Date of Award

2001

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Joseph Dolan

Second Advisor

Gillian Galbraith

Third Advisor

Jean-Michel Goust

Fourth Advisor

John Hildebrandt

Fifth Advisor

Kathryn E. Meier

Abstract

Phospholipase D (PLD) enzymes play significant roles in phospholipid metabolism, secretion, and lipid signal transduction. The yeast Candida albicans possesses a PLD activity that is designated PLD1. This enzyme has been implicated in the regulation of dimorphic transition. In vivo assays using sphinganine, propranolol, and staurosporine were used to test the importance of phosphatidic acid (PA) and diacylglycerol (DAG), two downstream products of PLDI activity. A concentration of 5 µM sphinganine was sufficient to decrease the appearance of germ tubes with no effect on culture doubling times. However, at higher concentrations sphinganine was inhibitory to cell growth. Propranolol concentrations up to 1 mM were able to inhibit germ tube formation without increasing doubling times. Increasing concentrations of propranolol (e.g. 2 mM) did inhibit yeast cell growth. Varying concentrations of staurosporine had no effect on either germ tube formation or cell growth. In vitro assays demonstrated a decrease in the conversion of PA to DAG with increasing amounts of propranolol. Since DAG kinase assays showed that DAG levels were not affected, in vivo, by the presence of propranolol, phosphatidic acid phosphohydrolase (PAP) activity was measured in the presence of propranolol. No significant change in PAPase function was observed. Wildtype albicans, SC5314, showed numerous, invasive hyphae when plated on Spider medium, in contrast to a confirmed pld1Δ mutant which showed no visible hyphae. DAG kinase assays comparing DAG levels between the wild type and mutant strain showed significantly higher levels in the null mutant. These results suggest that C. albicans may be compensating for a loss of PLD-derived DAG, possibly through up-regulation of phospholipase C (PLC). The enzymes monoacyl-glycerol-acyl-transferase (MAGAT) and inositol-phosphoryl ceramide synthase (IPC synthase) may participate in replacement of the DAG pool. The data also implicate PA, not DAG, as the enzyme needed for morphogenesis.

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