Characterization of the Effects of Exogenous Camp Combined on C. Albicans Morphogenesis in Strains Lacking NRG1P, RFG1P, or TUP1P (Poster)

The opportunistic human pathogen Candida albicans causes both superficial and lifethreatening systemic infections and is a leading cause of fungal disease in immunocompromised individuals.  C. albicans can grow in different cell shapes, or morphologies, including yeast-like cells and a variety of filamentous forms, such as true hyphae and pseudohyphae. Yeast, hyphae and pseudohyphae have been observed at the sites of Candida infection and there is strong evidence that morphogenesis, the transition between yeast and filamentous growth forms, is essential for virulence. Many studies have implicated cAMP in the regulation of morphogenesis. cAMP acts to activate filamentation. Our lab and others have previously characterized the impact of the negative regulators, Nrg1, Rfg1, and Tup1 on the expression of HWP1, a hyphal specific gene.  The goal of this project is to characterize whether the addition of exogenous cAMP will increase the expression oHWP1 in the absence of each of the negative regulators. This will help us better understand the signal transduction cascade that controls morphogenesis in C. albicans

Analysis of BH31-1 Derivative's Effect on Candida Species

Candida species are the most common and arguably the most important causative agents of human fungal infections. Oropharyngeal, esophageal, vulvovaginal, and cutaneous candidiasis leads to significant morbidity while systemic infections in immunocompromised patients (patients with AIDS, tissue transplants, central venous catheters, or those undergoing chemotherapy) has a 35% mortality rate. During infection, it is essential that the dimorphic Candida species switch between different morphological states including transitions between budded or yeast-like cells and hyphal forms. The small molecule BH3I-1 has shown promising results at inhibiting hyphal formation in several Candida species. The goal of this study is to find a BH3I-1 derivative that inhibits hyphal formation in several Candida species at a lower minimum inhibitory concentration (MIC) than BH3I-1. A derivative with a low MIC that affects several Candida species may have a potential to be a broad-spectrum antifungal drug. The Candida species being tested against the BH3I-1 derivatives are: C. albicans, C. glabrata, C. rugosa, C. krusei, C. tropicalis, C. lusitaniae, C. dubliniesis, and C. parapsilosis. Currently, 36 BH3I-1 derivatives have been tested. Molecule 25 has an MIC about 4 times lower than BH3I-1 in Candida albicans and has also been shown to work in other Candida species at inhibiting hyphal formation. Other derivatives such as molecule #10 did not inhibit many of the tested Candida species, but showed a much lower MIC than molecule #25 in C. rugosa. Out of the 36 tested derivatives, molecule #25 has shown the promise for a broad-ranged antifungal drug

Characteristics of the Effect of Exogenous Camp on C. Albicans Morphogenesis in Strains Lacking NRG1P, RFG1P, or TUP1P

The opportunistic human pathogen Candida albicans causes both superficial and life threatening systemic infections and is a leading cause of fungal disease in immunocompromised individuals such as those with AIDS. C. albicans can grow in different cell shapes, also known as morphologies, including yeast-like cells and a variety of filamentous forms, such as true hyphae and pseudohyphae. Yeast, hyphae and pseudohyphae, have been observed at the sites of Candida infection and there is strong evidence that morphogenesis, the transition between yeast and filamentous growth forms, is essential for its virulence. Many studies have implicated the second messenger molecule cAMP in the regulation of morphogenesis due to its role in activating filamentation. Our lab and others have previously characterized the impact of the negative regulators, Nrg1, Rfg1, and Tup1 on the expression of HWP1, a hyphal specific gene. The goal of this project is to characterize whether the addition of exogenous cAMP will increase the expression of HWP1 in the absence of each of the negative regulators as well as test a small molecule derivative of BH3I’s effects in conjunction with the exogenous cAMP. This will help us better understand the signal transduction cascade that controls morphogenesis in C. albicans

IDENTIFICATION OF POTENTIAL TARGETS OF THE GRR1P SCF UBIQUITIN LIGASE IN FUNGI

The opportunistic human pathogen Candida albicans causes both superficial and life-threatening systemic infections and is a leading cause of fungal disease in immunocompromised individuals.  C. albicans can grow in different cell shapes, or morphologies, including yeast-like cells and a variety of filamentous forms, such as true hyphae and pseudohyphae.   Yeast, hyphae and pseudohyphae have been observed at the sites of Candida infection and there is strong evidence that morphogenesis, the transition between yeast and filamentous growth forms, is essential for virulence. Several studies have implicated ubiquitin-dependent proteolysis in the regulation of morphogenesis, yet the mechanism by which this pathway does so is largely unknown.  Previously, we have shown that deletion of the GRR1 gene results in the constitutive formation of filamentous growth forms.  The Grr1 protein is a component of an SCF ubiquitin ligase system that selectively targets proteins for degradation.  Thus, the loss of Grr1-mediated proteolysis presumably leads to the aberrant accumulation, and inappropriate activity, of a protein or proteins that induce filamentous growth.  The spectrum of proteins targeted for degradation by Grr1 is not known.  The goal of this project is to identify Grr1 targets in Saccharomyces cerevisiae, an experimentally tractable model system for pathogenic fungi.  We are using a novel proteomics-based approach to isolate and characterize proteins that are ubiquitinated in a Grr1-dependent fashion. The successful identification of Grr1p targets will be important for developing a working model of the pathways involved in the yeast to filamentous growth transition in pathogenic fungi

BH3I-1 DERIVATIVES INHIBIT THE FILAMENTOUS GROWTH OF THE CEA10 STRAIN OF ASPERGILLUS FUMIGATUS

Recent and exciting advances in medical therapies for cancer and organ failures have greatly extended the life span of afflicted patients. However, these therapies often place the patient at risk for potentially lethal fungal infections. As the number of immunocompromised patients continues to rise, there has been an increase in associated opportunistic fungal infections. Treatment options for invasive mycoses caused by Candida albicans and Aspergillus fumigatus are surprisingly limited. A. fumigatus is the most common Aspergillus species associated with invasive pulmonary aspergillosis, accounting for over 60% of cases. Aspergillus grows as a filamentous mold with true hyphae originating from the germination of asexual conidia.  A. fumigatus is not a dimorphic fungi as is the case with C. albicans, however, as both grow in hyphal form it seems possible that small molecules that inhibit the transition of C. albicans budded cells to hyphal growth (often referred to as the germination of blastoconidia) may also inhibit the germination of Aspergillus conidia. We tested BH3I-1 and derivatives against A. fumigatus strain CEA10 in YPD media. BH3I-1 and five of the derivatives inhibited at a 200?M concentration based on general observation via microscopy as well as eleven showing promising inhibition at possible different concentrations. Out of these inhibiting molecules, seven also shown inhibition within the prior C. albicans assay. We are currently employing a micro-plate reader to obtain quantitative levels of inhibition with increasing concentrations of molecule. Molecule 54 at the 300?M concentration showed similar inhibition to that of BH3I-1 at the same concentration

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Cdc42p GTPase Regulates the Budded-to-Hyphal-Form Transition and Expression of Hypha-Specific Transcripts in Candida albicans

The yeast Candida albicans is a major opportunistic pathogen of immunocompromised individuals. It can grow in several distinct morphological states, including budded and hyphal forms, and the ability to make the dynamic transition between these forms is strongly correlated with virulence. Recent studies implicating the Cdc42p GTPase in hypha formation relied on cdc42 mutations that affected the mitotic functions of the protein, thereby precluding any substantive conclusions about the specific role of Cdc42p in the budded-to-hypha-form transition and virulence. Therefore, we took advantage of several Saccharomyces cerevisiae cdc42 mutants that separated Cdc42p's mitotic functions away from its role in filamentous growth. The homologous cdc42-S26I, cdc42-E100G, and cdc42-S158T mutations in C. albicans Cdc42p caused a dramatic defect in the budded-to-hypha-form transition in response to various hypha-inducing signals without affecting normal budded growth, strongly supporting the conclusion that Cdc42p has an integral function in orchestrating the morphological transition in C. albicans. In addition, the cdc42-S26I and cdc42-E100G mutants demonstrated a reduced ability to damage endothelial cells, a process that is strongly correlated to virulence. The three mutants also had reduced expression of several hypha-specific genes, including those under the regulation of the Efg1p transcription factor. These data indicate that Cdc42p-dependent signaling pathways regulate the budded-to-hypha-form transition and the expression of hypha-specific genes

Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.

Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other