carbon limitation) yet did not affect cell viability or growth. These molecules had a wide range of mammalian targets, from acting upon calcium ion homeostasis to receptor antagonists, though their targets in C. albieans have yet to be identified. As the cAMP/PKA is the primary morphogenic pathway in C. albieans we formed the hypothesis that the target for most ofthese molecules was a component ofthis pathway.
To test this our lab designed a series of chemical epistasis experiments using hyperfilamentous mutants of the MAPK and cAMP/PKA pathways, along with different hyphal-inducing media to identify if the molecule was acting upon the cAMP/PKA pathway to block hyphae formation and, if so, to identify potential targets. The principle behind these chemical epistasis experiments using the small molecule inhibitors was that if the molecule was epistatic to (budded phenotype was observed) to the constitutive mutant, then the molecule acted downstream of the protein or in another pathway. Conversely, if the mutant phenotype was observed (hyphal phenotype observed) then the molecule was likely-acting upstream of this protein.
The results from these experiments indicated that most of the molecules tested acted upon either the Cekl-mediated MAPK or cAMP/PKA pathways, with several molecules indicating that they either act upon both pathways separately or a common component to both pathways. In addition, several molecules were identified to have targets that were downstream of both of these pathways, indicating a target in a conserved process. Further analysis with the G-protein-coupled receptor antagonist clozapine, a FDA-approved drug treatment for atypical schizophrenia, along with several of its analog was shown to act at the level of the C. albieans Gprl G-protein-coupled receptor. Taken together, these studies represent the initial step in identifying the target and mechanism responsible for the molecules inhibitory activity.
The budded-to-hyphal transition is stimulated by diverse environmental signals and stresses, such as the presence ofserum, amino acids, CO₂, glucose and nitrogen starvation, and these signals are transmitted a several parallel, yet intercommunicating signaling pathways. Each of the pathways are responsible for the detection and transmission of a subset of the total signals and activate specific transcription factors that then generate a hyphal-specific response. The two primary morphogenic pathways are the CekI-mediated MAPK and the cAMP/PKA pathways, and these pathways activate the transcription factors Cph1 and Efg1, respectively. Deletion or mutation of genes in these two pathways may cause a loss of filamentation, hyperfilamentation, or have no morphological phenotype, depending upon or refractory to the environmental conditions in which the cells are grown.
Previously, our lab identified 16 small molecule inhibitors of the budded-to-hyphal transition that were able to block filamentation under inducing condition (carbon limitation) yet did not affect cell viability or growth. These molecules had a wide range of mammalian targets, from acting upon calcium ion homeostasis to receptor antagonists, though their targets in C. albieans have yet to be identified. As the cAMP/PKA is the primary morphogenic pathway in C. albieans we formed the hypothesis that the target for most ofthese molecules was a component ofthis pathway.
To test this our lab designed a series of chemical epistasis experiments using hyperfilamentous mutants of the MAPK and cAMP/PKA pathways, along with different hyphal-inducing media to identify if the molecule was acting upon the cAMP/PKA pathway to block hyphae formation and, if so, to identify potential targets. The principle behind these chemical epistasis experiments using the small molecule inhibitors was that if the molecule was epistatic to (budded phenotype was observed) to the constitutive mutant, then the molecule acted downstream of the protein or in another pathway. Conversely, if the mutant phenotype was observed (hyphal phenotype observed) then the molecule was likely-acting upstream of this protein.
The results from these experiments indicated that most of the molecules tested acted upon either the Cekl-mediated MAPK or cAMP/PKA pathways, with several molecules indicating that they either act upon both pathways separately or a common component to both pathways. In addition, several molecules were identified to have targets that were downstream of both of these pathways, indicating a target in a conserved process. Further analysis with the G-protein-coupled receptor antagonist clozapine, a FDA-approved drug treatment for atypical schizophrenia, along with several of its analog was shown to act at the level of the C. albieans Gprl G-protein-coupled receptor. Taken together, these studies represent the initial step in identifying the target and mechanism responsible for the molecules inhibitory activity.