Human systemic fungal infections are increasing, and causing high morbidity and mortality. Treatment is challenging because fungi share many metabolic pathways with mammals. Current antifungals are losing effectiveness due to drug resistance. In immunocompromised patients Aspergillus fumigatus causes systemic aspergillosis, the most important airborne fungal disease. Mortality from aspergillosis exceeds 50% even with aggressive treatment. We need novel antifungal drug targets. Fungal cell wall components are promising targets for antifungal therapy as they are essential for fungi and absent from humans. The sugar galactofuranose (Galf) is a 5-memberd ring form of galactose that is found in the cell walls of many fungi, but not in mammals. I used molecular biology and microscopy techniques to characterize Galf biosynthesis enzymes in the model species A. nidulans. I studied three enzymes that catalyze sequential steps in Galf biosynthesis: UgmA, UgtA and UgeA. UDP-galactopyranose mutase (UgmA) creates UDP-galactofuranose (UDP-Galf) from UDP galactopyranose (UDP-Galp) in the cytoplasm. The UDP-Galf transporter (UgtA) moves UDP Galf into membrane bound organelles for incorporation into cell wall compartments. Upstream of UgmA, UDP-glucose/galactose epimerase (UgeA) interconverts UDP-glucose into UDP-Galp, the UgmA substrate. Neither UgmA nor UgtA has a human counterpart; UgeA is in the Leloir galactose metabolism pathway that found in many organisms from bacteria to humans. None of UgeA, UgmA and UgtA is essential for viability of A. nidulans, but deleting any one of them substantially reduces colony growth and sporulation (Figure i). Wild type and Galf defective strains (ugeA∆, ugmA∆ and ugtA∆) were quantified for colony growth, cell morphometry, spore formation and germination, as well as wall architecture. The abundance of these proteins was regulated using the alcA promoter. Galf content was assessed by immunolocalization in the Galf defective strains, showing that those strains lacked immunodetectable Galf. Gene products were localized with fluorescent protein tags; both UgmA and UgeA were cytoplasmic, whereas UgtA was Golgi localized. Wall surfaces were imaged and force-probed using transmission electron microscopy and atomic force microscopy. Overall, Galf deletion strains had aberrant wall maturation, and poorly consolidated surfaces. Our results indicate that Galf is necessary for abundant sporulation, wild type growth and full maturation of Aspergillus cell wall. Galf deletion strains were assessed for sensitivity to antifungal agents in clinical use. They were significantly more sensitive to caspofungin and amphotericin B that target cell wall synthesis and cell membrane chemistry, respectively. Thus, anti-Galf drugs (once created) may be useful in combination with existing antifungal drugs. In summary, Galf biosynthesis pathway appears to be promising as an antifungal drug development target.

Thesis (Ph. D.)(Botany)--University of Pretoria, 2001. / Acrobat Adobe Reder needed to open files.

No description available.

Fungal pathogens are a leading cause of human mortality, at least in part due to their ability to thwart therapeutic regimens by rapidly evolving resistance to antifungal drugs, and as a consequence of the increasing frequency of immunocompromised individuals most vulnerable to fungal infection. Candida albicans, the leading human fungal pathogen, has evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by the azoles, which are the most widely deployed class of antifungals and inhibit ergosterol biosynthesis, inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular stress response circuitry, including that regulated by the molecular chaperone Hsp90 and its client protein calcineurin. My doctoral research focuses on three aspects of the role of fungal stress responses in regulation of azole resistance. First, I establish a novel role for nutrients and nutrient signalling in azole resistance of C. albicans and the model yeast Saccharomyces cerevisiae. Compromising a global regulator that couples growth to environmental cues, Tor kinase, provides a powerful strategy to abrogate fungal drug resistance with broad therapeutic potential. Second, I implicate the molecular chaperone Hsp90 as a key regulator of biofilm drug resistance in C. albicans. Compromising Hsp90 function transforms the azoles from ineffective to highly efficacious at eradicating biofilms in vitro and in vivo. Depletion of Hsp90 leads to reduction of client proteins’ calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Third, I establish that inhibition of lysine deacetylases (KDACs) blocks the emergence and maintenance of Hsp90-dependent azole resistance in C. albicans and S. cerevisiae. S. cerevisiae Hsp90 is acetylated on lysine 27 and 270, and key KDACs for drug resistance are Hda1 and Rpd3. Compromising KDACs alters stability and function of Hsp90 client proteins, including drug resistance regulator calcineurin. Overall, this work provides novel insight into the mechanisms by which cellular stress responses mediate azole resistance, and establishes acetylation as a novel mechanism of post-translational control of Hsp90 function in fungi; ultimately, this unveils numerous targets that could be exploited for therapeutic benefit in the treatment of fungal disease.

Fungal pathogens are a leading cause of human mortality, at least in part due to their ability to thwart therapeutic regimens by rapidly evolving resistance to antifungal drugs, and as a consequence of the increasing frequency of immunocompromised individuals most vulnerable to fungal infection. Candida albicans, the leading human fungal pathogen, has evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by the azoles, which are the most widely deployed class of antifungals and inhibit ergosterol biosynthesis, inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular stress response circuitry, including that regulated by the molecular chaperone Hsp90 and its client protein calcineurin. My doctoral research focuses on three aspects of the role of fungal stress responses in regulation of azole resistance. First, I establish a novel role for nutrients and nutrient signalling in azole resistance of C. albicans and the model yeast Saccharomyces cerevisiae. Compromising a global regulator that couples growth to environmental cues, Tor kinase, provides a powerful strategy to abrogate fungal drug resistance with broad therapeutic potential. Second, I implicate the molecular chaperone Hsp90 as a key regulator of biofilm drug resistance in C. albicans. Compromising Hsp90 function transforms the azoles from ineffective to highly efficacious at eradicating biofilms in vitro and in vivo. Depletion of Hsp90 leads to reduction of client proteins’ calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Third, I establish that inhibition of lysine deacetylases (KDACs) blocks the emergence and maintenance of Hsp90-dependent azole resistance in C. albicans and S. cerevisiae. S. cerevisiae Hsp90 is acetylated on lysine 27 and 270, and key KDACs for drug resistance are Hda1 and Rpd3. Compromising KDACs alters stability and function of Hsp90 client proteins, including drug resistance regulator calcineurin. Overall, this work provides novel insight into the mechanisms by which cellular stress responses mediate azole resistance, and establishes acetylation as a novel mechanism of post-translational control of Hsp90 function in fungi; ultimately, this unveils numerous targets that could be exploited for therapeutic benefit in the treatment of fungal disease.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: G.D. Wright. Includes bibliographical references.

Thesis (M.S.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on August 11, 2009). Research advisor: Jegdish P. Babu, Ph.D. Document formatted into pages (ix, 32 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 28-31).

Thesis (M. Sc.)--Carleton University, 2001. / Includes bibliographical references (p. 85-94). Also available in electronic format on the Internet.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Douglass F. Taber, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (Ph. D.)--University of Idaho, 2005. / Also available online in PDF format. Abstract. "May 2005." Includes bibliographical references.